Peptide and protein delivery using new drug delivery systems.

Science.gov (United States)

Jain, Ashish; Jain, Aviral; Gulbake, Arvind; Shilpi, Satish; Hurkat, Pooja; Jain, Sanjay K

2013-01-01

Pharmaceutical and biotechnological research sorts protein drug delivery systems by importance based on their various therapeutic applications. The effective and potent action of the proteins/peptides makes them the drugs of choice for the treatment of numerous diseases. Major research issues in protein delivery include the stabilization of proteins in delivery devices and the design of appropriate target-specific protein carriers. Many efforts have been made for effective delivery of proteins/peptidal drugs through various routes of administrations for successful therapeutic effects. Nanoparticles made of biodegradable polymers such as poly lactic acid, polycaprolactone, poly(lactic-co-glycolic acid), the poly(fumaric-co-sebacic) anhydride chitosan, and modified chitosan, as well as solid lipids, have shown great potential in the delivery of proteins/peptidal drugs. Moreover, scientists also have used liposomes, PEGylated liposomes, niosomes, and aquasomes, among others, for peptidal drug delivery. They also have developed hydrogels and transdermal drug delivery systems for peptidal drug delivery. A receptor-mediated delivery system is another attractive strategy to overcome the limitation in drug absorption that enables the transcytosis of the protein across the epithelial barrier. Modification such as PEGnology is applied to various proteins and peptides of the desired protein and peptides also increases the circulating life, solubility and stability, pharmacokinetic properties, and antigenicity of protein. This review focuses on various approaches for effective protein/peptidal drug delivery, with special emphasis on insulin delivery.

Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector

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Caplan, Jeffrey L.; Mamillapalli, Padmavathi; Burch-Smith, Tessa M.; Czymmek, Kirk; Dinesh-Kumar, S.P.

2008-01-01

Summary Plant innate immunity relies on the recognition of pathogen effector molecules by nucleotide-binding-leucine-rich repeat (NB-LRR) immune receptor families. Previously we have shown the N immune receptor, a member of TIR-NB-LRR family, indirectly recognizes the 50-kDa helicase (p50) domain of Tobacco mosaic virus (TMV) through its TIR domain. We have identified an N receptor-interacting protein, NRIP1, that directly interacts with both N's TIR domain and p50. NRIP1 is a functional rhodanese sulfurtransferase and is required for N to provide complete resistance to TMV. Interestingly, NRIP1 that normally localizes to the chloroplasts is recruited to the cytoplasm and nucleus by the p50 effector. As a consequence, NRIP1 interacts with N only in the presence of the p50 effector. Our findings show that a chloroplastic protein is intimately involved in pathogen recognition. We propose that N's activation requires a pre-recognition complex containing the p50 effector and NRIP1. PMID:18267075

TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA targeting proteins

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Doyle, Erin L.; Stoddard, Barry L.; Voytas, Daniel F.; Bogdanove, Adam J.

2013-01-01

Transcription activator-like (TAL) effectors are transcription factors injected into plant cells by pathogenic bacteria in the genus Xanthomonas. They function as virulence factors by activating host genes important for disease, or as avirulence factors by turning on genes that provide resistance. DNA binding specificity is encoded by polymorphic repeats in each protein that correspond one-to-one with different nucleotides. This code has facilitated target identification and opened new avenues for engineering disease resistance. It has also enabled TAL effector customization for targeted gene control, genome editing, and other applications. This article reviews the structural basis for TAL effector-DNA specificity, the impact of the TAL effector-DNA code on plant pathology and engineered resistance, and recent accomplishments and future challenges in TAL effector-based DNA targeting. PMID:23707478

A Legionella pneumophila effector protein encoded in a region of genomic plasticity binds to Dot/Icm-modified vacuoles.

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Shira Ninio

2009-01-01

Full Text Available Legionella pneumophila is an opportunistic pathogen that can cause a severe pneumonia called Legionnaires' disease. In the environment, L. pneumophila is found in fresh water reservoirs in a large spectrum of environmental conditions, where the bacteria are able to replicate within a variety of protozoan hosts. To survive within eukaryotic cells, L. pneumophila require a type IV secretion system, designated Dot/Icm, that delivers bacterial effector proteins into the host cell cytoplasm. In recent years, a number of Dot/Icm substrate proteins have been identified; however, the function of most of these proteins remains unknown, and it is unclear why the bacterium maintains such a large repertoire of effectors to promote its survival. Here we investigate a region of the L. pneumophila chromosome that displays a high degree of plasticity among four sequenced L. pneumophila strains. Analysis of GC content suggests that several genes encoded in this region were acquired through horizontal gene transfer. Protein translocation studies establish that this region of genomic plasticity encodes for multiple Dot/Icm effectors. Ectopic expression studies in mammalian cells indicate that one of these substrates, a protein called PieA, has unique effector activities. PieA is an effector that can alter lysosome morphology and associates specifically with vacuoles that support L. pneumophila replication. It was determined that the association of PieA with vacuoles containing L. pneumophila requires modifications to the vacuole mediated by other Dot/Icm effectors. Thus, the localization properties of PieA reveal that the Dot/Icm system has the ability to spatially and temporally control the association of an effector with vacuoles containing L. pneumophila through activities mediated by other effector proteins.

Hitting the Sweet Spot: Glycans as Targets of Fungal Defense Effector Proteins

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Markus Künzler

2015-05-01

Full Text Available Organisms which rely solely on innate defense systems must combat a large number of antagonists with a comparatively low number of defense effector molecules. As one solution of this problem, these organisms have evolved effector molecules targeting epitopes that are conserved between different antagonists of a specific taxon or, if possible, even of different taxa. In order to restrict the activity of the defense effector molecules to physiologically relevant taxa, these target epitopes should, on the other hand, be taxon-specific and easily accessible. Glycans fulfill all these requirements and are therefore a preferred target of defense effector molecules, in particular defense proteins. Here, we review this defense strategy using the example of the defense system of multicellular (filamentous fungi against microbial competitors and animal predators.

Identification and Initial Characterization of the Effectors of an Anther Smut Fungus and Potential Host Target Proteins

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Venkata S. Kuppireddy

2017-11-01

Full Text Available (1 Background: Plant pathogenic fungi often display high levels of host specificity and biotrophic fungi; in particular, they must manipulate their hosts to avoid detection and to complete their obligate pathogenic lifecycles. One important strategy of such fungi is the secretion of small proteins that serve as effectors in this process. Microbotryum violaceum is a species complex whose members infect members of the Caryophyllaceae; M. lychnidis-dioicae, a parasite on Silene latifolia, is one of the best studied interactions. We are interested in identifying and characterizing effectors of the fungus and possible corresponding host targets; (2 Methods: In silico analysis of the M. lychnidis-dioicae genome and transcriptomes allowed us to predict a pool of small secreted proteins (SSPs with the hallmarks of effectors, including a lack of conserved protein family (PFAM domains and also localized regions of disorder. Putative SSPs were tested for secretion using a yeast secretion trap method. We then used yeast two-hybrid analyses for candidate-secreted effectors to probe a cDNA library from a range of growth conditions of the fungus, including infected plants; (3 Results: Roughly 50 SSPs were identified by in silico analysis. Of these, 4 were studied further and shown to be secreted, as well as examined for potential host interactors. One of the putative effectors, MVLG_01732, was found to interact with Arabidopsis thaliana calcium-dependent lipid binding protein (AtCLB and with cellulose synthase interactive protein 1 orthologues; and (4 Conclusions: The identification of a pool of putative effectors provides a resource for functional characterization of fungal proteins that mediate the delicate interaction between pathogen and host. The candidate targets of effectors, e.g., AtCLB, involved in pollen germination suggest tantalizing insights that could drive future studies.

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence1[OPEN

Science.gov (United States)

2017-01-01

Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M. persicae-host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. PMID:28100451

Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

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Fernando Navarro-Garcia

2013-01-01

Full Text Available The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology.

Site-directed mutagenesis of the Arabidopsis heterotrimeric G protein β subunit suggests divergent mechanisms of effector activation between plant and animal G proteins.

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Chakravorty, David; Trusov, Yuri; Botella, José Ramón

2012-03-01

Heterotrimeric G proteins are integral components of signal transduction in humans and other mammals and have been therefore extensively studied. However, while they are known to mediate many processes, much less is currently known about the effector pathways and molecular mechanisms used by these proteins to regulate effectors in plants. We designed a complementation strategy to study G protein signaling in Arabidopsis thaliana, particularly the mechanism of action of AGB1, the sole identified β subunit. We used biochemical and effector regulation data from human G protein studies to identify four potentially important residues for site-directed mutagenesis (T65, M111, D250 and W361 of AGB1). Each residue was individually mutated and the resulting mutated protein introduced in the agb1-2 mutant background under the control of the native AGB1 promoter. Interestingly, even though these mutations have been shown to have profound effects on effector signaling in humans, all the mutated subunits were able to restore thirteen of the fifteen Gβ-deficient phenotypes characterized in this study. Only one mutated protein, T65A was unable to complement the hypersensitivity to mannitol during germination observed in agb1 mutants; while only D250A failed to restore lateral root numbers in the agb1 mutant to wild-type levels. Our results suggest that the mechanisms used in mammalian G protein signaling are not well conserved in plant G protein signaling, and that either the effectors used by plant G proteins, or the mechanisms used to activate them, are at least partially divergent from the well-studied mammalian G proteins.

Effector protein translocation by the Coxiella burnetii Dot/Icm type IV secretion system requires endocytic maturation of the pathogen-occupied vacuole.

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Hayley J Newton

Full Text Available The human pathogen Coxiella burnetii encodes a type IV secretion system called Dot/Icm that is essential for intracellular replication. The Dot/Icm system delivers bacterial effector proteins into the host cytosol during infection. The effector proteins delivered by C. burnetii are predicted to have important functions during infection, but when these proteins are needed during infection has not been clearly defined. Here, we use a reporter system consisting of fusion proteins that have a β-lactamase enzyme (BlaM fused to C. burnetii effector proteins to study protein translocation by the Dot/Icm system. Translocation of BlaM fused to the effector proteins CBU0077, CBU1823 and CBU1524 was not detected until 8-hours after infection of HeLa cells, which are permissive for C. burnetii replication. Translocation of these effector fusion proteins by the Dot/Icm system required acidification of the Coxiella-containing vacuole. Silencing of the host genes encoding the membrane transport regulators Rab5 or Rab7 interfered with effector translocation, which indicates that effectors are not translocated until bacteria traffic to a late endocytic compartment in the host cell. Similar requirements for effector translocation were discerned in bone marrow macrophages derived from C57BL/6 mice, which are primary cells that restrict the intracellular replication of C. burnetii. In addition to requiring endocytic maturation of the vacuole for Dot/Icm-mediated translocation of effectors, bacterial transcription was required for this process. Thus, translocation of effector proteins by the C. burnetii Dot/Icm system occurs after acidification of the CCV and maturation of this specialized organelle to a late endocytic compartment. This indicates that creation of the specialized vacuole in which C. burnetii replicates represents a two-stage process mediated initially by host factors that regulate endocytic maturation and then by bacterial effectors delivered into

The Salmonella Effector Protein SopA Modulates Innate Immune Responses by Targeting TRIM E3 Ligase Family Members.

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Jana Kamanova

2016-04-01

Full Text Available Salmonella Typhimurium stimulates inflammatory responses in the intestinal epithelium, which are essential for its ability to replicate within the intestinal tract. Stimulation of these responses is strictly dependent on the activity of a type III secretion system encoded within its pathogenicity island 1, which through the delivery of effector proteins, triggers signaling pathways leading to inflammation. One of these effectors is SopA, a HECT-type E3 ligase, which is required for the efficient stimulation of inflammation in an animal model of Salmonella Typhimurium infection. We show here that SopA contributes to the stimulation of innate immune responses by targeting two host E3 ubiquitin ligases, TRIM56 and TRIM65. We also found that TRIM65 interacts with the innate immune receptor MDA5 enhancing its ability to stimulate interferon-β signaling. Therefore, by targeting TRIM56 and TRIM65, SopA can stimulate signaling through two innate immune receptors, RIG-I and MDA5. These findings describe a Salmonella mechanism to modulate inflammatory responses by directly targeting innate immune signaling mechanisms.

Type VI secretion system MIX-effectors carry both antibacterial and anti-eukaryotic activities.

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Ray, Ann; Schwartz, Nika; de Souza Santos, Marcela; Zhang, Junmei; Orth, Kim; Salomon, Dor

2017-11-01

Most type VI secretion systems (T6SSs) described to date are protein delivery apparatuses that mediate bactericidal activities. Several T6SSs were also reported to mediate virulence activities, although only few anti-eukaryotic effectors have been described. Here, we identify three T6SSs in the marine bacterium Vibrio proteolyticus and show that T6SS1 mediates bactericidal activities under warm marine-like conditions. Using comparative proteomics, we find nine potential T6SS1 effectors, five of which belong to the polymorphic MIX-effector class. Remarkably, in addition to six predicted bactericidal effectors, the T6SS1 secretome includes three putative anti-eukaryotic effectors. One of these is a MIX-effector containing a cytotoxic necrotizing factor 1 domain. We demonstrate that T6SS1 can use this MIX-effector to target phagocytic cells, resulting in morphological changes and actin cytoskeleton rearrangements. In conclusion, the V. proteolyticus T6SS1, a system homologous to one found in pathogenic vibrios, uses a suite of polymorphic effectors that target both bacteria and eukaryotic neighbors. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

SPRYSEC effector proteins in Globodera rostochiensis

NARCIS (Netherlands)

Rehman, S.

2008-01-01

Plant pathogens inject so-called effector molecules into the cells of a host plant to promote their growth and reproduction in these hosts. In plant parasitic nematodes, these effector molecules are produced in the salivary glands. The objective of this thesis was to identify and characterize

Long-Term Live Cell Imaging Reveals New Roles For Salmonella Effector Proteins SseG and SteA

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McQuate, Sarah E.; Young, Alexandra M.; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S.; Palmer, Amy E.

2016-01-01

Summary Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here we establish a pipeline for long-term (16 hours) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages, and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyperreplication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. PMID:27376507

Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on diots and monocots

NARCIS (Netherlands)

Stergiopoulos, I.; Burg, van den H.A.; Ökmen, B.; Beenen, H.G.; Liere, van S.; Kema, G.H.J.; Wit, de P.J.G.M.

2010-01-01

Most fungal effectors characterized so far are species-specific and facilitate virulence on a particular host plant. During infection of its host tomato, Cladosporium fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce

Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

Science.gov (United States)

Zhou, Li; Wang, Hsuan-Yao; Tong, Shanshan; Okamoto, Curtis T; Shen, Wei-Chiang; Zaro, Jennica L

2017-02-01

Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc) 2 ), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc) 2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc) 2 -mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc) 2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structure and thermodynamics of effector molecule binding to the nitrogen signal transduction PII protein GlnZ from Azospirillum brasilense.

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Truan, Daphné; Bjelić, Saša; Li, Xiao-Dan; Winkler, Fritz K

2014-07-29

The trimeric PII signal transduction proteins regulate the function of a variety of target proteins predominantly involved in nitrogen metabolism. ATP, ADP and 2-oxoglutarate (2-OG) are key effector molecules influencing PII binding to targets. Studies of PII proteins have established that the 20-residue T-loop plays a central role in effector sensing and target binding. However, the specific effects of effector binding on T-loop conformation have remained poorly documented. We present eight crystal structures of the Azospirillum brasilense PII protein GlnZ, six of which are cocrystallized and liganded with ADP or ATP. We find that interaction with the diphosphate moiety of bound ADP constrains the N-terminal part of the T-loop in a characteristic way that is maintained in ADP-promoted complexes with target proteins. In contrast, the interactions with the triphosphate moiety in ATP complexes are much more variable and no single predominant interaction mode is apparent except for the ternary MgATP/2-OG complex. These conclusions can be extended to most investigated PII proteins of the GlnB/GlnK subfamily. Unlike reported for other PII proteins, microcalorimetry reveals no cooperativity between the three binding sites of GlnZ trimers for any of the three effectors under carefully controlled experimental conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on dicots and monocots.

Science.gov (United States)

Stergiopoulos, Ioannis; van den Burg, Harrold A; Okmen, Bilal; Beenen, Henriek G; van Liere, Sabine; Kema, Gert H J; de Wit, Pierre J G M

2010-04-20

Most fungal effectors characterized so far are species-specific and facilitate virulence on a particular host plant. During infection of its host tomato, Cladosporium fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce effector-triggered immunity in their presence. Here we show that homologs of the C. fulvum Avr4 and Ecp2 effectors are present in other pathogenic fungi of the Dothideomycete class, including Mycosphaerella fijiensis, the causal agent of black Sigatoka disease of banana. We demonstrate that the Avr4 homolog of M. fijiensis is a functional ortholog of C. fulvum Avr4 that protects fungal cell walls against hydrolysis by plant chitinases through binding to chitin and, despite the low overall sequence homology, triggers a Cf-4-mediated hypersensitive response (HR) in tomato. Furthermore, three homologs of C. fulvum Ecp2 are found in M. fijiensis, one of which induces different levels of necrosis or HR in tomato lines that lack or contain a putative cognate Cf-Ecp2 protein, respectively. In contrast to Avr4, which acts as a defensive virulence factor, M. fijiensis Ecp2 likely promotes virulence by interacting with a putative host target causing host cell necrosis, whereas Cf-Ecp2 could possibly guard the virulence target of Ecp2 and trigger a Cf-Ecp2-mediated HR. Overall our data suggest that Avr4 and Ecp2 represent core effectors that are collectively recognized by single cognate Cf-proteins. Transfer of these Cf genes to plant species that are attacked by fungi containing these cognate core effectors provides unique ways for breeding disease-resistant crops.

Protein-Based Drug-Delivery Materials

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Dave Jao

2017-05-01

Full Text Available There is a pressing need for long-term, controlled drug release for sustained treatment of chronic or persistent medical conditions and diseases. Guided drug delivery is difficult because therapeutic compounds need to survive numerous transport barriers and binding targets throughout the body. Nanoscale protein-based polymers are increasingly used for drug and vaccine delivery to cross these biological barriers and through blood circulation to their molecular site of action. Protein-based polymers compared to synthetic polymers have the advantages of good biocompatibility, biodegradability, environmental sustainability, cost effectiveness and availability. This review addresses the sources of protein-based polymers, compares the similarity and differences, and highlights characteristic properties and functionality of these protein materials for sustained and controlled drug release. Targeted drug delivery using highly functional multicomponent protein composites to guide active drugs to the site of interest will also be discussed. A systematical elucidation of drug-delivery efficiency in the case of molecular weight, particle size, shape, morphology, and porosity of materials will then be demonstrated to achieve increased drug absorption. Finally, several important biomedical applications of protein-based materials with drug-delivery function—including bone healing, antibiotic release, wound healing, and corneal regeneration, as well as diabetes, neuroinflammation and cancer treatments—are summarized at the end of this review.

Preparing and evaluating delivery systems for proteins

DEFF Research Database (Denmark)

Jorgensen, L; Moeller, E H; van de Weert, M

2006-01-01

From a formulation perspective proteins are complex and therefore challenging molecules to develop drug delivery systems for. The success of a formulation depends on the ability of the protein to maintain the native structure and activity during preparation and delivery as well as during shipping...... and long-term storage of the formulation. Therefore, the development and evaluation of successful and promising drug delivery systems is essential. In the present review, some of the particulate drug delivery systems for parenteral delivery of protein are presented and discussed. The challenge...... for incorporation of protein in particulate delivery systems is exemplified by water-in-oil emulsions....

Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

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Peter eMoffett

2015-08-01

Full Text Available Potato cyst nematodes (PCNs, including Globodera rostochiensis (Woll., are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC family. SPRYSEC proteins are unique to members of the genera Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense response in N. tabacum, and tobacco was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system.

Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

KAUST Repository

Ali, Shawkat

2015-08-11

Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins delivered to the apoplast and to the host cytoplasm. A number of effectors from G. rostochiensis predicted to be delivered to the host cytoplasm have been identified, including several belonging to the secreted SPRY domain (SPRYSEC) family. SPRYSEC proteins are unique to members of the genus Globodera and have been implicated in both the induction and the repression of host defense responses. We have tested the properties of six different G. rostochiensis SPRYSEC proteins by expressing them in Nicotiana benthamiana and N. tabacum. We have found that all SPRYSEC proteins tested are able to suppress defense responses induced by NB-LRR proteins as well as cell death induced by elicitors, suggesting that defense repression is a common characteristic of members of this effector protein family. At the same time, GrSPRYSEC-15 elicited a defense responses in N. tabacum, which was found to be resistant to a virus expressing GrSPRYSEC-15. These results suggest that SPRYSEC proteins may possess characteristics that allow them to be recognized by the plant immune system.

TAL effectors and the executor R genes.

Science.gov (United States)

Zhang, Junli; Yin, Zhongchao; White, Frank

2015-01-01

Transcription activator-like (TAL) effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL effector associated resistance (R) genes have been characterized-recessive, dominant non-transcriptional, and dominant TAL effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL effector binding to discrete effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have catalytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance.

TAL effectors and the executor R genes

Directory of Open Access Journals (Sweden)

Junli eZhang

2015-08-01

Full Text Available Transcription activation-like (TAL effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL effector associated resistance (R genes have been characterized - recessive, dominant non-transcriptional and dominant TAL effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL effector binding to discrete effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have catalytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance.

Three Antagonistic Cyclic di-GMP-Catabolizing Enzymes Promote Differential Dot/Icm Effector Delivery and Intracellular Survival at the Early Steps of Legionella pneumophila Infection

Science.gov (United States)

Allombert, Julie; Lazzaroni, Jean-Claude; Baïlo, Nathalie; Gilbert, Christophe; Charpentier, Xavier; Doublet, Patricia

2014-01-01

Legionella pneumophila is an intracellular pathogen which replicates within protozoan cells and can accidently infect alveolar macrophages, causing an acute pneumonia in humans. The second messenger cyclic di-GMP (c-di-GMP) has been shown to play key roles in the regulation of various bacterial processes, including virulence. While investigating the function of the 22 potential c-di-GMP-metabolizing enzymes of the L. pneumophila Lens strain, we found three that directly contribute to its ability to infect both protozoan and mammalian cells. These three enzymes display diguanylate cyclase (Lpl0780), phosphodiesterase (Lpl1118), and bifunctional diguanylate cyclase/phosphodiesterase (Lpl0922) activities, which are all required for the survival and intracellular replication of L. pneumophila. Mutants with deletions of the corresponding genes are efficiently taken up by phagocytic cells but are partially defective for the escape of the Legionella-containing vacuole (LCV) from the host degradative endocytic pathway and result in lower survival. In addition, Lpl1118 is required for efficient endoplasmic reticulum recruitment to the LCV. Trafficking and biogenesis of the LCV are dependent upon the orchestrated actions of several type 4 secretion system Dot/Icm effectors proteins, which exhibit differentially altered translocation in the three mutants. While translocation of some effectors remained unchanged, others appeared over- and undertranslocated. A general translocation offset of the large repertoire of Dot/Icm effectors may be responsible for the observed defects in the trafficking and biogenesis of the LCV. Our results suggest that L. pneumophila uses cyclic di-GMP signaling to fine-tune effector delivery and ensure effective evasion of the host degradative pathways and establishment of a replicative vacuole. PMID:24379287

Stimuli-responsive nanomaterials for therapeutic protein delivery.

Science.gov (United States)

Lu, Yue; Sun, Wujin; Gu, Zhen

2014-11-28

Protein therapeutics have emerged as a significant role in treatment of a broad spectrum of diseases, including cancer, metabolic disorders and autoimmune diseases. The efficacy of protein therapeutics, however, is limited by their instability, immunogenicity and short half-life. In order to overcome these barriers, tremendous efforts have recently been made in developing controlled protein delivery systems. Stimuli-triggered release is an appealing and promising approach for protein delivery and has made protein delivery with both spatiotemporal- and dosage-controlled manners possible. This review surveys recent advances in controlled protein delivery of proteins or peptides using stimuli-responsive nanomaterials. Strategies utilizing both physiological and external stimuli are introduced and discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

An optimal set of features for predicting type IV secretion system effector proteins for a subset of species based on a multi-level feature selection approach.

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Zhila Esna Ashari

Full Text Available Type IV secretion systems (T4SS are multi-protein complexes in a number of bacterial pathogens that can translocate proteins and DNA to the host. Most T4SSs function in conjugation and translocate DNA; however, approximately 13% function to secrete proteins, delivering effector proteins into the cytosol of eukaryotic host cells. Upon entry, these effectors manipulate the host cell's machinery for their own benefit, which can result in serious illness or death of the host. For this reason recognition of T4SS effectors has become an important subject. Much previous work has focused on verifying effectors experimentally, a costly endeavor in terms of money, time, and effort. Having good predictions for effectors will help to focus experimental validations and decrease testing costs. In recent years, several scoring and machine learning-based methods have been suggested for the purpose of predicting T4SS effector proteins. These methods have used different sets of features for prediction, and their predictions have been inconsistent. In this paper, an optimal set of features is presented for predicting T4SS effector proteins using a statistical approach. A thorough literature search was performed to find features that have been proposed. Feature values were calculated for datasets of known effectors and non-effectors for T4SS-containing pathogens for four genera with a sufficient number of known effectors, Legionella pneumophila, Coxiella burnetii, Brucella spp, and Bartonella spp. The features were ranked, and less important features were filtered out. Correlations between remaining features were removed, and dimensional reduction was accomplished using principal component analysis and factor analysis. Finally, the optimal features for each pathogen were chosen by building logistic regression models and evaluating each model. The results based on evaluation of our logistic regression models confirm the effectiveness of our four optimal sets of

Oomycetes, effectors, and all that jazz.

Science.gov (United States)

Bozkurt, Tolga O; Schornack, Sebastian; Banfield, Mark J; Kamoun, Sophien

2012-08-01

Plant pathogenic oomycetes secrete a diverse repertoire of effector proteins that modulate host innate immunity and enable parasitic infection. Understanding how effectors evolve, translocate and traffic inside host cells, and perturb host processes are major themes in the study of oomycete-plant interactions. The last year has seen important progress in the study of oomycete effectors with, notably, the elucidation of the 3D structures of five RXLR effectors, and novel insights into how cytoplasmic effectors subvert host cells. In this review, we discuss these and other recent advances and highlight the most important open questions in oomycete effector biology. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of effector-like proteins in Trichoderma spp. and role of a hydrophobin in the plant-fungus interaction and mycoparasitism.

Science.gov (United States)

Guzmán-Guzmán, Paulina; Alemán-Duarte, Mario Iván; Delaye, Luis; Herrera-Estrella, Alfredo; Olmedo-Monfil, Vianey

2017-02-15

Trichoderma spp. can establish beneficial interactions with plants by promoting plant growth and defense systems, as well as, antagonizing fungal phytopathogens in mycoparasitic interactions. Such interactions depend on signal exchange between both participants and can be mediated by effector proteins that alter the host cell structure and function, allowing the establishment of the relationship. The main purpose of this work was to identify, using computational methods, candidates of effector proteins from T. virens, T. atroviride and T. reesei, validate the expression of some of the genes during a beneficial interaction and mycoparasitism and to define the biological function for one of them. We defined a catalogue of putative effector proteins from T. virens, T. atroviride and T. reesei. We further validated the expression of 16 genes encoding putative effector proteins from T. virens and T. atroviride during the interaction with the plant Arabidopsis thaliana, and with two anastomosis groups of the phytopathogenic fungus Rhizoctonia solani. We found genes which transcript levels are modified in response to the presence of both plant fungi, as well as genes that respond only to either a plant or a fungal host. Further, we show that overexpression of the gene tvhydii1, a Class II hydrophobin family member, enhances the antagonistic activity of T. virens against R. solani AG2. Further, deletion of tvhydii1 results in reduced colonization of plant roots, while its overexpression increases it. Our results show that Trichoderma is able to respond in different ways to the presence of a plant or a fungal host, and it can even distinguish between different strains of fungi of a given species. The putative effector proteins identified here may play roles in preventing perception of the fungus by its hosts, favoring host colonization or protecting it from the host's defense response. Finally, the novel effector protein TVHYDII1 plays a role in plant root colonization by T

The phytopathogenic virulent effector protein RipI induces apoptosis in budding yeast Saccharomyces cerevisiae.

Science.gov (United States)

Deng, Meng-Ying; Sun, Yun-Hao; Li, Pai; Fu, Bei; Shen, Dong; Lu, Yong-Jun

2016-10-01

Virulent protein toxins secreted by the bacterial pathogens can cause cytotoxicity by various molecular mechanisms to combat host cell defense. On the other hand, these proteins can also be used as probes to investigate the defense pathway of host innate immunity. Ralstonia solanacearum, one of the most virulent bacterial phytopathogens, translocates more than 70 effector proteins via type III secretion system during infection. Here, we characterized the cytotoxicity of effector RipI in budding yeast Saccharomyce scerevisiae, an alternative host model. We found that over-expression of RipI resulted in severe growth defect and arginine (R) 117 within the predicted integrase motif was required for inhibition of yeast growth. The phenotype of death manifested the hallmarks of apoptosis. Our data also revealed that RipI-induced apoptosis was independent of Yca1 and mitochondria-mediated apoptotic pathways because Δyca1 and Δaif1 were both sensitive to RipI as compared with the wild type. We further demonstrated that RipI was localized in the yeast nucleus and the N-terminal 1-174aa was required for the localization. High-throughput RNA sequencing analysis showed that upon RipI over-expression, 101 unigenes of yeast ribosome presented lower expression level, and 42 GO classes related to the nucleus or recombination were enriched with differential expression levels. Taken together, our data showed that a nuclear-targeting effector RipI triggers yeast apoptosis, potentially dependent on its integrase function. Our results also provided an alternative strategy to dissect the signaling pathway of cytotoxicity induced by the protein toxins. Copyright © 2016 Elsevier Ltd. All rights reserved.

Long-term delivery of protein therapeutics.

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Vaishya, Ravi; Khurana, Varun; Patel, Sulabh; Mitra, Ashim K

2015-03-01

Proteins are effective biotherapeutics with applications in diverse ailments. Despite being specific and potent, their full clinical potential has not yet been realized. This can be attributed to short half-lives, complex structures, poor in vivo stability, low permeability, frequent parenteral administrations and poor adherence to treatment in chronic diseases. A sustained release system, providing controlled release of proteins, may overcome many of these limitations. This review focuses on recent development in approaches, especially polymer-based formulations, which can provide therapeutic levels of proteins over extended periods. Advances in particulate, gel-based formulations and novel approaches for extended protein delivery are discussed. Emphasis is placed on dosage form, method of preparation, mechanism of release and stability of biotherapeutics. Substantial advancements have been made in the field of extended protein delivery via various polymer-based formulations over last decade despite the unique delivery-related challenges posed by protein biologics. A number of injectable sustained-release formulations have reached market. However, therapeutic application of proteins is still hampered by delivery-related issues. A large number of protein molecules are under clinical trials, and hence, there is an urgent need to develop new methods to deliver these highly potent biologics.

Nematode effector proteins: an emerging paradigm of parasitism

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Phytonematodes use a stylet and secreted effectors to invade host tissues and extract nutrients to support their growth and development. The molecular function of nematode effectors is currently the subject of intense investigation. In this review, we summarize our current understanding of nematode ...

Differential expression of candidate salivary effector proteins in field collections of Hessian fly, Mayetiola destructor

Science.gov (United States)

Johnson, A J; Shukle, R H; Chen, M-S; Srivastava, S; Subramanyam, S; Schemerhorn, B J; Weintraub, P G; Abdel Moniem, H E M; Flanders, K L; Buntin, G D; Williams, C E

2015-01-01

Evidence is emerging that some proteins secreted by gall-forming parasites of plants act as effectors responsible for systemic changes in the host plant, such as galling and nutrient tissue formation. A large number of secreted salivary gland proteins (SSGPs) that are the putative effectors responsible for the physiological changes elicited in susceptible seedling wheat by Hessian fly, Mayetiola destructor (Say), larvae have been documented. However, how the genes encoding these candidate effectors might respond under field conditions is unknown. The goal of this study was to use microarray analysis to investigate variation in SSGP transcript abundance amongst field collections from different geographical regions (southeastern USA, central USA, and the Middle East). Results revealed significant variation in SSGP transcript abundance amongst the field collections studied. The field collections separated into three distinct groups that corresponded to the wheat classes grown in the different geographical regions as well as to recently described Hessian fly populations. These data support previous reports correlating Hessian fly population structure with micropopulation differences owing to agro-ecosystem parameters such as cultivation of regionally adapted wheat varieties, deployment of resistance genes and variation in climatic conditions. PMID:25528896

Dual roles for the variable domain in protein trafficking and host-specific recognition of Heterodera glycines CLE effector proteins

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Soybean cyst nematodes (Heterodera glycines) produce secreted effector proteins that function as peptide mimics of plant CLAVATA3 / ESR (CLE)-like peptides probably involved in the developmental reprogramming of root cells to form specialized feeding cells called syncytia. The site of action and me...

Complex structure of type VI peptidoglycan muramidase effector and a cognate immunity protein

International Nuclear Information System (INIS)

Wang, Tianyu; Ding, Jinjing; Zhang, Ying; Wang, Da-Cheng; Liu, Wei

2013-01-01

The structure of the Tse3–Tsi3 complex associated with the bacterial type VI secretion system of P. aeruginosa has been solved and refined at 1.9 Å resolution. The structural basis of the recognition of the muramidase effector and its inactivation by its cognate immunity protein is revealed. The type VI secretion system (T6SS) is a bacterial protein-export machine that is capable of delivering virulence effectors between Gram-negative bacteria. The T6SS of Pseudomonas aeruginosa transports two lytic enzymes, Tse1 and Tse3, to degrade cell-wall peptidoglycan in the periplasm of rival bacteria that are competing for niches via amidase and muramidase activities, respectively. Two cognate immunity proteins, Tsi1 and Tsi3, are produced by the bacterium to inactivate the two antibacterial effectors, thereby protecting its siblings from self-intoxication. Recently, Tse1–Tsi1 has been structurally characterized. Here, the structure of the Tse3–Tsi3 complex is reported at 1.9 Å resolution. The results reveal that Tse3 contains a C-terminal catalytic domain that adopts a soluble lytic transglycosylase (SLT) fold in which three calcium-binding sites were surprisingly observed close to the catalytic Glu residue. The electrostatic properties of the substrate-binding groove are also distinctive from those of known structures with a similar fold. All of these features imply that a unique catalytic mechanism is utilized by Tse3 in cleaving glycosidic bonds. Tsi3 comprises a single domain showing a β-sandwich architecture that is reminiscent of the immunoglobulin fold. Three loops of Tsi3 insert deeply into the groove of Tse3 and completely occlude its active site, which forms the structural basis of Tse3 inactivation. This work is the first crystallographic report describing the three-dimensional structure of the Tse3–Tsi3 effector–immunity pair

Complex structure of type VI peptidoglycan muramidase effector and a cognate immunity protein

Energy Technology Data Exchange (ETDEWEB)

Wang, Tianyu [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Ding, Jinjing; Zhang, Ying; Wang, Da-Cheng, E-mail: dcwang@ibp.ac.cn [Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China); Liu, Wei, E-mail: dcwang@ibp.ac.cn [The Third Military Medical University, Chongqing 400038 (China); Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

2013-10-01

The structure of the Tse3–Tsi3 complex associated with the bacterial type VI secretion system of P. aeruginosa has been solved and refined at 1.9 Å resolution. The structural basis of the recognition of the muramidase effector and its inactivation by its cognate immunity protein is revealed. The type VI secretion system (T6SS) is a bacterial protein-export machine that is capable of delivering virulence effectors between Gram-negative bacteria. The T6SS of Pseudomonas aeruginosa transports two lytic enzymes, Tse1 and Tse3, to degrade cell-wall peptidoglycan in the periplasm of rival bacteria that are competing for niches via amidase and muramidase activities, respectively. Two cognate immunity proteins, Tsi1 and Tsi3, are produced by the bacterium to inactivate the two antibacterial effectors, thereby protecting its siblings from self-intoxication. Recently, Tse1–Tsi1 has been structurally characterized. Here, the structure of the Tse3–Tsi3 complex is reported at 1.9 Å resolution. The results reveal that Tse3 contains a C-terminal catalytic domain that adopts a soluble lytic transglycosylase (SLT) fold in which three calcium-binding sites were surprisingly observed close to the catalytic Glu residue. The electrostatic properties of the substrate-binding groove are also distinctive from those of known structures with a similar fold. All of these features imply that a unique catalytic mechanism is utilized by Tse3 in cleaving glycosidic bonds. Tsi3 comprises a single domain showing a β-sandwich architecture that is reminiscent of the immunoglobulin fold. Three loops of Tsi3 insert deeply into the groove of Tse3 and completely occlude its active site, which forms the structural basis of Tse3 inactivation. This work is the first crystallographic report describing the three-dimensional structure of the Tse3–Tsi3 effector–immunity pair.

A Family of Salmonella Type III Secretion Effector Proteins Selectively Targets the NF-κB Signaling Pathway to Preserve Host Homeostasis.

Science.gov (United States)

Sun, Hui; Kamanova, Jana; Lara-Tejero, Maria; Galán, Jorge E

2016-03-01

Microbial infections usually lead to host innate immune responses and inflammation. These responses most often limit pathogen replication although they can also result in host-tissue damage. The enteropathogenic bacteria Salmonella Typhimurium utilizes a type III secretion system to induce intestinal inflammation by delivering specific effector proteins that stimulate signal transduction pathways resulting in the production of pro-inflammatory cytokines. We show here that a family of related Salmonella Typhimurium effector proteins PipA, GogA and GtgA redundantly target components of the NF-κB signaling pathway to inhibit transcriptional responses leading to inflammation. We show that these effector proteins are proteases that cleave both the RelA (p65) and RelB transcription factors but do not target p100 (NF-κB2) or p105 (NF-κB1). A Salmonella Typhimurium strain lacking these effectors showed increased ability to stimulate NF-κB and increased virulence in an animal model of infection. These results indicate that bacterial pathogens can evolve determinants to preserve host homeostasis and that those determinants can reduce the pathogen's virulence.

The TAL effector PthA4 interacts with nuclear factors involved in RNA-dependent processes including a HMG protein that selectively binds poly(U RNA.

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Tiago Antonio de Souza

Full Text Available Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC, a translin-associated factor X (CsTRAX, a VirE2-interacting protein (CsVIP2, a high mobility group (CsHMG and two poly(A-binding proteins (CsPABP1 and 2, interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control.

A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effectors of Bartonella.

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Harms, Alexander; Liesch, Marius; Körner, Jonas; Québatte, Maxime; Engel, Philipp; Dehio, Christoph

2017-10-01

Host-targeting type IV secretion systems (T4SS) evolved from conjugative T4SS machineries that mediate interbacterial plasmid transfer. However, the origins of effectors secreted by these virulence devices have remained largely elusive. Previous work showed that some effectors exhibit homology to toxins of bacterial toxin-antitoxin modules, but the evolutionary trajectories underlying these ties had not been resolved. We previously reported that FicT toxins of FicTA toxin-antitoxin modules disrupt cellular DNA topology via their enzymatic FIC (filamentation induced by cAMP) domain. Intriguingly, the FIC domain of the FicT toxin VbhT of Bartonella schoenbuchensis is fused to a type IV secretion signal-the BID (Bep intracellular delivery) domain-similar to the Bartonella effector proteins (Beps) that are secreted into eukaryotic host cells via the host-targeting VirB T4SS. In this study, we show that the VbhT toxin is an interbacterial effector protein secreted via the conjugative Vbh T4SS that is closely related to the VirB T4SS and encoded by plasmid pVbh of B. schoenbuchensis. We therefore propose that the Vbh T4SS together with its effector VbhT represent an evolutionary missing link on a path that leads from a regular conjugation system and FicTA toxin-antitoxin modules to the VirB T4SS and the Beps. Intriguingly, phylogenetic analyses revealed that the fusion of FIC and BID domains has probably occurred independently in VbhT and the common ancestor of the Beps, suggesting parallel evolutionary paths. Moreover, several other examples of TA module toxins that are bona fide substrates of conjugative T4SS indicate that their recruitment as interbacterial effectors is prevalent and serves yet unknown biological functions in the context of bacterial conjugation. We propose that the adaptation for interbacterial transfer favors the exaptation of FicT and other TA module toxins as inter-kingdom effectors and may thus constitute an important stepping stone in the

A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effectors of Bartonella.

Directory of Open Access Journals (Sweden)

Alexander Harms

2017-10-01

Full Text Available Host-targeting type IV secretion systems (T4SS evolved from conjugative T4SS machineries that mediate interbacterial plasmid transfer. However, the origins of effectors secreted by these virulence devices have remained largely elusive. Previous work showed that some effectors exhibit homology to toxins of bacterial toxin-antitoxin modules, but the evolutionary trajectories underlying these ties had not been resolved. We previously reported that FicT toxins of FicTA toxin-antitoxin modules disrupt cellular DNA topology via their enzymatic FIC (filamentation induced by cAMP domain. Intriguingly, the FIC domain of the FicT toxin VbhT of Bartonella schoenbuchensis is fused to a type IV secretion signal-the BID (Bep intracellular delivery domain-similar to the Bartonella effector proteins (Beps that are secreted into eukaryotic host cells via the host-targeting VirB T4SS. In this study, we show that the VbhT toxin is an interbacterial effector protein secreted via the conjugative Vbh T4SS that is closely related to the VirB T4SS and encoded by plasmid pVbh of B. schoenbuchensis. We therefore propose that the Vbh T4SS together with its effector VbhT represent an evolutionary missing link on a path that leads from a regular conjugation system and FicTA toxin-antitoxin modules to the VirB T4SS and the Beps. Intriguingly, phylogenetic analyses revealed that the fusion of FIC and BID domains has probably occurred independently in VbhT and the common ancestor of the Beps, suggesting parallel evolutionary paths. Moreover, several other examples of TA module toxins that are bona fide substrates of conjugative T4SS indicate that their recruitment as interbacterial effectors is prevalent and serves yet unknown biological functions in the context of bacterial conjugation. We propose that the adaptation for interbacterial transfer favors the exaptation of FicT and other TA module toxins as inter-kingdom effectors and may thus constitute an important stepping

Protein-Based Drug-Delivery Materials

OpenAIRE

Jao, Dave; Xue, Ye; Medina, Jethro; Hu, Xiao

2017-01-01

There is a pressing need for long-term, controlled drug release for sustained treatment of chronic or persistent medical conditions and diseases. Guided drug delivery is difficult because therapeutic compounds need to survive numerous transport barriers and binding targets throughout the body. Nanoscale protein-based polymers are increasingly used for drug and vaccine delivery to cross these biological barriers and through blood circulation to their molecular site of action. Protein-based pol...

Bacterial effector binding to ribosomal protein s3 subverts NF-kappaB function.

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Xiaofei Gao

2009-12-01

Full Text Available Enteric bacterial pathogens cause food borne disease, which constitutes an enormous economic and health burden. Enterohemorrhagic Escherichia coli (EHEC causes a severe bloody diarrhea following transmission to humans through various means, including contaminated beef and vegetable products, water, or through contact with animals. EHEC also causes a potentially fatal kidney disease (hemolytic uremic syndrome for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g., enteropathogenic E. coli (EPEC, Salmonella, Shigella, Yersinia utilize a type III secretion system (T3SS to inject virulence proteins (effectors into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, in many cases, the mechanisms by which these effectors bind to host proteins and disrupt the normal function of intestinal epithelial cells have not been completely characterized. In this study, we present evidence that the E. coli O157:H7 nleH1 and nleH2 genes encode T3SS effectors that bind to the human ribosomal protein S3 (RPS3, a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB transcriptional complexes. NleH1 and NleH2 co-localized with RPS3 in the cytoplasm, but not in cell nuclei. The N-terminal region of both NleH1 and NleH2 was required for binding to the N-terminus of RPS3. NleH1 and NleH2 are autophosphorylated Ser/Thr protein kinases, but their binding to RPS3 is independent of kinase activity. NleH1, but not NleH2, reduced the nuclear abundance of RPS3 without altering the p50 or p65 NF-kappaB subunits or affecting the phosphorylation state or abundance of the inhibitory NF-kappaB chaperone IkappaBalpha NleH1 repressed the transcription of a RPS3/NF-kappaB-dependent reporter plasmid, but did not inhibit the transcription of RPS3-independent reporters. In contrast, NleH2 stimulated RPS3-dependent transcription, as well

Secretion of Rhoptry and Dense Granule Effector Proteins by Nonreplicating Toxoplasma gondii Uracil Auxotrophs Controls the Development of Antitumor Immunity.

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Barbara A Fox

2016-07-01

Full Text Available Nonreplicating type I uracil auxotrophic mutants of Toxoplasma gondii possess a potent ability to activate therapeutic immunity to established solid tumors by reversing immune suppression in the tumor microenvironment. Here we engineered targeted deletions of parasite secreted effector proteins using a genetically tractable Δku80 vaccine strain to show that the secretion of specific rhoptry (ROP and dense granule (GRA proteins by uracil auxotrophic mutants of T. gondii in conjunction with host cell invasion activates antitumor immunity through host responses involving CD8α+ dendritic cells, the IL-12/interferon-gamma (IFN-γ TH1 axis, as well as CD4+ and CD8+ T cells. Deletion of parasitophorous vacuole membrane (PVM associated proteins ROP5, ROP17, ROP18, ROP35 or ROP38, intravacuolar network associated dense granule proteins GRA2 or GRA12, and GRA24 which traffics past the PVM to the host cell nucleus severely abrogated the antitumor response. In contrast, deletion of other secreted effector molecules such as GRA15, GRA16, or ROP16 that manipulate host cell signaling and transcriptional pathways, or deletion of PVM associated ROP21 or GRA3 molecules did not affect the antitumor activity. Association of ROP18 with the PVM was found to be essential for the development of the antitumor responses. Surprisingly, the ROP18 kinase activity required for resistance to IFN-γ activated host innate immunity related GTPases and virulence was not essential for the antitumor response. These data show that PVM functions of parasite secreted effector molecules, including ROP18, manipulate host cell responses through ROP18 kinase virulence independent mechanisms to activate potent antitumor responses. Our results demonstrate that PVM associated rhoptry effector proteins secreted prior to host cell invasion and dense granule effector proteins localized to the intravacuolar network and host nucleus that are secreted after host cell invasion coordinately

Analysis of Globodera rostochiensis effectors reveals conserved functions of SPRYSEC proteins in suppressing and eliciting plant immune responses

Science.gov (United States)

Potato cyst nematodes (PCNs), including Globodera rostochiensis (Woll.), are important pests of potato. Plant parasitic nematodes produce multiple effector proteins, secreted from their stylets, to successfully infect their hosts. These include proteins that are delivered to the apoplast, as well as...

Oral delivery of peptides and proteins using lipid-based drug delivery systems

DEFF Research Database (Denmark)

Li, Ping; Nielsen, Hanne Mørck; Müllertz, Anette

2012-01-01

INTRODUCTION: In order to successfully develop lipid-based drug delivery systems (DDS) for oral administration of peptides and proteins, it is important to gain an understanding of the colloid structures formed by these DDS, the mode of peptide and protein incorporation as well as the mechanism...... by which intestinal absorption of peptides and proteins is promoted. AREAS COVERED: The present paper reviews the literature on lipid-based DDS, employed for oral delivery of peptides and proteins and highlights the mechanisms by which the different lipid-based carriers are expected to overcome the two...... and proteins. EXPERT OPINION: Lipid-based DDS are safe and suitable for oral delivery of peptides and proteins. Significant progress has been made in this area with several technologies on clinical trials. However, a better understanding of the mechanism of action in vivo is needed in order to improve...

Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

Science.gov (United States)

Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

2017-04-01

Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (V H +C H1 ) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Characterization of particulate drug delivery systems for oral delivery of Peptide and protein drugs.

Science.gov (United States)

Christophersen, Philip Carsten; Fano, Mathias; Saaby, Lasse; Yang, Mingshi; Nielsen, Hanne Mørck; Mu, Huiling

2015-01-01

Oral drug delivery is a preferred route because of good patient compliance. However, most peptide/ protein drugs are delivered via parenteral routes because of the absorption barriers in the gastrointestinal (GI) tract such as enzymatic degradation by proteases and low permeability acrossthe biological membranes. To overcome these barriers, different formulation strategies for oral delivery of biomacromolecules have been proposed, including lipid based formulations and polymer-based particulate drug delivery systems (DDS). The aim of this review is to summarize the existing knowledge about oral delivery of peptide/protein drugs and to provide an overview of formulationand characterization strategies. For a better understanding of the challenges in oral delivery of peptide/protein drugs, the composition of GI fluids and the digestion processes of different kinds of excipients in the GI tract are summarized. Additionally, the paper provides an overview of recent studies on characterization of solid drug carriers for peptide/protein drugs, drug distribution in particles, drug release and stability in simulated GI fluids, as well as the absorption of peptide/protein drugs in cell-based models. The use of biorelevant media when applicable can increase the knowledge about the quality of DDS for oral protein delivery. Hopefully, the knowledge provided in this review will aid the establishment of improved biorelevant models capable of forecasting the performance of particulate DDS for oral peptide/protein delivery.

Effectors from Wheat Rust Fungi Suppress Multiple Plant Defense Responses.

Science.gov (United States)

Ramachandran, Sowmya R; Yin, Chuntao; Kud, Joanna; Tanaka, Kiwamu; Mahoney, Aaron K; Xiao, Fangming; Hulbert, Scot H

2017-01-01

Fungi that cause cereal rust diseases (genus Puccinia) are important pathogens of wheat globally. Upon infection, the fungus secretes a number of effector proteins. Although a large repository of putative effectors has been predicted using bioinformatic pipelines, the lack of available high-throughput effector screening systems has limited functional studies on these proteins. In this study, we mined the available transcriptomes of Puccinia graminis and P. striiformis to look for potential effectors that suppress host hypersensitive response (HR). Twenty small (wheat, confirming its activity in a homologous system. Overall, this study provides the first evidence for the presence of effectors in Puccinia species suppressing multiple plant defense responses.

Robotic end-effector for rewaterproofing shuttle tiles

Science.gov (United States)

Manouchehri, Davoud; Hansen, Joseph M.; Wu, Cheng M.; Yamamoto, Brian S.; Graham, Todd

1992-11-01

This paper summarizes work by Rockwell International's Space Systems Division's Robotics Group at Downey, California. The work is part of a NASA-led team effort to automate Space Shuttle rewaterproofing in the Orbiter Processing Facility at the Kennedy Space Center and the ferry facility at the Ames-Dryden Flight Research Facility. Rockwell's effort focuses on the rewaterproofing end-effector, whose function is to inject hazardous dimethylethyloxysilane into thousands of ceramic tiles on the underside of the orbiter after each flight. The paper has five sections. First, it presents background on the present manual process. Second, end-effector requirements are presented, including safety and interface control. Third, a design is presented for the five end-effector systems: positioning, delivery, containment, data management, and command and control. Fourth, end-effector testing and integrating to the total system are described. Lastly, future applications for this technology are discussed.

Stimuli-Responsive Polymeric Systems for Controlled Protein and Peptide Delivery: Future Implications for Ocular Delivery.

Science.gov (United States)

Mahlumba, Pakama; Choonara, Yahya E; Kumar, Pradeep; du Toit, Lisa C; Pillay, Viness

2016-07-30

Therapeutic proteins and peptides have become notable in the drug delivery arena for their compatibility with the human body as well as their high potency. However, their biocompatibility and high potency does not negate the existence of challenges resulting from physicochemical properties of proteins and peptides, including large size, short half-life, capability to provoke immune responses and susceptibility to degradation. Various delivery routes and delivery systems have been utilized to improve bioavailability, patient acceptability and reduce biodegradation. The ocular route remains of great interest, particularly for responsive delivery of macromolecules due to the anatomy and physiology of the eye that makes it a sensitive and complex environment. Research in this field is slowly gaining attention as this could be the breakthrough in ocular drug delivery of macromolecules. This work reviews stimuli-responsive polymeric delivery systems, their use in the delivery of therapeutic proteins and peptides as well as examples of proteins and peptides used in the treatment of ocular disorders. Stimuli reviewed include pH, temperature, enzymes, light, ultrasound and magnetic field. In addition, it discusses the current progress in responsive ocular drug delivery. Furthermore, it explores future prospects in the use of stimuli-responsive polymers for ocular delivery of proteins and peptides. Stimuli-responsive polymers offer great potential in improving the delivery of ocular therapeutics, therefore there is a need to consider them in order to guarantee a local, sustained and ideal delivery of ocular proteins and peptides, evading tissue invasion and systemic side-effects.

Oral delivery of peptides and proteins using lipid-based drug delivery systems.

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Li, Ping; Nielsen, Hanne Mørck; Müllertz, Anette

2012-10-01

In order to successfully develop lipid-based drug delivery systems (DDS) for oral administration of peptides and proteins, it is important to gain an understanding of the colloid structures formed by these DDS, the mode of peptide and protein incorporation as well as the mechanism by which intestinal absorption of peptides and proteins is promoted. The present paper reviews the literature on lipid-based DDS, employed for oral delivery of peptides and proteins and highlights the mechanisms by which the different lipid-based carriers are expected to overcome the two most important barriers (extensive enzymatic degradation and poor transmucosal permeability). This paper also gives a clear-cut idea about advantages and drawbacks of using different lipidic colloidal carriers ((micro)emulsions, solid lipid core particles and liposomes) for oral delivery of peptides and proteins. Lipid-based DDS are safe and suitable for oral delivery of peptides and proteins. Significant progress has been made in this area with several technologies on clinical trials. However, a better understanding of the mechanism of action in vivo is needed in order to improve the design and development of lipid-based DDS with the desired bioavailability and therapeutic profile.

Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence.

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Shi-qi An

2014-10-01

Full Text Available Bis-(3',5' cyclic di-guanylate (cyclic di-GMP is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc. This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K(d∼2 µM. Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

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Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Côté, Olivier; Stare, Barbara Gerič; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

2015-01-01

The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.

Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

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Shawkat Ali

Full Text Available The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12 and an expansin-like protein (GrEXPB2, suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.

Nanochemistry of protein-based delivery agents

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Rajendran, Subin; Udenigwe, Chibuike; Yada, Rickey

2016-07-01

The past decade has seen an increased interest in the conversion of food proteins into functional biomaterials, including their use for loading and delivery of physiologically active compounds such as nutraceuticals and pharmaceuticals. Proteins possess a competitive advantage over other platforms for the development of nanodelivery systems since they are biocompatible, amphipathic, and widely available. Proteins also have unique molecular structures and diverse functional groups that can be selectively modified to alter encapsulation and release properties. A number of physical and chemical methods have been used for preparing protein nanoformulations, each based on different underlying protein chemistry. This review focuses on the chemistry of the reorganization and/or modification of proteins into functional nanostructures for delivery, from the perspective of their preparation, functionality, stability and physiological behavior.

New kids on the block: The Popeye domain containing (POPDC) protein family acting as a novel class of cAMP effector proteins in striated muscle.

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Brand, Thomas; Schindler, Roland

2017-12-01

The cyclic 3',5'-adenosine monophosphate (cAMP) signalling pathway constitutes an ancient signal transduction pathway present in prokaryotes and eukaryotes. Previously, it was thought that in eukaryotes three effector proteins mediate cAMP signalling, namely protein kinase A (PKA), exchange factor directly activated by cAMP (EPAC) and the cyclic-nucleotide gated channels. However, recently a novel family of cAMP effector proteins emerged and was termed the Popeye domain containing (POPDC) family, which consists of three members POPDC1, POPDC2 and POPDC3. POPDC proteins are transmembrane proteins, which are abundantly present in striated and smooth muscle cells. POPDC proteins bind cAMP with high affinity comparable to PKA. Presently, their biochemical activity is poorly understood. However, mutational analysis in animal models as well as the disease phenotype observed in patients carrying missense mutations suggests that POPDC proteins are acting by modulating membrane trafficking of interacting proteins. In this review, we will describe the current knowledge about this gene family and also outline the apparent gaps in our understanding of their role in cAMP signalling and beyond. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

RNAi effector diversity in nematodes.

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Johnathan J Dalzell

2011-06-01

Full Text Available While RNA interference (RNAi has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (dsRNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii The Argonautes (AGOs responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii Secondary Argonautes (SAGOs are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research

Recent developments in protein and peptide parenteral delivery approaches

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Patel, Ashaben; Cholkar, Kishore; Mitra, Ashim K

2014-01-01

Discovery of insulin in the early 1900s initiated the research and development to improve the means of therapeutic protein delivery in patients. In the past decade, great emphasis has been placed on bringing protein and peptide therapeutics to market. Despite tremendous efforts, parenteral delivery still remains the major mode of administration for protein and peptide therapeutics. Other routes such as oral, nasal, pulmonary and buccal are considered more opportunistic rather than routine application. Improving biological half-life, stability and therapeutic efficacy is central to protein and peptide delivery. Several approaches have been tried in the past to improve protein and peptide in vitro/in vivo stability and performance. Approaches may be broadly categorized as chemical modification and colloidal delivery systems. In this review we have discussed various chemical approaches such as PEGylation, hyperglycosylation, mannosylation, and colloidal carriers including microparticles, nanoparticles, liposomes, carbon nanotubes and micelles for improving protein and peptide delivery. Recent developments on in situ thermosensitive gel-based protein and peptide delivery have also been described. This review summarizes recent developments on some currently existing approaches to improve stability, bioavailability and bioactivity of peptide and protein therapeutics following parenteral administration. PMID:24592957

Protein based therapeutic delivery agents: Contemporary developments and challenges.

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Yin, Liming; Yuvienco, Carlo; Montclare, Jin Kim

2017-07-01

As unique biopolymers, proteins can be employed for therapeutic delivery. They bear important features such as bioavailability, biocompatibility, and biodegradability with low toxicity serving as a platform for delivery of various small molecule therapeutics, gene therapies, protein biologics and cells. Depending on size and characteristic of the therapeutic, a variety of natural and engineered proteins or peptides have been developed. This, coupled to recent advances in synthetic and chemical biology, has led to the creation of tailor-made protein materials for delivery. This review highlights strategies employing proteins to facilitate the delivery of therapeutic matter, addressing the challenges for small molecule, gene, protein and cell transport. Copyright © 2017 Elsevier Ltd. All rights reserved.

Nanochemistry of protein-based delivery agents

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Subin R.C.K. Rajendran

2016-07-01

Full Text Available The past decade has seen an increased interest in the conversion of food proteins into functional biomaterials, including their use for loading and delivery of physiologically active compounds such as nutraceuticals and pharmaceuticals. Proteins possess a competitive advantage over other platforms for the development of nanodelivery systems since they are biocompatible, amphipathic, and widely available. Proteins also have unique molecular structures and diverse functional groups that can be selectively modified to alter encapsulation and release properties. A number of physical and chemical methods have been used for preparing protein nanoformulations, each based on different underlying protein chemistry. This review focuses on the chemistry of the reorganization and/or modification of proteins into functional nanostructures for delivery, from the perspective of their preparation, functionality, stability and physiological behavior.

Modulation of innate immune responses by Yersinia type III secretion system translocators and effectors.

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Bliska, James B; Wang, Xiaoying; Viboud, Gloria I; Brodsky, Igor E

2013-10-01

The innate immune system of mammals responds to microbial infection through detection of conserved molecular determinants called 'pathogen-associated molecular patterns' (PAMPs). Pathogens use virulence factors to counteract PAMP-directed responses. The innate immune system can in turn recognize signals generated by virulence factors, allowing for a heightened response to dangerous pathogens. Many Gram-negative bacterial pathogens encode type III secretion systems (T3SSs) that translocate effector proteins, subvert PAMP-directed responses and are critical for infection. A plasmid-encoded T3SS in the human-pathogenic Yersinia species translocates seven effectors into infected host cells. Delivery of effectors by the T3SS requires plasma membrane insertion of two translocators, which are thought to form a channel called a translocon. Studies of the Yersinia T3SS have provided key advances in our understanding of how innate immune responses are generated by perturbations in plasma membrane and other signals that result from translocon insertion. Additionally, studies in this system revealed that effectors function to inhibit innateimmune responses resulting from insertion of translocons into plasma membrane. Here, we review these advances with the goal of providing insight into how a T3SS can activate and inhibit innate immune responses, allowing a virulent pathogen to bypass host defences. © 2013 John Wiley & Sons Ltd.

The Burkholderia pseudomallei Proteins BapA and BapC Are Secreted TTSS3 Effectors and BapB Levels Modulate Expression of BopE.

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Puthayalai Treerat

Full Text Available Many Gram-negative pathogens use a type III secretion system (TTSS for the injection of bacterial effector proteins into host cells. The injected effector proteins play direct roles in modulation of host cell pathways for bacterial benefit. Burkholderia pseudomallei, the causative agent of melioidosis, expresses three different TTSSs. One of these systems, the TTSS3, is essential for escape from host endosomes and therefore intracellular survival and replication. Here we have characterized three putative TTSS3 proteins; namely BapA, BapB and BapC. By employing a tetracysteine (TC-FlAsH™ labelling technique to monitor the secretion of TC-tagged fusion proteins, BapA and BapC were shown to be secreted during in vitro growth in a TTSS3-dependant manner, suggesting a role as TTSS3 effectors. Furthermore, we constructed B. pseudomallei bapA, bapB and bapC mutants and used the well-characterized TTSS3 effector BopE as a marker of secretion to show that BapA, BapB and BapC are not essential for the secretion process. However, BopE transcription and secretion were significantly increased in the bapB mutant, suggesting that BapB levels modulate BopE expression. In a BALB/c mouse model of acute melioidosis, the bapA, bapB and bapC mutants showed a minor reduction of in vivo fitness. Thus, this study defines BapA and BapC as novel TTSS3 effectors, BapB as a regulator of BopE production, and all three as necessary for full B. pseudomallei in vivo fitness.

Characterization of particulate drug delivery systems for oral delivery of Peptide and protein drugs

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Christophersen, Philip Carsten; Fano, Mathias; Saaby, Lasse

2015-01-01

Oral drug delivery is a preferred route because of good patient compliance. However, most peptide/ protein drugs are delivered via parenteral routes because of the absorption barriers in the gastrointestinal (GI) tract such as enzymatic degradation by proteases and low permeability acrossthe...... delivery of peptide/protein drugs and to provide an overview of formulationand characterization strategies. For a better understanding of the challenges in oral delivery of peptide/protein drugs, the composition of GI fluids and the digestion processes of different kinds of excipients in the GI tract...... biological membranes. To overcome these barriers, different formulation strategies for oral delivery of biomacromolecules have been proposed, including lipid based formulations and polymer-based particulate drug delivery systems (DDS). The aim of this review is to summarize the existing knowledge about oral...

Multiplexed Quantitation of Intraphagocyte Mycobacterium tuberculosis Secreted Protein Effectors

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Fadel Sayes

2018-04-01

Full Text Available Summary: The pathogenic potential of Mycobacterium tuberculosis largely depends on ESX secretion systems exporting members of the multigenic Esx, Esp, and PE/PPE protein families. To study the secretion and regulation patterns of these proteins while circumventing immune cross-reactions due to their extensive sequence homologies, we developed an approach that relies on the recognition of their MHC class II epitopes by highly discriminative T cell receptors (TCRs of a panel of T cell hybridomas. The latter were engineered so that each expresses a unique fluorescent reporter linked to specific antigen recognition. The resulting polychromatic and multiplexed imaging assay enabled us to measure the secretion of mycobacterial effectors inside infected host cells. We applied this novel technology to a large panel of mutants, clinical isolates, and host-cell types to explore the host-mycobacteria interplay and its impact on the intracellular bacterial secretome, which also revealed the unexpected capacity of phagocytes from lung granuloma to present mycobacterial antigens via MHC class II. : Sayes et al. develop an approach to express distinct fluorescent reporters that is based on the recognition of specific Mycobacterium tuberculosis MHC class II epitopes by highly discriminative T cell hybridomas. This multiplexed technology allows the study of secretion, subcellular location, and regulation patterns of these instrumental protein members. Keywords: mycobacterium tuberculosis, type VII secretion systems, intracellular bacteria, T-cell hybridomas, mycobacterial virulence factors, bacterial antigen presentation, lentiviral vectors, reporter T cells, in vivo antigen presentation, protein localization

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses.

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Rodríguez-Herva, José J; González-Melendi, Pablo; Cuartas-Lanza, Raquel; Antúnez-Lamas, María; Río-Alvarez, Isabel; Li, Ziduo; López-Torrejón, Gema; Díaz, Isabel; Del Pozo, Juan C; Chakravarthy, Suma; Collmer, Alan; Rodríguez-Palenzuela, Pablo; López-Solanilla, Emilia

2012-05-01

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His(6) -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1(D299A) non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression. © 2012 Blackwell Publishing Ltd.

Peptides, proteins and peptide/protein-polymer conjugates as drug delivery system.

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Mukherjee, Biswajit; Karmakar, Swapna D; Hossain, Chowdhury M; Bhattacharya, Sanchari

2014-01-01

In the last few decades, novel drug delivery strategies have been a big priority to the formulation scientists. Peptides and proteins have drawn a special attention for their wide scope in the area. Serum albumin, transferrin, recom- binant proteins, virus capsids etc. are used as carrier for drug and biomolecules. Conjugates of polymers with proteins have also shown strong potency in the field of drug delivery. Polyethylene glycol is one of the most successful polymers that has been used extensively to develop protein conjugated formulations. Besides, polyvinyl pyrrolidone, polylactic-co- glycolic acid, N-(2-hydroxypropyl) methacrylamide copolymer, polyglutamic acid have also been investigated. In this re- view, we will highlight on the most recent overview of various advantages, limitations and marketed products of proteins, peptides and protein/peptide-polymer conjugates as drug carriers, such products in clinical trials and their various uses in the field of modern drug delivery. Understanding the key features of these materials and the vigorous research in this field will develop new drug formulations that will combat various types of life-threatening diseases.

Subtle variation within conserved effector operon gene products contributes to T6SS-mediated killing and immunity.

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Alteri, Christopher J; Himpsl, Stephanie D; Zhu, Kevin; Hershey, Haley L; Musili, Ninette; Miller, Jessa E; Mobley, Harry L T

2017-11-01

Type VI secretion systems (T6SS) function to deliver lethal payloads into target cells. Many studies have shown that protection against a single, lethal T6SS effector protein requires a cognate antidote immunity protein, both of which are often encoded together in a two-gene operon. The T6SS and an effector-immunity pair is sufficient for both killing and immunity. HereIn this paper we describe a T6SS effector operon that differs from conventional effector-immunity pairs in that eight genes are necessary for lethal effector function, yet can be countered by a single immunity protein. In this study, we investigated the role that the PefE T6SS immunity protein plays in recognition between two strains harboring nearly identical effector operons. Interestingly, despite containing seven of eight identical effector proteins, the less conserved immunity proteins only provided protection against their native effectors, suggesting that specificity and recognition could be dependent on variation within an immunity protein and one effector gene product. The variable effector gene product, PefD, is encoded upstream from pefE, and displays toxic activity that can be countered by PefE independent of T6SS-activity. Interestingly, while the entire pef operon was necessary to exert toxic activity via the T6SS in P. mirabilis, production of PefD and PefE alone was unable to exert this effector activity. Chimeric PefE proteins constructed from two P. mirabilis strains were used to localize immunity function to three amino acids. A promiscuous immunity protein was created using site-directed mutagenesis to change these residues from one variant to another. These findings support the notion that subtle differences between conserved effectors are sufficient for T6SS-mediated kin discrimination and that PefD requires additional factors to function as a T6SS-dependent effector.

A multi-pronged search for a common structural motif in the secretion signal of Salmonella enterica serovar Typhimurium type III effector proteins

Energy Technology Data Exchange (ETDEWEB)

Buchko, Garry W.; Niemann, George; Baker, Erin Shammel; Belov, Mikhail E.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.; McDermott, Jason E.

2010-11-08

Many pathogenic Gram-negative bacteria use a type III secretion system (T3SS) to deliver effector proteins into the host cell where they reprogram host defenses and facilitate pathogenesis. While it has been determined that the first 20 - 30 N-terminal residues usually contain the ‘secretion signal’ that targets effector proteins for translocation, the molecular basis for recognition of this signal is not understood. Recent machine-learning approaches, such as SVM-based Identification and Evaluation of Virulence Effectors (SIEVE), have improved the ability to identify effector proteins from genomics sequence information. While these methods all suggest that the T3SS secretion signal has a characteristic amino acid composition bias, it is still unclear if the amino acid pattern is important and if there are any unifying structural properties that direct recognition. To address these issues a peptide corresponding to the secretion signal for Salmonella enterica serovar Typhimurium effector SseJ was synthesized (residues 1-30, SseJ) along with scrambled peptides of the same amino acid composition that produced high (SseJ-H) and low (SseJ-L) SIEVE scores. The secretion properties of these three peptides were tested using a secretion signal-CyaA fusion assay and their structures systematically probed using circular dichroism, nuclear magnetic resonance, and ion mobility spectrometry-mass spectrometry. The signal-CyaA fusion assay showed that the native and SseJ-H fusion constructs were secreted into J774 macrophage at similar levels via the SPI-2 secretion pathway while secretion of the SseJ-L fusion construct was substantially retarded, suggesting that the SseJ secretion signal was sequence order dependent. The structural studies showed that the SseJ, SseJ-H, and SseJ-L peptides were intrinsically disordered in aqueous solution with only a small predisposition to adopt nascent helical structure in the presence of the powerful structure stabilizing agent, 1

The Potato Aphid Salivary Effector Me47 Is a Glutathione-S-Transferase Involved in Modifying Plant Responses to Aphid Infestation.

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Kettles, Graeme J; Kaloshian, Isgouhi

2016-01-01

Polyphagous aphid pests cause considerable economic damage to crop plants, primarily through the depletion of photoassimilates and transfer of viruses. The potato aphid (Macrosiphum euphorbiae) is a notable pest of solanaceous crops, however, the molecular mechanisms that underpin the ability to colonize these hosts are unknown. It has recently been demonstrated that like other aphid species, M. euphorbiae injects a battery of salivary proteins into host plants during feeding. It is speculated that these proteins function in a manner analagous to secreted effectors from phytopathogenic bacteria, fungi and oomycetes. Here, we describe a novel aphid effector (Me47) which was identified from the potato aphid salivary secretome as a putative glutathione-S-transferase (GST). Expression of Me47 in Nicotiana benthamiana enhanced reproductive performance of green peach aphid (Myzus persicae). Similarly, delivery of Me47 into leaves of tomato (Solanum lycopersicum) by Pseudomonas spp. enhanced potato aphid fecundity. In contrast, delivery of Me47 into Arabidopsis thaliana reduced GPA reproductive performance, indicating that Me47 impacts the outcome of plant-aphid interactions differently depending on the host species. Delivery of Me47 by the non-pathogenic Pseudomonas fluorescens revealed that Me47 protein or activity triggers defense gene transcriptional upregulation in tomato but not Arabidopsis. Recombinant Me47 was purified and demonstrated to have GST activity against two specific isothiocyanates (ITCs), compounds implicated in herbivore defense. Whilst GSTs have previously been associated with development of aphid resistance to synthetic insecticides, the findings described here highlight a novel function as both an elicitor and suppressor of plant defense when delivered into host tissues.

Substantial conformational change mediated by charge-triad residues of the death effector domain in protein-protein interactions.

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Edward C Twomey

Full Text Available Protein conformational changes are commonly associated with the formation of protein complexes. The non-catalytic death effector domains (DEDs mediate protein-protein interactions in a variety of cellular processes, including apoptosis, proliferation and migration, and glucose metabolism. Here, using NMR residual dipolar coupling (RDC data, we report a conformational change in the DED of the phosphoprotein enriched in astrocytes, 15 kDa (PEA-15 protein in the complex with a mitogen-activated protein (MAP kinase, extracellular regulated kinase 2 (ERK2, which is essential in regulating ERK2 cellular distribution and function in cell proliferation and migration. The most significant conformational change in PEA-15 happens at helices α2, α3, and α4, which also possess the highest flexibility among the six-helix bundle of the DED. This crucial conformational change is modulated by the D/E-RxDL charge-triad motif, one of the prominent structural features of DEDs, together with a number of other electrostatic and hydrogen bonding interactions on the protein surface. Charge-triad motif promotes the optimal orientation of key residues and expands the binding interface to accommodate protein-protein interactions. However, the charge-triad residues are not directly involved in the binding interface between PEA-15 and ERK2.

Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

Energy Technology Data Exchange (ETDEWEB)

Sontag, Ryan L.; Nakayasu, Ernesto S.; Brown, Roslyn N.; Niemann, George S.; Sydor, Michael A.; Sanchez, Octavio; Ansong, Charles; Lu, Shao-Yeh; Choi, Hyungwon; Valleau, Dylan; Weitz, Karl K.; Savchenko, Alexei; Cambronne, Eric D.; Adkins, Joshua N.; McFall-Ngai, Margaret J.

2016-07-12

Many pathogenic bacteria of the familyEnterobacteriaceaeuse type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from theEnterobacteriaceaeintracellular pathogensSalmonella entericaserovar Typhimurium andCitrobacter rodentium. We identified 54 high-confidence host interactors for theSalmonellaeffectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for theCitrobactereffectors EspT, NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfHSalmonellaprotein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction.

IMPORTANCEDuring infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets ofSalmonellaandCitrobactereffectors, which will help elucidate their mechanisms of

A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

Science.gov (United States)

Sarris, Panagiotis F; Duxbury, Zane; Huh, Sung Un; Ma, Yan; Segonzac, Cécile; Sklenar, Jan; Derbyshire, Paul; Cevik, Volkan; Rallapalli, Ghanasyam; Saucet, Simon B; Wirthmueller, Lennart; Menke, Frank L H; Sohn, Kee Hoon; Jones, Jonathan D G

2015-05-21

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain. Copyright © 2015 Elsevier Inc. All rights reserved.

Nanostructured Mineral Coatings Stabilize Proteins for Therapeutic Delivery.

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Yu, Xiaohua; Biedrzycki, Adam H; Khalil, Andrew S; Hess, Dalton; Umhoefer, Jennifer M; Markel, Mark D; Murphy, William L

2017-09-01

Proteins tend to lose their biological activity due to their fragile structural conformation during formulation, storage, and delivery. Thus, the inability to stabilize proteins in controlled-release systems represents a major obstacle in drug delivery. Here, a bone mineral inspired protein stabilization strategy is presented, which uses nanostructured mineral coatings on medical devices. Proteins bound within the nanostructured coatings demonstrate enhanced stability against extreme external stressors, including organic solvents, proteases, and ethylene oxide gas sterilization. The protein stabilization effect is attributed to the maintenance of protein conformational structure, which is closely related to the nanoscale feature sizes of the mineral coatings. Basic fibroblast growth factor (bFGF) released from a nanostructured mineral coating maintains its biological activity for weeks during release, while it maintains activity for less than 7 d during release from commonly used polymeric microspheres. Delivery of the growth factors bFGF and vascular endothelial growth factor using a mineral coated surgical suture significantly improves functional Achilles tendon healing in a rabbit model, resulting in increased vascularization, more mature collagen fiber organization, and a two fold improvement in mechanical properties. The findings of this study demonstrate that biomimetic interactions between proteins and nanostructured minerals provide a new, broadly applicable mechanism to stabilize proteins in the context of drug delivery and regenerative medicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protein instability and immunogenicity: roadblocks to clinical application of injectable protein delivery systems for sustained release.

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Jiskoot, Wim; Randolph, Theodore W; Volkin, David B; Middaugh, C Russell; Schöneich, Christian; Winter, Gerhard; Friess, Wolfgang; Crommelin, Daan J A; Carpenter, John F

2012-03-01

Protein instability and immunogenicity are two main roadblocks to the clinical success of novel protein drug delivery systems. In this commentary, we discuss the need for more extensive analytical characterization in relation to concerns about protein instability in injectable drug delivery systems for sustained release. We then will briefly address immunogenicity concerns and outline current best practices for using state-of-the-art analytical assays to monitor protein stability for both conventional and novel therapeutic protein dosage forms. Next, we provide a summary of the stresses on proteins arising during preparation of drug delivery systems and subsequent in vivo release. We note the challenges and difficulties in achieving the absolute requirement of quantitatively assessing the degradation of protein molecules in a drug delivery system. We describe the potential roles for academic research in further improving protein stability and developing new analytical technologies to detect protein degradation byproducts in novel drug delivery systems. Finally, we provide recommendations for the appropriate approaches to formulation design and assay development to ensure that stable, minimally immunogenic formulations of therapeutic proteins are created. These approaches should help to increase the probability that novel drug delivery systems for sustained protein release will become more readily available as effective therapeutic agents to treat and benefit patients. Copyright © 2011 Wiley Periodicals, Inc.

Structure and evolution of barley powdery mildew effector candidates

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Pedersen Carsten

2012-12-01

Full Text Available Abstract Background Protein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses Blumeria graminis causes one of the most important diseases of cereal crops. B. graminis is an obligate biotrophic pathogen and as such has an absolute requirement to suppress or avoid host immunity if it is to survive and cause disease. Results Here we characterise a superfamily predicted to be the full complement of Candidates for Secreted Effector Proteins (CSEPs in the fungal barley powdery mildew parasite B. graminis f.sp. hordei. The 491 genes encoding these proteins constitute over 7% of this pathogen’s annotated genes and most were grouped into 72 families of up to 59 members. They were predominantly expressed in the intracellular feeding structures called haustoria, and proteins specifically associated with the haustoria were identified by large-scale mass spectrometry-based proteomics. There are two major types of effector families: one comprises shorter proteins (100–150 amino acids, with a high relative expression level in the haustoria and evidence of extensive diversifying selection between paralogs; the second type consists of longer proteins (300–400 amino acids, with lower levels of differential expression and evidence of purifying selection between paralogs. An analysis of the predicted protein structures underscores their overall similarity to known fungal effectors, but also highlights unexpected structural affinities to ribonucleases throughout the entire effector super-family. Candidate effector genes belonging to the same family are loosely clustered in the genome and are associated with repetitive DNA derived from retro-transposons. Conclusions We employed the full complement of genomic, transcriptomic and proteomic analyses as well as structural prediction methods to identify and characterize the members of the CSEPs superfamily in B. graminis f

Phytoplasma effector SAP54 induces indeterminate leaf-like flower development in Arabidopsis plants.

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MacLean, Allyson M; Sugio, Akiko; Makarova, Olga V; Findlay, Kim C; Grieve, Victoria M; Tóth, Réka; Nicolaisen, Mogens; Hogenhout, Saskia A

2011-10-01

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches' Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches' broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host.

New clues in the nucleus: Transcriptional reprogramming in effector-triggered immunity

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SAIKAT eBHATTACHARJEE

2013-09-01

Full Text Available The robustness of plant effector-triggered immunity is correlated with massive alterations of the host transcriptome. Yet the molecular mechanisms that cause and underlie this reprogramming remain obscure. Here we will review recent advances in deciphering nuclear functions of plant immune receptors and of associated proteins. Important open questions remain, such as the identities of the primary transcription factors involved in control of effector-triggered immune responses, and indeed whether this can be generalized or whether particular effector-resistance protein interactions impinge on distinct sectors in the transcriptional response web. Multiple lines of evidence have implicated WRKY transcription factors at the core of responses to microbe-associated molecular patterns and in intersections with effector-triggered immunity. Recent findings from yeast two-hybrid studies suggest that members of the TCP transcription factor family are targets of several effectors from diverse pathogens. Additional transcription factor families that are directly or indirectly involved in effector-triggered immunity are likely to be identified.

Epigenetic control of effectors in plant pathogens

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Mark eGijzen

2014-11-01

Full Text Available Plant pathogens display impressive versatility in adapting to host immune systems. Pathogen effector proteins facilitate disease but can become avirulence (Avr factors when the host acquires discrete recognition capabilities that trigger immunity. The mechanisms that lead to changes to pathogen Avr factors that enable escape from host immunity are diverse, and include epigenetic switches that allow for reuse or recycling of effectors. This perspective outlines possibilities of how epigenetic control of Avr effector gene expression may have arisen and persisted in plant pathogens, and how it presents special problems for diagnosis and detection of specific pathogen strains or pathotypes.

Yersinia type III effectors perturb host innate immune responses

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Pha, Khavong; Navarro, Lorena

2016-01-01

The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type III secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gram-negative bacteria that share in common a 70 kb virulence plasmid which encodes the T3SS. Translocation of the Yersinia effector proteins (YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia

Defining essential processes in plant pathogenesis with Pseudomonas syringae pv. tomato DC3000 disarmed polymutants and a subset of key type III effectors.

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Wei, Hai-Lei; Collmer, Alan

2017-12-25

Pseudomonas syringae pv. tomato DC3000 and its derivatives cause disease in tomato, Arabidopsis and Nicotiana benthamiana. The primary virulence factors include a repertoire of 29 effector proteins injected into plant cells by the type III secretion system and the phytotoxin coronatine. The complete repertoire of effector genes and key coronatine biosynthesis genes have been progressively deleted and minimally reassembled to reconstitute basic pathogenic ability in N. benthamiana, and in Arabidopsis plants that have mutations in target genes that mimic effector actions. This approach and molecular studies of effector activities and plant immune system targets have highlighted a small subset of effectors that contribute to essential processes in pathogenesis. Most notably, HopM1 and AvrE1 redundantly promote an aqueous apoplastic environment, and AvrPtoB and AvrPto redundantly block early immune responses, two conditions that are sufficient for substantial bacterial growth in planta. In addition, disarmed DC3000 polymutants have been used to identify the individual effectors responsible for specific activities of the complete repertoire and to more effectively study effector domains, effector interplay and effector actions on host targets. Such work has revealed that AvrPtoB suppresses cell death elicitation in N. benthamiana that is triggered by another effector in the DC3000 repertoire, highlighting an important aspect of effector interplay in native repertoires. Disarmed DC3000 polymutants support the natural delivery of test effectors and infection readouts that more accurately reveal effector functions in key pathogenesis processes, and enable the identification of effectors with similar activities from a broad range of other pathogens that also defeat plants with cytoplasmic effectors. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Protein Nanoparticles as Drug Delivery Carriers for Cancer Therapy

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Warangkana Lohcharoenkal

2014-01-01

Full Text Available Nanoparticles have increasingly been used for a variety of applications, most notably for the delivery of therapeutic and diagnostic agents. A large number of nanoparticle drug delivery systems have been developed for cancer treatment and various materials have been explored as drug delivery agents to improve the therapeutic efficacy and safety of anticancer drugs. Natural biomolecules such as proteins are an attractive alternative to synthetic polymers which are commonly used in drug formulations because of their safety. In general, protein nanoparticles offer a number of advantages including biocompatibility and biodegradability. They can be prepared under mild conditions without the use of toxic chemicals or organic solvents. Moreover, due to their defined primary structure, protein-based nanoparticles offer various possibilities for surface modifications including covalent attachment of drugs and targeting ligands. In this paper, we review the most significant advancements in protein nanoparticle technology and their use in drug delivery arena. We then examine the various sources of protein materials that have been used successfully for the construction of protein nanoparticles as well as their methods of preparation. Finally, we discuss the applications of protein nanoparticles in cancer therapy.

Protein nanoparticles as drug delivery carriers for cancer therapy.

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Lohcharoenkal, Warangkana; Wang, Liying; Chen, Yi Charlie; Rojanasakul, Yon

2014-01-01

Nanoparticles have increasingly been used for a variety of applications, most notably for the delivery of therapeutic and diagnostic agents. A large number of nanoparticle drug delivery systems have been developed for cancer treatment and various materials have been explored as drug delivery agents to improve the therapeutic efficacy and safety of anticancer drugs. Natural biomolecules such as proteins are an attractive alternative to synthetic polymers which are commonly used in drug formulations because of their safety. In general, protein nanoparticles offer a number of advantages including biocompatibility and biodegradability. They can be prepared under mild conditions without the use of toxic chemicals or organic solvents. Moreover, due to their defined primary structure, protein-based nanoparticles offer various possibilities for surface modifications including covalent attachment of drugs and targeting ligands. In this paper, we review the most significant advancements in protein nanoparticle technology and their use in drug delivery arena. We then examine the various sources of protein materials that have been used successfully for the construction of protein nanoparticles as well as their methods of preparation. Finally, we discuss the applications of protein nanoparticles in cancer therapy.

The potato aphid salivary effector Me47 is a glutathione-S-transferase involved in modifying plant responses to aphid infestation

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Graeme James Kettles

2016-08-01

Full Text Available Polyphagous aphid pests cause considerable economic damage to crop plants, primarily through the depletion of photoassimilates and transfer of viruses. The potato aphid (Macrosiphum euphorbiae is a notable pest of solanaceous crops, however the molecular mechanisms that underpin the ability to colonize these hosts are unknown. It has recently been demonstrated that like other aphid species, M. euphorbiae injects a battery of salivary proteins into host plants during feeding. It is speculated that these proteins function in a manner analagous to secreted effectors from phytopathogenic bacteria, fungi and oomycetes. Here we describe a novel aphid effector (Me47 which was identified from the potato aphid salivary secretome as a putative glutathione-S-transferase (GST. Expression of Me47 in Nicotiana benthamiana enhanced reproductive performance of green peach aphid (Myzus persicae. Similarly, delivery of Me47 into leaves of tomato (Solanum lycopersicum by Pseudomonas spp. enhanced potato aphid fecundity. In contrast, delivery of Me47 into Arabidopsis thaliana reduced GPA reproductive performance, indicating that Me47 impacts the outcome of plant-aphid interactions differently depending on the host species. Delivery of Me47 by the non-pathogenic Pseudomonas fluorescens revealed that Me47 protein or activity triggers defense gene transcriptional upregulation in tomato but not Arabidopsis. Recombinant Me47 was purified and demonstrated to have GST activity against two specific isothiocyanates (ITCs, compounds implicated in herbivore defense. Whilst GSTs have previously been associated with development of aphid resistance to synthetic insecticides, the findings described here highlight a novel function as both an elicitor and suppressor of plant defense when delivered into host tissues.

Effector candidates in the secretome of Piriformospora indica, a ubiquitous plant-associated fungus

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Maryam eRafiqi

2013-07-01

Full Text Available One of the emerging systems in plant-microbe interaction is the study of proteins, referred to as effectors, secreted by microbes in order to modulate host cells function and structure and to promote microbial growth on plant tissue. Current knowledge on fungal effectors derives mainly from biotrophic and hemibiotrophic plant fungal pathogens that have a limited host range. Here, we focus on effectors of Piriformospora indica, a soil borne endophyte forming intimate associations with roots of a wide range of plant species. Complete genome sequencing provides an opportunity to investigate the role of effectors during the interaction of this mutualistic fungus with plants. We describe in silico analyses to predict effectors of P. indica and we explore effector features considered here to mine a high priority protein list for functional analysis.

Lentiviral Delivery of Proteins for Genome Engineering.

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Cai, Yujia; Mikkelsen, Jacob Giehm

2016-01-01

Viruses have evolved to traverse cellular barriers and travel to the nucleus by mechanisms that involve active transport through the cytoplasm and viral quirks to resist cellular restriction factors and innate immune responses. Virus-derived vector systems exploit the capacity of viruses to ferry genetic information into cells, and now - more than three decades after the discovery of HIV - lentiviral vectors based on HIV-1 have become instrumental in biomedical research and gene therapies that require genomic insertion of transgenes. By now, the efficacy of lentiviral gene delivery to stem cells, cells of the immune system including T cells, hepatic cells, and many other therapeutically relevant cell types is well established. Along with nucleic acids, HIV-1 virions carry the enzymatic tools that are essential for early steps of infection. Such capacity to package enzymes, even proteins of nonviral origin, has unveiled new ways of exploiting cellular intrusion of HIV-1. Based on early findings demonstrating the packaging of heterologous proteins into virus particles as part of the Gag and GagPol polypeptides, we have established lentiviral protein transduction for delivery of DNA transposases and designer nucleases. This strategy for delivering genome-engineering proteins facilitates high enzymatic activity within a short time frame and may potentially improve the safety of genome editing. Exploiting the full potential of lentiviral vectors, incorporation of foreign protein can be combined with the delivery of DNA transposons or a donor sequence for homology-directed repair in so-called 'all-in-one' lentiviral vectors. Here, we briefly describe intracellular restrictions that may affect lentiviral gene and protein delivery and review the current status of lentiviral particles as carriers of tool kits for genome engineering.

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors.

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Lopez, David; Ribeiro, Sébastien; Label, Philippe; Fumanal, Boris; Venisse, Jean-Stéphane; Kohler, Annegret; de Oliveira, Ricardo R; Labutti, Kurt; Lipzen, Anna; Lail, Kathleen; Bauer, Diane; Ohm, Robin A; Barry, Kerrie W; Spatafora, Joseph; Grigoriev, Igor V; Martin, Francis M; Pujade-Renaud, Valérie

2018-01-01

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species ( Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca , and Botrosphaeria dothidea ) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors

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David Lopez

2018-03-01

Full Text Available Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca, and Botrosphaeria dothidea sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector

Expression, purification and preliminary crystallographic analysis of the T6SS effector protein Tse3 from Pseudomonas aeruginosa

International Nuclear Information System (INIS)

Lu, Defen; Shang, Guijun; Yu, Qian; Zhang, Heqiao; Zhao, Yanyu; Cang, Huaixing; Gu, Lichuan; Xu, Sujuan; Huang, Yan

2013-01-01

Tse3, one of the effectors of the type VI secretion system in Pseudomonas aeruginosa, has been crystallized and diffracted to 1.5 Å resolution. Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to inject effector proteins into rival cells in niche competition. Tse3, one of the effectors of T6SS, is delivered into the periplasm of recipient cells. Tse3 functions as a muramidase that degrades the β-1,4-linkage between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan, thus leading to lysis of the recipient cells and providing a competitive advantage to the donor cells. Here, the preliminary crystallographic study of Tse3 is reported. A crystal of Tse3 diffracted to 1.5 Å resolution. It belonged to space group C121, with unit-cell parameters a = 166.99, b = 70.13, c = 41.94 Å, α = 90.00, β = 90.52, γ = 90.00° and one molecule per asymmetric unit

Functionalization of protein-based nanocages for drug delivery applications.

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Schoonen, Lise; van Hest, Jan C M

2014-07-07

Traditional drug delivery strategies involve drugs which are not targeted towards the desired tissue. This can lead to undesired side effects, as normal cells are affected by the drugs as well. Therefore, new systems are now being developed which combine targeting functionalities with encapsulation of drug cargo. Protein nanocages are highly promising drug delivery platforms due to their perfectly defined structures, biocompatibility, biodegradability and low toxicity. A variety of protein nanocages have been modified and functionalized for these types of applications. In this review, we aim to give an overview of different types of modifications of protein-based nanocontainers for drug delivery applications.

Convergent Evolution of Pathogen Effectors toward Reactive Oxygen Species Signaling Networks in Plants.

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Jwa, Nam-Soo; Hwang, Byung Kook

2017-01-01

Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS) act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs) as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs) responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants.

Convergent Evolution of Pathogen Effectors toward Reactive Oxygen Species Signaling Networks in Plants

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Nam-Soo Jwa

2017-09-01

Full Text Available Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP-triggered immunity (PTI and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants.

Uncovering the Legionella genus effector repertoire - strength in diversity and numbers

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Burstein, David; Amaro, Francisco; Zusman, Tal; Lifshitz, Ziv; Cohen, Ofir; Gilbert, Jack A; Pupko, Tal; Shuman, Howard A; Segal, Gil

2016-01-01

Infection by the human pathogen Legionella pneumophila relies on the translocation of ~300 virulence proteins, termed effectors, which manipulate host-cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species, and predicted their effector repertoire using a previously validated machine-learning approach. This analysis revealed a treasure trove of 5,885 predicted effectors. The effector repertoire of different Legionella species was found to be largely non-overlapping, and only seven core-effectors were shared among all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from their natural protozoan hosts. Furthermore, we detected numerous novel conserved effector domains, and discovered new domain combinations, which allowed inferring yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution. PMID:26752266

Distinct activities of Bartonella henselae type IV secretion effector proteins modulate capillary-like sprout formation.

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Scheidegger, F; Ellner, Y; Guye, P; Rhomberg, T A; Weber, H; Augustin, H G; Dehio, C

2009-07-01

The zoonotic pathogen Bartonella henselae (Bh) can lead to vasoproliferative tumour lesions in the skin and inner organs known as bacillary angiomatosis and bacillary peliosis. The knowledge on the molecular and cellular mechanisms involved in this pathogen-triggered angiogenic process is confined by the lack of a suitable animal model and a physiologically relevant cell culture model of angiogenesis. Here we employed a three-dimensional in vitro angiogenesis assay of collagen gel-embedded endothelial cell (EC) spheroids to study the angiogenic properties of Bh. Spheroids generated from Bh-infected ECs displayed a high capacity to form sprouts, which represent capillary-like projections into the collagen gel. The VirB/VirD4 type IV secretion system and a subset of its translocated Bartonella effector proteins (Beps) were found to profoundly modulate this Bh-induced sprouting activity. BepA, known to protect ECs from apoptosis, strongly promoted sprout formation. In contrast, BepG, triggering cytoskeletal rearrangements, potently inhibited sprouting. Hence, the here established in vitro model of Bartonella- induced angiogenesis revealed distinct and opposing activities of type IV secretion system effector proteins, which together with a VirB/VirD4-independent effect may control the angiogenic activity of Bh during chronic infection of the vasculature.

Exploitation of the host cell ubiquitin machinery by microbial effector proteins.

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Lin, Yi-Han; Machner, Matthias P

2017-06-15

Pathogenic bacteria are in a constant battle for survival with their host. In order to gain a competitive edge, they employ a variety of sophisticated strategies that allow them to modify conserved host cell processes in ways that favor bacterial survival and growth. Ubiquitylation, the covalent attachment of the small modifier ubiquitin to target proteins, is such a pathway. Ubiquitylation profoundly alters the fate of a myriad of cellular proteins by inducing changes in their stability or function, subcellular localization or interaction with other proteins. Given the importance of ubiquitylation in cell development, protein homeostasis and innate immunity, it is not surprising that this post-translational modification is exploited by a variety of effector proteins from microbial pathogens. Here, we highlight recent advances in our understanding of the many ways microbes take advantage of host ubiquitylation, along with some surprising deviations from the canonical theme. The lessons learned from the in-depth analyses of these host-pathogen interactions provide a fresh perspective on an ancient post-translational modification that we thought was well understood.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Mechanisms of regulation and diversification of deubiquitylating enzyme function' by Pawel Leznicki and Yogesh Kulathu ( J. Cell Sci. 130 , 1997-2006). 'Cell scientist to watch - Mads Gyrd-Hansen' ( J. Cell Sci. 130 , 1981-1983). © 2017. Published by The Company of Biologists Ltd.

A novel Meloidogyne graminicola effector, MgMO237, interacts with multiple host defence-related proteins to manipulate plant basal immunity and promote parasitism.

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Chen, Jiansong; Hu, Lili; Sun, Longhua; Lin, Borong; Huang, Kun; Zhuo, Kan; Liao, Jinling

2018-02-27

Plant-parasitic nematodes can secrete effector proteins into the host tissue to facilitate their parasitism. In this study, we report a novel effector protein, MgMO237, from Meloidogyne graminicola, which is exclusively expressed within the dorsal oesophageal gland cell and markedly up-regulated in parasitic third-/fourth-stage juveniles of M. graminicola. Transient expression of MgMO237 in protoplasts from rice roots showed that MgMO237 was localized in the cytoplasm and nucleus of the host cells. Rice plants overexpressing MgMO237 showed an increased susceptibility to M. graminicola. In contrast, rice plants expressing RNA interference vectors targeting MgMO237 showed an increased resistance to M. graminicola. In addition, yeast two-hybrid and co-immunoprecipitation assays showed that MgMO237 interacted specifically with three rice endogenous proteins, i.e. 1,3-β-glucan synthase component (OsGSC), cysteine-rich repeat secretory protein 55 (OsCRRSP55) and pathogenesis-related BetvI family protein (OsBetvI), which are all related to host defences. Moreover, MgMO237 can suppress host defence responses, including the expression of host defence-related genes, cell wall callose deposition and the burst of reactive oxygen species. These results demonstrate that the effector MgMO237 probably promotes the parasitism of M. graminicola by interacting with multiple host defence-related proteins and suppressing plant basal immunity in the later parasitic stages of nematodes. © 2018 BSPP AND JOHN WILEY & SONS LTD.

Identification and functional analysis of secreted effectors from phytoparasitic nematodes.

Science.gov (United States)

Rehman, Sajid; Gupta, Vijai K; Goyal, Aakash K

2016-03-21

Plant parasitic nematodes develop an intimate and long-term feeding relationship with their host plants. They induce a multi-nucleate feeding site close to the vascular bundle in the roots of their host plant and remain sessile for the rest of their life. Nematode secretions, produced in the oesophageal glands and secreted through a hollow stylet into the host plant cytoplasm, are believed to play key role in pathogenesis. To combat these persistent pathogens, the identity and functional analysis of secreted effectors can serve as a key to devise durable control measures. In this review, we will recapitulate the knowledge over the identification and functional characterization of secreted nematode effector repertoire from phytoparasitic nematodes. Despite considerable efforts, the identity of genes encoding nematode secreted proteins has long been severely hampered because of their microscopic size, long generation time and obligate biotrophic nature. The methodologies such as bioinformatics, protein structure modeling, in situ hybridization microscopy, and protein-protein interaction have been used to identify and to attribute functions to the effectors. In addition, RNA interference (RNAi) has been instrumental to decipher the role of the genes encoding secreted effectors necessary for parasitism and genes attributed to normal development. Recent comparative and functional genomic approaches have accelerated the identification of effectors from phytoparasitic nematodes and offers opportunities to control these pathogens. Plant parasitic nematodes pose a serious threat to global food security of various economically important crops. There is a wealth of genomic and transcriptomic information available on plant parasitic nematodes and comparative genomics has identified many effectors. Bioengineering crops with dsRNA of phytonematode genes can disrupt the life cycle of parasitic nematodes and therefore holds great promise to develop resistant crops against plant

Peptide-chaperone-directed transdermal protein delivery requires energy.

Science.gov (United States)

Ruan, Renquan; Jin, Peipei; Zhang, Li; Wang, Changli; Chen, Chuanjun; Ding, Weiping; Wen, Longping

2014-11-03

The biologically inspired transdermal enhanced peptide TD1 has been discovered to specifically facilitate transdermal delivery of biological macromolecules. However, the biological behavior of TD1 has not been fully defined. In this study, we find that energy is required for the TD1-mediated transdermal protein delivery through rat and human skins. Our results show that the permeation activity of TD1-hEGF, a fusion protein composed of human epidermal growth factor (hEGF) and the TD1 sequence connected with a glycine-serine linker (GGGGS), can be inhibited by the energy inhibitor, rotenone or oligomycin. In addition, adenosine triphosphate (ATP), the essential energetic molecule in organic systems, can effectively facilitate the TD1 directed permeation of the protein-based drug into the skin in a dose-dependent fashion. Our results here demonstrate a novel energy-dependent permeation process during the TD1-mediated transdermal protein delivery that could be valuable for the future development of promising new transdermal drugs.

Bioengineered protein-based nanocage for drug delivery.

Science.gov (United States)

Lee, Eun Jung; Lee, Na Kyeong; Kim, In-San

2016-11-15

Nature, in its wonders, presents and assembles the most intricate and delicate protein structures and this remarkable phenomenon occurs in all kingdom and phyla of life. Of these proteins, cage-like multimeric proteins provide spatial control to biological processes and also compartmentalizes compounds that may be toxic or unstable and avoids their contact with the environment. Protein-based nanocages are of particular interest because of their potential applicability as drug delivery carriers and their perfect and complex symmetry and ideal physical properties, which have stimulated researchers to engineer, modify or mimic these qualities. This article reviews various existing types of protein-based nanocages that are used for therapeutic purposes, and outlines their drug-loading mechanisms and bioengineering strategies via genetic and chemical functionalization. Through a critical evaluation of recent advances in protein nanocage-based drug delivery in vitro and in vivo, an outlook for de novo and in silico nanocage design, and also protein-based nanocage preclinical and future clinical applications will be presented. Copyright © 2016 Elsevier B.V. All rights reserved.

Cationic polymers for intracellular delivery of proteins

NARCIS (Netherlands)

Coué, G.M.J.P.C.; Engbersen, Johannes F.J.; Samal, Sangram; Dubruel, Peter

2015-01-01

Many therapeutic proteins exert their pharmaceutical action inside the cytoplasm or onto individual organelles inside the cell. Intracellular protein delivery is considered to be the most direct, fastest and safest approach for curing gene-deficiency diseases, enhancing vaccination and triggering

Liposome-based Formulation for Intracellular Delivery of Functional Proteins

Directory of Open Access Journals (Sweden)

Benoît Chatin

2015-01-01

Full Text Available The intracellular delivery of biologically active protein represents an important emerging strategy for both fundamental and therapeutic applications. Here, we optimized in vitro delivery of two functional proteins, the β-galactosidase (β-gal enzyme and the anti-cytokeratin8 (K8 antibody, using liposome-based formulation. The guanidinium-cholesterol cationic lipid bis (guanidinium-tren-cholesterol (BGTC (bis (guanidinium-tren-cholesterol combined to the colipid dioleoyl phosphatidylethanolamine (DOPE (dioleoyl phosphatidylethanolamine was shown to efficiently deliver the β-gal intracellularly without compromising its activity. The lipid/protein molar ratio, protein amount, and culture medium were demonstrated to be key parameters affecting delivery efficiency. The protein itself is an essential factor requiring selection of the appropriate cationic lipid as illustrated by low K8 binding activity of the anti-K8 antibody using guanidinium-based liposome. Optimization of various lipids led to the identification of the aminoglycoside lipid dioleyl succinyl paromomycin (DOSP associated with the imidazole-based helper lipid MM27 as a potent delivery system for K8 antibody, achieving delivery in 67% of HeLa cells. Cryo-transmission electron microscopy showed that the structure of supramolecular assemblies BGTC:DOPE/β-gal and DOSP:MM27/K8 were different depending on liposome types and lipid/protein molar ratio. Finally, we observed that K8 treatment with DOSP:MM27/K8 rescues the cyclic adenosine monophosphate (cAMP-dependent chloride efflux in F508del-CFTR expressing cells, providing a new tool for the study of channelopathies.

The effector repertoire of Fusarium oxysporum determines the tomato xylem proteome composition following infection

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Fleur eGawehns

2015-11-01

Full Text Available Plant pathogens secrete small proteins, of which some are effectors that promote infection. During colonization of the tomato xylem vessels the fungus Fusarium oxysporum f. sp. lycopersici (Fol secretes small proteins that are referred to as SIX (Secreted In Xylem proteins. Of these, Six1 (Avr3, Six3 (Avr2, Six5 and Six6 are required for full virulence, denoting them as effectors. To investigate their activities in the plant, the xylem sap proteome of plants inoculated with Fol wild-type or either AVR2, AVR3, SIX2, SIX5 or SIX6 knockout strains was analyzed with nano-Liquid Chromatography-Mass Spectrometry (nLC-MSMS. Compared to mock-inoculated sap 12 additional plant proteins appeared while 45 proteins were no longer detectable in the xylem sap of Fol-infected plants. Of the 285 proteins found in both uninfected and infected plants the abundance of 258 proteins changed significantly following infection. The xylem sap proteome of plants infected with four Fol effector knockout strains differed significantly from plants infected with wild-type Fol, while that of the SIX2-knockout inoculated plants remained unchanged. Besides an altered abundance of a core set of 24 differentially accumulated proteins (DAPs, each of the four effector knockout strains affected specifically the abundance of a subset of DAPs. Hence, Fol effectors have both unique and shared effects on the composition of the tomato xylem sap proteome.

Modular Study of the Type III Effector Repertoire in Pseudomonas syringae pv. tomato DC3000 Reveals a Matrix of Effector Interplay in Pathogenesis.

Science.gov (United States)

Wei, Hai-Lei; Zhang, Wei; Collmer, Alan

2018-05-08

The bacterial pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered innate immune system of Nicotiana benthamiana and other plants by injecting a complex repertoire of type III secretion effector (T3E) proteins. Effectorless polymutant DC3000D36E was used with a modularized system for native delivery of the 29 DC3000 T3Es singly and in pairs. Assays of the performance of this T3E library in N. benthamiana leaves revealed a matrix of T3E interplay, with six T3Es eliciting death and eight others variously suppressing the death activity of the six. The T3E library was also interrogated for effects on DC3000D36E elicitation of a reactive oxygen species burst, for growth in planta, and for T3Es that reversed these effects. Pseudomonas fluorescens and Agrobacterium tumefaciens heterologous delivery systems yielded notably different sets of death-T3Es. The DC3000D36E T3E library system highlights the importance of 13 T3Es and their interplay in interactions with N. benthamiana. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

pH-responsive and enzymatically-responsive hydrogel microparticles for the oral delivery of therapeutic proteins: Effects of protein size, crosslinking density, and hydrogel degradation on protein delivery.

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Koetting, Michael Clinton; Guido, Joseph Frank; Gupta, Malvika; Zhang, Annie; Peppas, Nicholas A

2016-01-10

Two potential platform technologies for the oral delivery of protein therapeutics were synthesized and tested. pH-responsive poly(itaconic acid-co-N-vinyl-2-pyrrolidone) (P(IA-co-NVP)) hydrogel microparticles were tested in vitro with model proteins salmon calcitonin, urokinase, and rituximab to determine the effects of particle size, protein size, and crosslinking density on oral delivery capability. Particle size showed no significant effect on overall delivery potential but did improve percent release of encapsulated protein over the micro-scale particle size range studied. Protein size was shown to have a significant impact on the delivery capability of the P(IA-co-NVP) hydrogel. We show that when using P(IA-co-NVP) hydrogel microparticles with 3 mol% tetra(ethylene glycol) dimethacrylate crosslinker, a small polypeptide (salmon calcitonin) loads and releases up to 45 μg/mg hydrogel while the mid-sized protein urokinase and large monoclonal antibody rituximab load and release only 19 and 24 μg/mg hydrogel, respectively. We further demonstrate that crosslinking density offers a simple method for tuning hydrogel properties to variously sized proteins. Using 5 mol% TEGDMA crosslinker offers optimal performance for the small peptide, salmon calcitonin, whereas lower crosslinking density of 1 mol% offers optimal performance for the much larger protein rituximab. Finally, an enzymatically-degradable hydrogels of P(MAA-co-NVP) crosslinked with the peptide sequence MMRRRKK were synthesized and tested in simulated gastric and intestinal conditions. These hydrogels offer ideal loading and release behavior, showing no degradative release of encapsulated salmon calcitonin in gastric conditions while yielding rapid and complete release of encapsulated protein within 1h in intestinal conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Nanostructures for protein drug delivery.

Science.gov (United States)

Pachioni-Vasconcelos, Juliana de Almeida; Lopes, André Moreni; Apolinário, Alexsandra Conceição; Valenzuela-Oses, Johanna Karina; Costa, Juliana Souza Ribeiro; Nascimento, Laura de Oliveira; Pessoa, Adalberto; Barbosa, Leandro Ramos Souza; Rangel-Yagui, Carlota de Oliveira

2016-02-01

Use of nanoscale devices as carriers for drugs and imaging agents has been extensively investigated and successful examples can already be found in therapy. In parallel, recombinant DNA technology together with molecular biology has opened up numerous possibilities for the large-scale production of many proteins of pharmaceutical interest, reflecting in the exponentially growing number of drugs of biotechnological origin. When we consider protein drugs, however, there are specific criteria to take into account to select adequate nanostructured systems as drug carriers. In this review, we highlight the main features, advantages, drawbacks and recent developments of nanostructures for protein encapsulation, such as nanoemulsions, liposomes, polymersomes, single-protein nanocapsules and hydrogel nanoparticles. We also discuss the importance of nanoparticle stabilization, as well as future opportunities and challenges in nanostructures for protein drug delivery.

Injectable nanocomposite cryogels for versatile protein drug delivery.

Science.gov (United States)

Koshy, Sandeep T; Zhang, David K Y; Grolman, Joshua M; Stafford, Alexander G; Mooney, David J

2018-01-01

Sustained, localized protein delivery can enhance the safety and activity of protein drugs in diverse disease settings. While hydrogel systems are widely studied as vehicles for protein delivery, they often suffer from rapid release of encapsulated cargo, leading to a narrow duration of therapy, and protein cargo can be denatured by incompatibility with the hydrogel crosslinking chemistry. In this work, we describe injectable nanocomposite hydrogels that are capable of sustained, bioactive, release of a variety of encapsulated proteins. Injectable and porous cryogels were formed by bio-orthogonal crosslinking of alginate using tetrazine-norbornene coupling. To provide sustained release from these hydrogels, protein cargo was pre-adsorbed to charged Laponite nanoparticles that were incorporated within the walls of the cryogels. The presence of Laponite particles substantially hindered the release of a number of proteins that otherwise showed burst release from these hydrogels. By modifying the Laponite content within the hydrogels, the kinetics of protein release could be precisely tuned. This versatile strategy to control protein release simplifies the design of hydrogel drug delivery systems. Here we present an injectable nanocomposite hydrogel for simple and versatile controlled release of therapeutic proteins. Protein release from hydrogels often requires first entrapping the protein in particles and embedding these particles within the hydrogel to allow controlled protein release. This pre-encapsulation process can be cumbersome, can damage the protein's activity, and must be optimized for each protein of interest. The strategy presented in this work simply premixes the protein with charged nanoparticles that bind strongly with the protein. These protein-laden particles are then placed within a hydrogel and slowly release the protein into the surrounding environment. Using this method, tunable release from an injectable hydrogel can be achieved for a variety of

BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures.

Science.gov (United States)

Truttmann, Matthias C; Guye, Patrick; Dehio, Christoph

2011-01-01

The gram-negative, zoonotic pathogen Bartonella henselae (Bhe) translocates seven distinct Bartonella effector proteins (Beps) via the VirB/VirD4 type IV secretion system (T4SS) into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.

An overview of site-specific delivery of orally administered proteins ...

African Journals Online (AJOL)

Oral delivery of proteins and peptides poses one of the greatest challenges in controlled drug delivery due to degradation by proteolytic enzymes, poor membrane permeability and large molecular size. Therapeutic proteins/peptides are useful in correcting metabolic disorders (e.g., insulin in diabetes mellitus), ...

In vitro and in vivo delivery of therapeutic proteins using cell penetrating peptides.

Science.gov (United States)

Bolhassani, Azam; Jafarzade, Behnaz Sadat; Mardani, Golnaz

2017-01-01

The failure of proteins to penetrate mammalian cells or target tumor cells restricts their value as therapeutic tools in a variety of diseases such as cancers. Recently, protein transduction domains (PTDs) or cell penetrating peptides (CPPs) have been shown to promote the delivery of therapeutic proteins or peptides into live cells. The successful delivery of proteins mainly depends on their physicochemical properties. Although, linear cell penetrating peptides are one of the most effective delivery vehicles; but currently, cyclic CPPs has been developed to potently transport bioactive full-length proteins into cells. Up to now, several small protein transduction domains from viral proteins including Tat or VP22 could be fused to other peptides or proteins to entry them in various cell types at a dose-dependent approach. A major disadvantage of PTD-fusion proteins is primary uptake into endosomal vesicles leading to inefficient release of the fusion proteins into the cytosol. Recently, non-covalent complex formation (Chariot) between proteins and CPPs has attracted a special interest to overcome some delivery limitations (e.g., toxicity). Many preclinical and clinical trials of CPP-based delivery are currently under evaluation. Generally, development of more efficient protein transduction domains would significantly increase the potency of protein therapeutics. Moreover, the synergistic or combined effects of CPPs with other delivery systems for protein/peptide drug delivery would promote their therapeutic effects in cancer and other diseases. In this review, we will describe the functions and implications of CPPs for delivering the therapeutic proteins or peptides in preclinical and clinical studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification, structure, and function of a novel type VI secretion peptidoglycan glycoside hydrolase effector-immunity pair.

Science.gov (United States)

Whitney, John C; Chou, Seemay; Russell, Alistair B; Biboy, Jacob; Gardiner, Taylor E; Ferrin, Michael A; Brittnacher, Mitchell; Vollmer, Waldemar; Mougous, Joseph D

2013-09-13

Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity.

Identification, Structure, and Function of a Novel Type VI Secretion Peptidoglycan Glycoside Hydrolase Effector-Immunity Pair*

Science.gov (United States)

Whitney, John C.; Chou, Seemay; Russell, Alistair B.; Biboy, Jacob; Gardiner, Taylor E.; Ferrin, Michael A.; Brittnacher, Mitchell; Vollmer, Waldemar; Mougous, Joseph D.

2013-01-01

Bacteria employ type VI secretion systems (T6SSs) to facilitate interactions with prokaryotic and eukaryotic cells. Despite the widespread identification of T6SSs among Gram-negative bacteria, the number of experimentally validated substrate effector proteins mediating these interactions remains small. Here, employing an informatics approach, we define novel families of T6S peptidoglycan glycoside hydrolase effectors. Consistent with the known intercellular self-intoxication exhibited by the T6S pathway, we observe that each effector gene is located adjacent to a hypothetical open reading frame encoding a putative periplasmically localized immunity determinant. To validate our sequence-based approach, we functionally investigate a representative family member from the soil-dwelling bacterium Pseudomonas protegens. We demonstrate that this protein is secreted in a T6SS-dependent manner and that it confers a fitness advantage in growth competition assays with Pseudomonas putida. In addition, we determined the 1.4 Å x-ray crystal structure of this effector in complex with its cognate immunity protein. The structure reveals the effector shares highest overall structural similarity to a glycoside hydrolase family associated with peptidoglycan N-acetylglucosaminidase activity, suggesting that T6S peptidoglycan glycoside hydrolase effector families may comprise significant enzymatic diversity. Our structural analyses also demonstrate that self-intoxication is prevented by the immunity protein through direct occlusion of the effector active site. This work significantly expands our current understanding of T6S effector diversity. PMID:23878199

GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

Science.gov (United States)

Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

2015-01-01

Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

Intracellular cargo delivery by virus capsid protein-based vehicles: From nano to micro.

Science.gov (United States)

Gao, Ding; Lin, Xiu-Ping; Zhang, Zhi-Ping; Li, Wei; Men, Dong; Zhang, Xian-En; Cui, Zong-Qiang

2016-02-01

Cellular delivery is an important concern for the efficiency of medicines and sensors for disease diagnoses and therapy. However, this task is quite challenging. Self-assembly virus capsid proteins might be developed as building blocks for multifunctional cellular delivery vehicles. In this work, we found that SV40 VP1 (Simian virus 40 major capsid protein) could function as a new cell-penetrating protein. The VP1 protein could carry foreign proteins into cells in a pentameric structure. A double color structure, with red QDs (Quantum dots) encapsulated by viral capsids fused with EGFP, was created for imaging cargo delivery and release from viral capsids. The viral capsids encapsulating QDs were further used for cellular delivery of micron-sized iron oxide particles (MPIOs). MPIOs were efficiently delivered into live cells and controlled by a magnetic field. Therefore, our study built virus-based cellular delivery systems for different sizes of cargos: protein molecules, nanoparticles, and micron-sized particles. Much research is being done to investigate methods for efficient and specific cellular delivery of drugs, proteins or genetic material. In this article, the authors describe their approach in using self-assembly virus capsid proteins SV40 VP1 (Simian virus 40 major capsid protein). The cell-penetrating behavior provided excellent cellular delivery and should give a new method for biomedical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Effector Protein Cig2 Decreases Host Tolerance of Infection by Directing Constitutive Fusion of Autophagosomes with the Coxiella-Containing Vacuole

Directory of Open Access Journals (Sweden)

Lara J. Kohler

2016-07-01

Full Text Available Coxiella burnetii replicates in an acidified lysosome-derived vacuole. Biogenesis of the Coxiella-containing vacuole (CCV requires bacterial effector proteins delivered into host cells by the Dot/Icm secretion system. Genetic and cell biological analysis revealed that an effector protein called Cig2 promotes constitutive fusion of autophagosomes with the CCV to maintain this compartment in an autolysosomal stage of maturation. This distinguishes the CCV from other pathogen-containing vacuoles that are targeted by the host autophagy pathway, which typically confers host resistance to infection by delivering the pathogen to a toxic lysosomal environment. By maintaining the CCV in an autolysosomal stage of maturation, Cig2 enabled CCV homotypic fusion and enhanced bacterial virulence in the Galleria mellonella (wax moth model of infection by a mechanism that decreases host tolerance. Thus, C. burnetii residence in an autolysosomal organelle alters host tolerance of infection, which indicates that Cig2-dependent manipulation of a lysosome-derived vacuole influences the host response to infection.

Characterization of the largest effector gene cluster of Ustilago maydis.

Directory of Open Access Journals (Sweden)

Thomas Brefort

2014-07-01

Full Text Available In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function.

BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures.

Directory of Open Access Journals (Sweden)

Matthias C Truttmann

Full Text Available The gram-negative, zoonotic pathogen Bartonella henselae (Bhe translocates seven distinct Bartonella effector proteins (Beps via the VirB/VirD4 type IV secretion system (T4SS into human cells, thereby interfering with host cell signaling [1], [2]. In particular, the effector protein BepG alone or the combination of effector proteins BepC and BepF trigger massive F-actin rearrangements that lead to the establishment of invasome structures eventually resulting in the internalization of entire Bhe aggregates [2], [3]. In this report, we investigate the molecular function of the effector protein BepF in the eukaryotic host cell. We show that the N-terminal [E/T]PLYAT tyrosine phosphorylation motifs of BepF get phosphorylated upon translocation but do not contribute to invasome-mediated Bhe uptake. In contrast, we found that two of the three BID domains of BepF are capable to trigger invasome formation together with BepC, while a mutation of the WxxxE motif of the BID-F1 domain inhibited its ability to contribute to the formation of invasome structures. Next, we show that BepF function during invasome formation can be replaced by the over-expression of constitutive-active Rho GTPases Rac1 or Cdc42. Finally we demonstrate that BID-F1 and BID-F2 domains promote the formation of filopodia-like extensions in NIH 3T3 and HeLa cells as well as membrane protrusions in HeLa cells, suggesting a role for BepF in Rac1 and Cdc42 activation during the process of invasome formation.

Highly Efficient Intracellular Protein Delivery by Cationic Polyethyleneimine-Modified Gelatin Nanoparticles

Directory of Open Access Journals (Sweden)

Ming-Ju Chou

2018-02-01

Full Text Available Intracellular protein delivery may provide a safe and non-genome integrated strategy for targeting abnormal or specific cells for applications in cell reprogramming therapy. Thus, highly efficient intracellular functional protein delivery would be beneficial for protein drug discovery. In this study, we generated a cationic polyethyleneimine (PEI-modified gelatin nanoparticle and evaluated its intracellular protein delivery ability in vitro and in vivo. The experimental results showed that the PEI-modified gelatin nanoparticle had a zeta potential of approximately +60 mV and the particle size was approximately 135 nm. The particle was stable at different biological pH values and temperatures and high protein loading efficiency was observed. The fluorescent image results revealed that large numbers of particles were taken up into the mammalian cells and escaped from the endosomes into the cytoplasm. In a mouse C26 cell-xenograft cancer model, particles accumulated in cancer cells. In conclusion, the PEI-modified gelatin particle may provide a biodegradable and highly efficient protein delivery system for use in regenerative medicine and cancer therapy.

The potential of protein-nanomaterial interaction for advanced drug delivery

DEFF Research Database (Denmark)

Peng, Qiang; Mu, Huiling

2016-01-01

Nanomaterials, like nanoparticles, micelles, nano-sheets, nanotubes and quantum dots, have great potentials in biomedical fields. However, their delivery is highly limited by the formation of protein corona upon interaction with endogenous proteins. This new identity, instead of nanomaterial itself...... of such interaction for advanced drug delivery are presented........ Therefore, protein-nanomaterial interaction is a great challenge for nanomaterial systems and should be inhibited. However, this interaction can also be used to functionalize nanomaterials by forming a selected protein corona. Unlike other decoration using exogenous molecules, nanomaterials functionalized...

Co-delivery of chemotherapeutics and proteins for synergistic therapy.

Science.gov (United States)

He, Chaoliang; Tang, Zhaohui; Tian, Huayu; Chen, Xuesi

2016-03-01

Combination therapy with chemotherapeutics and protein therapeutics, typically cytokines and antibodies, has been a type of crucial approaches for synergistic cancer treatment. However, conventional approaches by simultaneous administration of free chemotherapeutic drugs and proteins lead to limitations for further optimizing the synergistic effects, due to the distinct in vivo pharmacokinetics and distribution of small drugs and proteins, insufficient tumor selectivity and tumor accumulation, unpredictable drug/protein ratios at tumor sites, short half-lives, and serious systemic adverse effects. Consequently, to obtain optimal synergistic anti-tumor efficacy, considerable efforts have been devoted to develop the co-delivery systems for co-incorporating chemotherapeutics and proteins into a single carrier system and subsequently releasing the dual or multiple payloads at desired target sites in a more controllable manner. The co-delivery systems result in markedly enhanced blood stability and in vivo half-lives of the small drugs and proteins, elevated tumor accumulation, as well as the capability of delivering the multiple agents to the same target sites with rational drug/protein ratios, which may facilitate maximizing the synergistic effects and therefore lead to optimal antitumor efficacy. This review emphasizes the recent advances in the co-delivery systems for chemotherapeutics and proteins, typically cytokines and antibodies, for systemic or localized synergistic cancer treatment. Moreover, the proposed mechanisms responsible for the synergy of chemotherapeutic drugs and proteins are discussed. Copyright © 2015 Elsevier B.V. All rights reserved.

Evaluation of secretion prediction highlights differing approaches needed for oomycete and fungal effectors

Directory of Open Access Journals (Sweden)

Jana eSperschneider

2015-12-01

Full Text Available The steadily increasing number of sequenced fungal and oomycete genomes has enabled detailed studies of how these eukaryotic microbes infect plants and cause devastating losses in food crops. During infection, fungal and oomycete pathogens secrete effector molecules which manipulate host plant cell processes to the pathogen’s advantage. Proteinaceous effectors are synthesised intracellularly and must be externalised to interact with host cells. Computational prediction of secreted proteins from genomic sequences is an important technique to narrow down the candidate effector repertoire for subsequent experimental validation. In this study, we benchmark secretion prediction tools on experimentally validated fungal and oomycete effectors. We observe that for a set of fungal SwissProt protein sequences, SignalP 4 and the neural network predictors of SignalP 3 (D-score and SignalP 2 perform best. For effector prediction in particular, the use of a sensitive method can be desirable to obtain the most complete candidate effector set. We show that the neural network predictors of SignalP 2 and 3, as well as TargetP were the most sensitive tools for fungal effector secretion prediction, whereas the hidden Markov model predictors of SignalP 2 and 3 were the most sensitive tools for oomycete effectors. Thus, previous versions of SignalP retain value for oomycete effector prediction, as the current version, SignalP 4, was unable to reliably predict the signal peptide of the oomycete Crinkler effectors in the test set. Our assessment of subcellular localisation predictors shows that cytoplasmic effectors are often predicted as not extracellular. This limits the reliability of secretion predictions that depend on these tools. We present our assessment with a view to informing future pathogenomics studies and suggest revised pipelines for secretion prediction to obtain optimal effector predictions in fungi and oomycetes.

Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

International Nuclear Information System (INIS)

Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

2008-01-01

Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs

Comparative genomics of the type VI secretion systems of Pantoea and Erwinia species reveals the presence of putative effector islands that may be translocated by the VgrG and Hcp proteins

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De Maayer Pieter

2011-11-01

Full Text Available Abstract Background The Type VI secretion apparatus is assembled by a conserved set of proteins encoded within a distinct locus. The putative effector proteins Hcp and VgrG are also encoded within these loci. We have identified numerous distinct Type VI secretion system (T6SS loci in the genomes of several ecologically diverse Pantoea and Erwinia species and detected the presence of putative effector islands associated with the hcp and vgrG genes. Results Between two and four T6SS loci occur among the Pantoea and Erwinia species. While two of the loci (T6SS-1 and T6SS-2 are well conserved among the various strains, the third (T6SS-3 locus is not universally distributed. Additional orthologous loci are present in Pantoea sp. aB-valens and Erwinia billingiae Eb661. Comparative analysis of the T6SS-1 and T6SS-3 loci showed non-conserved islands associated with the vgrG and hcp, and vgrG genes, respectively. These regions had a G+C content far lower than the conserved portions of the loci. Many of the proteins encoded within the hcp and vgrG islands carry conserved domains, which suggests they may serve as effector proteins for the T6SS. A number of the proteins also show homology to the C-terminal extensions of evolved VgrG proteins. Conclusions Extensive diversity was observed in the number and content of the T6SS loci among the Pantoea and Erwinia species. Genomic islands could be observed within some of T6SS loci, which are associated with the hcp and vgrG proteins and carry putative effector domain proteins. We propose new hypotheses concerning a role for these islands in the acquisition of T6SS effectors and the development of novel evolved VgrG and Hcp proteins.

The SecA2 pathway of Mycobacterium tuberculosis exports effectors that work in concert to arrest phagosome and autophagosome maturation.

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Zulauf, Katelyn E; Sullivan, Jonathan Tabb; Braunstein, Miriam

2018-04-30

To subvert host defenses, Mycobacterium tuberculosis (Mtb) avoids being delivered to degradative phagolysosomes in macrophages by arresting the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. The SecA2 dependent protein export system is required for phagosome maturation arrest and consequently growth of Mtb in macrophages. To better understand the role of the SecA2 pathway in phagosome maturation arrest, we identified two effectors exported by SecA2 that contribute to this process: the phosphatase SapM and the kinase PknG. Then, utilizing the secA2 mutant of Mtb as a platform to study effector functions, we identified specific steps in phagosome maturation inhibited by SapM and/or PknG. By identifying a histidine residue that is essential for SapM phosphatase activity, we confirmed for the first time that the phosphatase activity of SapM is required for its effects on phagosome maturation in macrophages. We further demonstrated that SecA2 export of SapM and PknG contributes to the ability of Mtb to replicate in macrophages. Finally, we extended our understanding of the SecA2 pathway, SapM, and PknG by revealing that their contribution goes beyond preventing Mtb delivery to mature phagolysosomes and includes inhibiting Mtb delivery to autophagolysosomes. Together, our results revealed SapM and PknG to be two effectors exported by the SecA2 pathway of Mtb with distinct as well as cumulative effects on phagosome and autophagosome maturation. Our results further reveal that Mtb must have additional mechanisms of limiting acidification of the phagosome, beyond inhibiting recruitment of the V-ATPase proton pump to the phagosome, and they indicate differences between effects of Mtb on phagosome and autophagosome maturation.

Phytoplasma protein effector SAP11 enhances insect vector reproduction by manipulating plant development and defense hormone biosynthesis.

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Sugio, Akiko; Kingdom, Heather N; MacLean, Allyson M; Grieve, Victoria M; Hogenhout, Saskia A

2011-11-29

Phytoplasmas are insect-transmitted phytopathogenic bacteria that can alter plant morphology and the longevity and reproduction rates and behavior of their insect vectors. There are various examples of animal and plant parasites that alter the host phenotype to attract insect vectors, but it is unclear how these parasites accomplish this. We hypothesized that phytoplasmas produce effectors that modulate specific targets in their hosts leading to the changes in plant development and insect performance. Previously, we sequenced and mined the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB) and identified 56 candidate effectors. Here, we report that the secreted AY-WB protein 11 (SAP11) effector modulates plant defense responses to the advantage of the AY-WB insect vector Macrosteles quadrilineatus. SAP11 binds and destabilizes Arabidopsis CINCINNATA (CIN)-related TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORS 1 and 2 (TCP) transcription factors, which control plant development and promote the expression of lipoxygenase (LOX) genes involved in jasmonate (JA) synthesis. Both the Arabidopsis SAP11 lines and AY-WB-infected plants produce less JA on wounding. Furthermore, the AY-WB insect vector produces more offspring on AY-WB-infected plants, SAP11 transgenic lines, and plants impaired in CIN-TCP and JA synthesis. Thus, SAP11-mediated destabilization of CIN-TCPs leads to the down-regulation of LOX2 expression and JA synthesis and an increase in M. quadrilineatus progeny. Phytoplasmas are obligate inhabitants of their plant host and insect vectors, in which the latter transmits AY-WB to a diverse range of plant species. This finding demonstrates that pathogen effectors can reach beyond the pathogen-host interface to modulate a third organism in the biological interaction.

Plant parasitic nematode effectors target host defence and nuclear functions to establish feeding cells

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Michaël eQuentin

2013-03-01

Full Text Available Plant parasitic nematodes are microscopic worms, the most damaging species of which have adopted a sedentary lifestyle within their hosts. These obligate endoparasites have a biotrophic relationship with plants, in which they induce the differentiation of root cells into hypertrophied, multinucleate feeding cells. Effectors synthesised in the oesophageal glands of the nematode are injected into the plant cells via the syringe-like stylet and play a key role in manipulating the host machinery. The establishment of specialized feeding cells requires these effectors to modulate many aspects of plant cell morphogenesis and physiology, including defence responses. This cell reprogramming requires changes to host nuclear processes. Some proteins encoded by parasitism genes target host nuclei. Several of these proteins were immunolocalised within feeding cell nuclei or shown to interact with host nuclear proteins. Comparative genomics and functional analyses are gradually revealing the roles of nematode effectors. We describe here these effectors and their hypothesised roles in the unique feeding behaviour of these pests.

Stabilization and delivery approaches for protein and peptide pharmaceuticals: an extensive review of patents.

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Swain, Suryakanta; Mondal, Debanik; Beg, Sarwar; Patra, Chinam Niranjan; Dinda, Subas Chandra; Sruti, Jammula; Rao, Muddana Eswara Bhanoji

2013-04-01

Proteins and peptides are the building blocks of human body and act as the arsenal to combat against the invading pathogenic organisms for treatment and management of diseases. Majority of such biomacromolecules are synthesized by the human body itself. However, entry of disease causing pathogens causes misleading in the synthesis of desired proteins for antibody formation. In such alarming situations, the delivery of requisite protein and peptide from external source helps in augmenting the body's immunity. The major drawbacks underlying poor biopharmaceutical performance of high molecular weight protein and peptide drugs are due to poor oral absorption, formulation stability, degradation in the gastric milieu, susceptible to presystemic metabolism. Numerous literature recounts the application of myriad drug delivery strategies for the effective delivery of protein and peptides viz. parentral, oral, transdermal, nasal, pulmonary, rectal, buccal and ocular drug delivery systems. There are many reviews on various delivery strategies for protein and peptide pharmaceuticals, but the present review article provides a bird's eye view on various novel drug delivery systems used for enhanced delivery of protein and peptide pharmaceuticals in the light of patent literature. Apart from this, the present manuscript endeavor provides idea on possible causes and major degradation pathways responsible for poor stability of protein and peptide drugs along with recent market instances on them utilizing novel drug delivery systems.

Phytoplasma Effector SAP54 Induces Indeterminate Leaf-Like Flower Development in Arabidopsis Plants1[C][W][OA

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MacLean, Allyson M.; Sugio, Akiko; Makarova, Olga V.; Findlay, Kim C.; Grieve, Victoria M.; Tóth, Réka; Nicolaisen, Mogens; Hogenhout, Saskia A.

2011-01-01

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches’ broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host. PMID:21849514

Intracellular Protein Delivery System Using a Target-Specific Repebody and Translocation Domain of Bacterial Exotoxin.

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Kim, Hee-Yeon; Kang, Jung Ae; Ryou, Jeong-Hyun; Lee, Gyeong Hee; Choi, Dae Seong; Lee, Dong Eun; Kim, Hak-Sung

2017-11-17

With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.

A translocated effector required for Bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors.

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Okujava, Rusudan; Guye, Patrick; Lu, Yun-Yueh; Mistl, Claudia; Polus, Florine; Vayssier-Taussat, Muriel; Halin, Cornelia; Rolink, Antonius G; Dehio, Christoph

2014-06-01

Numerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. Bartonellae are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of Bartonella effector proteins (Beps) into host cells. Based on in vitro infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for in vivo dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs) infected with a ΔbepE mutant of B. henselae (Bhe) displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A ΔbepCE mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of ΔbepE with BepEBhe or its homologues from other Bartonella species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal Bartonella intracellular delivery (BID) domain of BepEBhe (BID2.EBhe). Ectopic expression of BID2.EBhe impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An intradermal (i.d.) model for B. tribocorum (Btr) infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the Btr mutant ΔbepDE was abacteremic following i.d. inoculation, complementation with BepEBtr, BepEBhe or BIDs.EBhe restored bacteremia. Given that we observed a similar protective effect of BepEBhe on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we propose that

A translocated effector required for Bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors.

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Rusudan Okujava

2014-06-01

Full Text Available Numerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. Bartonellae are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of Bartonella effector proteins (Beps into host cells. Based on in vitro infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for in vivo dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs infected with a ΔbepE mutant of B. henselae (Bhe displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A ΔbepCE mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of ΔbepE with BepEBhe or its homologues from other Bartonella species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal Bartonella intracellular delivery (BID domain of BepEBhe (BID2.EBhe. Ectopic expression of BID2.EBhe impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An intradermal (i.d. model for B. tribocorum (Btr infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the Btr mutant ΔbepDE was abacteremic following i.d. inoculation, complementation with BepEBtr, BepEBhe or BIDs.EBhe restored bacteremia. Given that we observed a similar protective effect of BepEBhe on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we

Delivery of proteins to mammalian cells via gold nanoparticle mediated laser transfection

International Nuclear Information System (INIS)

Heinemann, D; Kalies, S; Schomaker, M; Ertmer, W; Meyer, H; Ripken, T; Murua Escobar, H

2014-01-01

Nanoparticle laser interactions are in widespread use in cell manipulation. In particular, molecular medicine needs techniques for the directed delivery of molecules into mammalian cells. Proteins are the final mediator of most cellular cascades. However, despite several methodical approaches, the efficient delivery of proteins to cells remains challenging. This paper presents a new protein transfection technique via laser scanning of cells previously incubated with gold nanoparticles. The laser-induced plasmonic effects on the gold nanoparticles cause a transient permeabilization of the cellular membrane, allowing proteins to enter the cell. Applying this technique, it was possible to deliver green fluorescent protein into mammalian cells with an efficiency of 43%, maintaining a high level of cell viability. Furthermore, a functional delivery of Caspase 3, an apoptosis mediating protein, was demonstrated and evaluated in several cellular assays. Compared to conventional protein transfection techniques such as microinjection, the methodical approach presented here enables high-throughput transfection of about 10 000 cells per second. Moreover, a well-defined point in time of delivery is guaranteed by gold nanoparticle mediated laser transfection, allowing the detailed temporal analysis of cellular pathways and protein trafficking. (papers)

Transcriptional programming and functional interactions within the Phytophthora sojae RXLR effector repertoire.

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Wang, Qunqing; Han, Changzhi; Ferreira, Adriana O; Yu, Xiaoli; Ye, Wenwu; Tripathy, Sucheta; Kale, Shiv D; Gu, Biao; Sheng, Yuting; Sui, Yangyang; Wang, Xiaoli; Zhang, Zhengguang; Cheng, Baoping; Dong, Suomeng; Shan, Weixing; Zheng, Xiaobo; Dou, Daolong; Tyler, Brett M; Wang, Yuanchao

2011-06-01

The genome of the soybean pathogen Phytophthora sojae contains nearly 400 genes encoding candidate effector proteins carrying the host cell entry motif RXLR-dEER. Here, we report a broad survey of the transcription, variation, and functions of a large sample of the P. sojae candidate effectors. Forty-five (12%) effector genes showed high levels of polymorphism among P. sojae isolates and significant evidence for positive selection. Of 169 effectors tested, most could suppress programmed cell death triggered by BAX, effectors, and/or the PAMP INF1, while several triggered cell death themselves. Among the most strongly expressed effectors, one immediate-early class was highly expressed even prior to infection and was further induced 2- to 10-fold following infection. A second early class, including several that triggered cell death, was weakly expressed prior to infection but induced 20- to 120-fold during the first 12 h of infection. The most strongly expressed immediate-early effectors could suppress the cell death triggered by several early effectors, and most early effectors could suppress INF1-triggered cell death, suggesting the two classes of effectors may target different functional branches of the defense response. In support of this hypothesis, misexpression of key immediate-early and early effectors severely reduced the virulence of P. sojae transformants.

Gene Delivery into Plant Cells for Recombinant Protein Production

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Qiang Chen

2015-01-01

Full Text Available Recombinant proteins are primarily produced from cultures of mammalian, insect, and bacteria cells. In recent years, the development of deconstructed virus-based vectors has allowed plants to become a viable platform for recombinant protein production, with advantages in versatility, speed, cost, scalability, and safety over the current production paradigms. In this paper, we review the recent progress in the methodology of agroinfiltration, a solution to overcome the challenge of transgene delivery into plant cells for large-scale manufacturing of recombinant proteins. General gene delivery methodologies in plants are first summarized, followed by extensive discussion on the application and scalability of each agroinfiltration method. New development of a spray-based agroinfiltration and its application on field-grown plants is highlighted. The discussion of agroinfiltration vectors focuses on their applications for producing complex and heteromultimeric proteins and is updated with the development of bridge vectors. Progress on agroinfiltration in Nicotiana and non-Nicotiana plant hosts is subsequently showcased in context of their applications for producing high-value human biologics and low-cost and high-volume industrial enzymes. These new advancements in agroinfiltration greatly enhance the robustness and scalability of transgene delivery in plants, facilitating the adoption of plant transient expression systems for manufacturing recombinant proteins with a broad range of applications.

IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation.

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Jefferis, R; Lund, J; Pound, J D

1998-06-01

The Fc region of human IgG expresses interaction sites for many effector ligands. In this review the topographical distributions of ten of these sites are discussed in relation to functional requirement. It is apparent that interaction sites localised to the inter-CH2-CH3 domain region of the Fc allow for functional divalency, whereas sites localised to the hinge proximal region of the CH2 domain are functionally monovalent, with expression of the latter sites being particularly dependent on glycosylation. All x-ray crystal structures for Fc and Fc-ligand complexes report that the protein structure of the hinge proximal region of the CH2 domain is "disordered", suggesting "internal mobility". We propose a model in which such "internal mobility" results in the generation of a dynamic equilibrium between multiple conformers, certain of which express interaction sites specific to individual ligands. The emerging understanding of the influence of oligosaccharide/protein interactions on protein conformation and biological function of IgG antibodies suggests a potential to generate novel glycoforms of antibody molecules having unique profiles of effector functions.

Recent advances in protein and Peptide drug delivery: a special emphasis on polymeric nanoparticles.

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Patel, Ashaben; Patel, Mitesh; Yang, Xiaoyan; Mitra, Ashim K

2014-01-01

Proteins and peptides are widely indicated in many diseased states. Parenteral route is the most commonly em- ployed method of administration for therapeutic proteins and peptides. However, requirement of frequent injections due to short in vivo half-life results in poor patient compliance. Non-invasive drug delivery routes such as nasal, transdermal, pulmonary, and oral offer several advantages over parenteral administration. Intrinsic physicochemical properties and low permeability across biological membrane limit protein delivery via non-invasive routes. One of the strategies to improve protein and peptide absorption is by delivering through nanostructured delivery carriers. Among nanocarriers, polymeric nanoparticles (NPs) have demonstrated significant advantages over other delivery systems. This article summarizes the application of polymeric NPs for protein and peptide drug delivery following oral, nasal, pulmonary, parenteral, transder mal, and ocular administrations.

The genome sequence and effector complement of the flax rust pathogen Melampsora lini

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Adnane eNemri

2014-03-01

Full Text Available Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp. Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimise parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analysed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote

The genome sequence and effector complement of the flax rust pathogen Melampsora lini.

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Nemri, Adnane; Saunders, Diane G O; Anderson, Claire; Upadhyaya, Narayana M; Win, Joe; Lawrence, Gregory J; Jones, David A; Kamoun, Sophien; Ellis, Jeffrey G; Dodds, Peter N

2014-01-01

Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed most extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimize parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analyzed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their

Functional heterogeneity of human effector CD8+ T cells.

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Takata, Hiroshi; Naruto, Takuya; Takiguchi, Masafumi

2012-02-09

Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.

Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins

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Grinberg, Yehudit; Benhar, Itai

2017-01-01

Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called “human cytotoxic fusion proteins”, in which antibodies are used to target human effector proteins. Here, we present three relevant approaches for reducing the immunogenicity of antibody-targeted protein therapeutics: (1) reducing the immunogenicity of the bacterial toxin, (2) fusing human cytokines to antibodies to generate immunocytokines and (3) addressing the immunogenicity of the targeting antibodies. PMID:28574434

Fructose 1-phosphate is the preferred effector of the metabolic regulator Cra of Pseudomonas putida.

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Chavarría, Max; Santiago, César; Platero, Raúl; Krell, Tino; Casasnovas, José M; de Lorenzo, Víctor

2011-03-18

The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5'-TTAAACGTTTCA-3' (K(D) = 26.3 ± 3.1 nM) and that F1P binds the protein with an apparent stoichiometry of 1.06 ± 0.06 molecules per Cra monomer and a K(D) of 209 ± 20 nM. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-Å resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida.

Fructose 1-Phosphate Is the Preferred Effector of the Metabolic Regulator Cra of Pseudomonas putida*

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Chavarría, Max; Santiago, César; Platero, Raúl; Krell, Tino; Casasnovas, José M.; de Lorenzo, Víctor

2011-01-01

The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of Gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5′-TTAAACGTTTCA-3′ (KD = 26.3 ± 3.1 nm) and that F1P binds the protein with an apparent stoichiometry of 1.06 ± 0.06 molecules per Cra monomer and a KD of 209 ± 20 nm. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-Å resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida. PMID:21239488

Recent trends in drug delivery system using protein nanoparticles.

Science.gov (United States)

Sripriyalakshmi, S; Jose, Pinkybel; Ravindran, Aswathy; Anjali, C H

2014-09-01

Engineered nanoparticles that can facilitate drug formulation and passively target tumours have been under extensive research in recent years. These successes have driven a new wave of significant innovation in the generation of advanced particles. The fate and transport of diagnostic nanoparticles would significantly depend on nonselective drug delivery, and hence the use of high drug dosage is implemented. In this perspective, nanocarrier-based drug targeting strategies can be used which improve the selective delivery of drugs to the site of action, i.e. drug targeting. Pharmaceutical industries majorly focus on reducing the toxicity and side effects of drugs but only recently it has been realised that carrier systems themselves may pose risks to the patient. Proteins are compatible with biological systems and they are biodegradable. They offer a multitude of moieties for modifications to tailor drug binding, imaging or targeting entities. Thus, protein nanoparticles provide outstanding contributions as a carrier for drug delivery systems. This review summarises recent progress in particle-based therapeutic delivery and discusses important concepts in particle design and biological barriers for developing the next generation of particles drug delivery systems.

Establishment of protein delivery systems targeting podocytes.

Directory of Open Access Journals (Sweden)

Wen Chih Chiang

2010-07-01

Full Text Available Podocytes are uniquely structured cells that are critical to the kidney filtration barrier. Their anatomic location on the outer side of the glomerular capillaries expose podocytes to large quantities of both plasma and urinary components and thus are reachable for drug delivery. Recent years have made clear that interference with podocyte-specific disease pathways can modulate glomerular function and influence severity and progression of glomerular disease.Here, we describe studies that show efficient transport of proteins into the mammalian cells mouse 3T3 fibroblasts and podocytes, utilizing an approach termed profection. We are using synthetic lipid structures that allow the safe packing of proteins or antibodies resulting in the subsequent delivery of protein into the cell. The uptake of lipid coated protein is facilitated by the intrinsic characteristic of cells such as podocytes to engulf particles that are physiologically retained in the extracellular matrix. Profection of the restriction enzyme MunI in 3T3 mouse fibroblasts caused an increase in DNA degradation. Moreover, purified proteins such as beta-galactosidase and the large GTPase dynamin could be profected into podocytes using two different profection reagents with the success rate of 95-100%. The delivered beta-galactosidase enzyme was properly folded and able to cleave its substrate X-gal in podocytes. Diseased podocytes are also potential recipients of protein cargo as we also delivered fluorophore labeled IgG into puromycin treated podocytes. We are currently optimizing our protocol for in vivo profection.Protein transfer is developing as an exciting tool to study and target highly differentiated cells such as podocytes.

Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein.

Science.gov (United States)

Kessler, P D; Podsakoff, G M; Chen, X; McQuiston, S A; Colosi, P C; Matelis, L A; Kurtzman, G J; Byrne, B J

1996-11-26

Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the beta-galactosidase (AAV-lacZ) gene into adult BALB/c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies.

Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein

Science.gov (United States)

Kessler, Paul D.; Podsakoff, Gregory M.; Chen, Xiaojuan; McQuiston, Susan A.; Colosi, Peter C.; Matelis, Laura A.; Kurtzman, Gary J.; Byrne, Barry J.

1996-01-01

Somatic gene therapy has been proposed as a means to achieve systemic delivery of therapeutic proteins. However, there is limited evidence that current methods of gene delivery can practically achieve this goal. In this study, we demonstrate that, following a single intramuscular administration of a recombinant adeno-associated virus (rAAV) vector containing the β-galactosidase (AAV-lacZ) gene into adult BALB/c mice, protein expression was detected in myofibers for at least 32 weeks. A single intramuscular administration of an AAV vector containing a gene for human erythropoietin (AAV-Epo) into mice resulted in dose-dependent secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks. Primary human myotubes transduced in vitro with the AAV-Epo vector also showed dose-dependent production of Epo. These results demonstrate that rAAV vectors are able to transduce skeletal muscle and are capable of achieving sustained expression and systemic delivery of a therapeutic protein following a single intramuscular administration. Gene therapy using AAV vectors may provide a practical strategy for the treatment of inherited and acquired protein deficiencies. PMID:8943064

The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules.

Science.gov (United States)

Simon, Katharina; Hennen, Stephanie; Merten, Nicole; Blättermann, Stefanie; Gillard, Michel; Kostenis, Evi; Gomeza, Jesus

2016-01-08

Recent studies have recognized G protein-coupled receptors as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan G protein-coupled receptor that has been identified as oligodendroglial maturation inhibitor because its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small molecule agonist MDL29,951 (2-carboxy-4,6-dichloro-1H-indole-3-propionic acid) decreases myelin basic protein expression levels mainly by triggering the Gαi/o signaling pathway, which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element-binding protein (CREB). In addition, we show that GPR17 activation also diminishes myelin basic protein abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Genome-scale identification of Legionella pneumophila effectors using a machine learning approach.

Directory of Open Access Journals (Sweden)

David Burstein

2009-07-01

Full Text Available A large number of highly pathogenic bacteria utilize secretion systems to translocate effector proteins into host cells. Using these effectors, the bacteria subvert host cell processes during infection. Legionella pneumophila translocates effectors via the Icm/Dot type-IV secretion system and to date, approximately 100 effectors have been identified by various experimental and computational techniques. Effector identification is a critical first step towards the understanding of the pathogenesis system in L. pneumophila as well as in other bacterial pathogens. Here, we formulate the task of effector identification as a classification problem: each L. pneumophila open reading frame (ORF was classified as either effector or not. We computationally defined a set of features that best distinguish effectors from non-effectors. These features cover a wide range of characteristics including taxonomical dispersion, regulatory data, genomic organization, similarity to eukaryotic proteomes and more. Machine learning algorithms utilizing these features were then applied to classify all the ORFs within the L. pneumophila genome. Using this approach we were able to predict and experimentally validate 40 new effectors, reaching a success rate of above 90%. Increasing the number of validated effectors to around 140, we were able to gain novel insights into their characteristics. Effectors were found to have low G+C content, supporting the hypothesis that a large number of effectors originate via horizontal gene transfer, probably from their protozoan host. In addition, effectors were found to cluster in specific genomic regions. Finally, we were able to provide a novel description of the C-terminal translocation signal required for effector translocation by the Icm/Dot secretion system. To conclude, we have discovered 40 novel L. pneumophila effectors, predicted over a hundred additional highly probable effectors, and shown the applicability of machine

Arginine-rich intracellular delivery peptides noncovalently transport protein into living cells

International Nuclear Information System (INIS)

Wang, Y.-H.; Chen, C.-P.; Chan, M.-H.; Chang, M.; Hou, Y.-W.; Chen, H.-H.; Hsu, H.-R.; Liu, Kevin; Lee, H.-J.

2006-01-01

Plasma membranes of plant or animal cells are generally impermeable to peptides or proteins. Many basic peptides have previously been investigated and covalently cross-linked with cargoes for cellular internalization. In the current study, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to deliver fluorescent proteins or β-galactosidase enzyme into animal and plant cells, as well as animal tissue. Cellular internalization and transdermal delivery of protein could be mediated by effective and nontoxic AID peptides in a neither fusion protein nor conjugation fashion. Therefore, noncovalent AID peptides may provide a useful strategy to have active proteins function in living cells and tissues in vivo

Shigella IpaH Family Effectors as a Versatile Model for Studying Pathogenic Bacteria.

Science.gov (United States)

Ashida, Hiroshi; Sasakawa, Chihiro

2015-01-01

Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis). Via the type III secretion system (T3SS), Shigella deliver a subset of virulence proteins (effectors) that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens, including Salmonella, Yersinia, and enteropathogenic Escherichia coli (EPEC). Therefore, studying Shigella T3SS effectors will not only improve our understanding of bacterial infection systems, but also provide a molecular basis for developing live bacterial vaccines and antibacterial drugs. One of Shigella T3SS effectors, IpaH family proteins, which have E3 ubiquitin ligase activity and are widely conserved among other bacterial pathogens, are very relevant because they promote bacterial survival by triggering cell death and modulating the host immune responses. Here, we describe selected examples of Shigella pathogenesis, with particular emphasis on the roles of IpaH family effectors, which shed new light on bacterial survival strategies and provide clues about how to overcome bacterial infections.

Shigella IpaH family effectors as a versatile model for studying pathogenic bacteria

Directory of Open Access Journals (Sweden)

Hiroshi eAshida

2016-01-01

Full Text Available Shigella spp. are highly adapted human pathogens that cause bacillary dysentery (shigellosis. Via the type III secretion system (T3SS, Shigella deliver a subset of virulence proteins (effectors that are responsible for pathogenesis, with functions including pyroptosis, invasion of the epithelial cells, intracellular survival, and evasion of host immune responses. Intriguingly, T3SS effector activity and strategies are not unique to Shigella, but are shared by many other bacterial pathogens, including Salmonella, Yersinia, and enteropathogenic Escherichia coli (EPEC. Therefore, studying Shigella T3SS effectors will not only improve our understanding of bacterial infection systems, but also provide a molecular basis for developing live bacterial vaccines and antibacterial drugs. One of Shigella T3SS effectors, IpaH family proteins, which have E3 ubiquitin ligase activity and are widely conserved among other bacterial pathogens, are very relevant because they promote bacterial survival by triggering cell death and modulating the host immune responses. Here, we describe selected examples of Shigella pathogenesis, with particular emphasis on the roles of IpaH family effectors, which shed new light on bacterial survival strategies and provide clues about how to overcome bacterial infections.

A sight on protein-based nanoparticles as drug/gene delivery systems.

Science.gov (United States)

Salatin, Sara; Jelvehgari, Mitra; Maleki-Dizaj, Solmaz; Adibkia, Khosro

2015-01-01

Polymeric nanomaterials have extensively been applied for the preparation of targeted and controlled release drug/gene delivery systems. However, problems involved in the formulation of synthetic polymers such as using of the toxic solvents and surfactants have limited their desirable applications. In this regard, natural biomolecules including proteins and polysaccharide are suitable alternatives due to their safety. According to literature, protein-based nanoparticles possess many advantages for drug and gene delivery such as biocompatibility, biodegradability and ability to functionalize with targeting ligands. This review provides a general sight on the application of biodegradable protein-based nanoparticles in drug/gene delivery based on their origins. Their unique physicochemical properties that help them to be formulated as pharmaceutical carriers are also discussed.

Identification and characterisation of a hyper-variable apoplastic effector gene family of the potato cyst nematodes.

Science.gov (United States)

Eves-van den Akker, Sebastian; Lilley, Catherine J; Jones, John T; Urwin, Peter E

2014-09-01

Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism.

How to conquer a tomato plant? Fusarium oxysporum effector targets

NARCIS (Netherlands)

de Sain, M.

2016-01-01

Pathogens secrete small proteins, called effectors, to alter the environment in their host to facilitate infection. The causal agent of Fusarium wilt on tomato, Fusarium oxysporum f. sp. lycopersici (Fol), secretes these proteins in the xylem sap of infected plants and hence they have been called

Multi-protein delivery by nanodiamonds promotes bone formation.

Science.gov (United States)

Moore, L; Gatica, M; Kim, H; Osawa, E; Ho, D

2013-11-01

Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE(®) for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation.

Delivery of bioactive peptides and proteins across oral (buccal) mucosa.

Science.gov (United States)

Senel, S; Kremer, M; Nagy, K; Squier, C

2001-06-01

The identification of an increasing array of highly potent, endogenous peptide and protein factors termed cytokines, that can be efficiently synthesized using recombinant DNA technology, offers exciting new approaches for drug therapy. However, the physico-chemical and biological properties of these agents impose limitations in formulation and development of optimum drug delivery systems as well as on the routes of delivery. Oral mucosa, including the lining of the cheek (buccal mucosa), floor of mouth and underside of tongue (sublingual mucosa) and gingival mucosa, has received much attention in the last decade because it offers excellent accessibility, is not easily traumatized and avoids degradation of proteins and peptides that occurs as a result of oral administration, gastrointestinal absorption and first-pass hepatic metabolism. Peptide absorption occurs across oral mucosa by passive diffusion and it is unlikely that there is a carrier-mediated transport mechanism. The principal pathway is probably via the intercellular route where the major permeability barrier is represented by organized array of neutral lipids in the superficial layers of the epithelium. The relative role of aqueous as opposed to the lipid pathway in drug transport is still under investigation; penetration is not necessarily enhanced by simply increasing lipophilicity, for other effects, such as charge and molecular size, also play an important role in absorption of peptide and protein drugs. Depending on the pharmacodynamics of the peptides, various oral mucosal delivery systems can be designed. Delivery of peptide/protein drugs by conventional means such as solutions has some limitations. The possibility of excluding a major part of drug from absorption by involuntary swallowing and the continuous dilution due to salivary flow limits a controlled release. However these limitations can be overcome by adhesive dosage forms such as gels, films, tablets, and patches. They can localize the

Hybrid protein-synthetic polymer nanoparticles for drug delivery.

Science.gov (United States)

Koseva, Neli S; Rydz, Joanna; Stoyanova, Ekaterina V; Mitova, Violeta A

2015-01-01

Among the most common nanoparticulate systems, the polymeric nanocarriers have a number of key benefits, which give a great choice of delivery platforms. Nevertheless, polymeric nanoparticles possess some limitations that include use of toxic solvents in the production process, polymer degradation, drug leakage outside the diseased tissue, and polymer cytotoxicity. The combination of polymers of biological and synthetic origin is an appealing modern strategy for the production of novel nanocarriers with unprecedented properties. Proteins' interface can play an important role in determining bioactivity and toxicity and gives perspective for future development of the polymer-based nanoparticles. The design of hybrid constructs composed of synthetic polymer and biological molecules such as proteins can be considered as a straightforward tool to integrate a broad spectrum of properties and biofunctions into a single device. This review discusses hybrid protein-synthetic polymer nanoparticles with different structures and levels in complexity and functionality, in view of their applications as drug delivery systems. © 2015 Elsevier Inc. All rights reserved.

Aminoacyl-tRNA-charged eukaryotic elongation factor 1A is a bona fide substrate for Legionelle pneumophila effector glucosyltransferases

DEFF Research Database (Denmark)

Tzivelekidis, Tina; Jank, Thomas; Pohl, Corinna

2011-01-01

Legionella pneumophila, which is the causative organism of Legionnaires disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (Lgt’s), which...

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy.

Science.gov (United States)

Kellner, Christian; Otte, Anna; Cappuzzello, Elisa; Klausz, Katja; Peipp, Matthias

2017-09-01

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

Electrospun fish protein fibers as a biopolymer-based carrier – implications for oral protein delivery

DEFF Research Database (Denmark)

Boutrup Stephansen, Karen; García-Díaz, María; Jessen, Flemming

2014-01-01

Purpose: Protein-based electrospun fibers have emerged as novel nanostructured materials for tissue engineering and drug delivery due to their unique structural characteristics, biocompatibility and biodegradability. The aim of this study was to explore the use of electrospun fibers based on fish...... sarcoplasmic proteins as an oral delivery platform for biopharmaceuticals, using insulin as a model protein. Methods: Fish sarcoplasmic proteins (FSP) were isolated from fresh cod and electrospun into nanomicrofibers using insulin as a model payload. The morphology of FSP fibers was characterized using...... differentiated Caco-2 cell monolayers was followed by RP-HPLC and ELISA, and the transepithelial electrical resistance (TEER) was measured before and after the experiment. Cell viability was assessed by the MTS/PMS assay. Results: Insulin was encapsulated in the electrospun FSP fibers with high efficiency, high...

Bioreducible Lipid-like Nanoparticles for Intracellular Protein Delivery

Science.gov (United States)

Arellano, Carlos Luis

Protein-based therapy is one of the most direct ways to manipulate cell function and treat human disease. Although protein therapeutics has made its way to clinical practice, with five of the top fifteen global pharmaceuticals being peptide or protein-based drugs, one common limitation is that the effects of protein therapy are only achieved through the targeting of cell surface receptors and intracellular domains. Due to the impermeability of the cell membrane to most foreign materials, entire classes of potentially therapeutic proteins cannot thoroughly be studied without a safe and efficient method of transporting proteins into the cytosol. We report the use of a combinatorially-designed bioreducible lipid-like material (termed "lipidoid") - based protein delivery platform for the transfection of human cancer cell lines. Lipidoid nanoparticles are synthesized through a thin film dispersion method. The degradation of the bioreducible nanoparticles was observed when exposed to glutathione, a highly reductive compound present in the cytosol. We demonstrate that the nanoparticles are capable of transfecting a dose-dependent concentration of our model protein, beta-galactosidase into HeLa cells. Furthermore, formulations of the lipidoid containing the cytotoxic proteins saporin and RNase-A are both capable of inhibiting tumor cell proliferation as observed in in vitro treatment of different human cancer cell lines. There was no observed loss in protein activity after lyophilization and long--term storage, indicating the potential of pre-clinical applications. Overall, we demonstrate an effective approach to protein formulation and intracellular delivery. We believe that our formulations will lead to the study of a whole class of previously untapped therapeutics that may generate new solutions for previously untreatable diseases.

Construction and characterization of a pure protein hydrogel for drug delivery application.

Science.gov (United States)

Xu, Xu; Xu, ZhaoKang; Yang, XiaoFeng; He, YanHao; Lin, Rong

2017-02-01

Injectable hydrogels have a variety of applications, including regenerative medicine, tissue engineering and controlled drug delivery. In this paper, we reported on a pure protein hydrogel based on tetrameric recombinant proteins for the potential drug delivery application. This protein hydrogel was formed instantly by simply mixing two recombinant proteins (ULD-TIP1 and ULD-GGGWRESAI) through the specific protein-peptide interaction. The protein hydrogel was characterized by rheology and scanning electron microscopy (SEM). In vitro cytotoxicity test indicated that the developed protein hydrogel had no apparent cytotoxicity against L-929 cells and HCEC cells after 48h incubation. The formed protein hydrogels was gradually degraded after incubation in phosphate buffered solution (PBS, pH=7.4) for a period of 144h study, as indicated by in vitro degradation test. Encapsulation of model drug (sodium diclofenac; DIC) were achieved by simple mixing of drugs with hydrogelator and the entrapped drugs was almost completely released from hydrogels within 24h via a diffusion manner. As a conclusion, the simple and mild preparation procedure and good biocompatibility of protein hydrogel would render its good promising candidate for drug delivery applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Bacterial effector HopF2 interacts with AvrPto and suppresses Arabidopsis innate immunity at the plasma membrane

Science.gov (United States)

Plant pathogenic bacteria inject a cocktail of effector proteins into host plant cells to modulate the host immune response, thereby promoting pathogenicity. How or whether these effectors work cooperatively is largely unknown. The Pseudomonas syringae DC3000 effector HopF2 suppresses the host plan...

A massive expansion of effector genes underlies gall-formation in the wheat pest Mayetiola destructor

DEFF Research Database (Denmark)

Zhao, Chaoyang; Escalante, Lucio Navarro; Chen, Hang

2015-01-01

Gall-forming arthropods are highly specialized herbivores that, in combination with their hosts, produce extended phenotypes with unique morphologies [1]. Many are economically important, and others have improved our understanding of ecology and adaptive radiation [2]. However, the mechanisms...... in plants and E3-ligase-mimicking effectors in plant pathogenic bacteria. SSGP-71 proteins and wheat Skp proteins interact in vivo. Mutations in different SSGP-71 genes avoid the effector-triggered immunity that is directed by the wheat resistance genes H6 and H9. Results point to effectors as the agents...

Identification and characterisation of a hyper-variable apoplastic effector gene family of the potato cyst nematodes.

Directory of Open Access Journals (Sweden)

Sebastian Eves-van den Akker

2014-09-01

Full Text Available Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism.

A highly conserved glycine within linker I and the extreme C terminus of G protein alpha subunits interact cooperatively in switching G protein-coupled receptor-to-effector specificity

DEFF Research Database (Denmark)

Kostenis, Evi; Martini, Lene; Ellis, James

2004-01-01

Numerous studies have attested to the importance of the extreme C terminus of G protein alpha subunits in determining their selectivity of receptor recognition. We have previously reported that a highly conserved glycine residue within linker I is important for constraining the fidelity of receptor...... recognition by Galpha(q) proteins. Herein, we explored whether both modules (linker I and extreme C terminus) interact cooperatively in switching G protein-coupled receptor (GPCR)-to-effector specificity and created as models mutant Galpha(q) proteins in which glycine was replaced with various amino acids...... and the C-terminal five Galpha(q) residues with the corresponding Galpha(i) or Galpha(s) sequence. Coupling properties of the mutated Galpha(q) proteins were determined after coexpression with a panel of 13 G(i)-and G(s) -selective receptors and compared with those of Galpha proteins modified in only one...

Structure-function analysis of the Fusarium oxysporum Avr2 effector allows uncoupling of its immune-suppressing activity from recognition

NARCIS (Netherlands)

Di, X.; Cao, L.; Hughes, R.K.; Tintor, N.; Banfield, M.J.; Takken, F.L.W.

2017-01-01

Plant pathogens employ effector proteins to manipulate their hosts. Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of tomato wilt disease, produces effector protein Avr2. Besides being a virulence factor, Avr2 triggers immunity in I-2 carrying tomato (Solanum lycopersicum). Fol

The potential of protein-nanomaterial interaction for advanced drug delivery.

Science.gov (United States)

Peng, Qiang; Mu, Huiling

2016-03-10

Nanomaterials, like nanoparticles, micelles, nano-sheets, nanotubes and quantum dots, have great potentials in biomedical fields. However, their delivery is highly limited by the formation of protein corona upon interaction with endogenous proteins. This new identity, instead of nanomaterial itself, would be the real substance the organs and cells firstly encounter. Consequently, the behavior of nanomaterials in vivo is uncontrollable and some undesired effects may occur, like rapid clearance from blood stream; risk of capillary blockage; loss of targeting capacity; and potential toxicity. Therefore, protein-nanomaterial interaction is a great challenge for nanomaterial systems and should be inhibited. However, this interaction can also be used to functionalize nanomaterials by forming a selected protein corona. Unlike other decoration using exogenous molecules, nanomaterials functionalized by selected protein corona using endogenous proteins would have greater promise for clinical use. In this review, we aim to provide a comprehensive understanding of protein-nanomaterial interaction. Importantly, a discussion about how to use such interaction is launched and some possible applications of such interaction for advanced drug delivery are presented. Copyright © 2016 Elsevier B.V. All rights reserved.

Multi-functional characteristics of the Pseudomonas aeruginosa type III needle-tip protein, PcrV; comparison to orthologs in other gram negative bacteria

Directory of Open Access Journals (Sweden)

Hiromi eSato

2011-07-01

Full Text Available Pseudomonas aeruginosa possesses a type III secretion system (T3SS to intoxicate host cells and evade innate immunity. This virulence-related machinery consists of a molecular syringe and needle assembled on the bacterial surface, which allows delivery of T3 effector proteins into infected cells. To accomplish a one-step effector translocation, a tip protein is required at the top end of the T3 needle structure. Strains lacking expression of the functional tip protein fail to intoxicate host cells.P. aeruginosa encodes a T3S that is highly homologous to the proteins encoded by Yersinia species. The needle tip proteins of Yersinia, LcrV, and P. aeruginosa, PcrV, share 37% identity and 65% similarity. Other known tip proteins are AcrV (Aeromonas, IpaD (Shigella, SipD (Salmonella, BipD (Burkholderia, EspA (EPEC, EHEC, Bsp22 (Bordetella, with additional proteins identified from various Gram negative species, such as Vibrio and Bordetella. The tip proteins can serve as a protective antigen or may be critical for sensing host cells and evading innate immune responses. Recognition of the host microenvironment transcriptionally activates synthesis of T3SS components. The machinery appears to be mechanically controlled by the assemblage of specific junctions within the apparatus. These junctions include the tip and base of the T3 apparatus, the needle proteins and components within the bacterial cytoplasm. The tip proteins likely have chaperone functions for translocon proteins, allowing the proper assembly of translocation channels in the host membrane and completing vectorial delivery of effector proteins into the host cytoplasm. Multifunctional features of the needle-tip proteins appear to be intricately controlled. In this review, we highlight the functional aspects and complex controls of T3 needle-tip proteins with particular emphasis on PcrV and LcrV.

In planta processing and glycosylation of a nematode CLE effector and its interaction with a CLV2-like receptor to promote parasitism

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Like other biotrophic plant pathogens, plant-parasitic nematodes secrete effector proteins into host cells to facilitate infection. Effector proteins that mimic plant CLAVATA3/ESR (CLE)-like proteins have been identified in several cyst nematodes including the potato cyst nematode (PCN); however, th...

Identification and Characterisation CRN Effectors in Phytophthora capsici Shows Modularity and Functional Diversity.

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Remco Stam

Full Text Available Phytophthora species secrete a large array of effectors during infection of their host plants. The Crinkler (CRN gene family encodes a ubiquitous but understudied class of effectors with possible but as of yet unknown roles in infection. To appreciate CRN effector function in Phytophthora, we devised a simple Crn gene identification and annotation pipeline to improve effector prediction rates. We predicted 84 full-length CRN coding genes and assessed CRN effector domain diversity in sequenced Oomycete genomes. These analyses revealed evidence of CRN domain innovation in Phytophthora and expansion in the Peronosporales. We performed gene expression analyses to validate and define two classes of CRN effectors, each possibly contributing to infection at different stages. CRN localisation studies revealed that P. capsici CRN effector domains target the nucleus and accumulate in specific sub-nuclear compartments. Phenotypic analyses showed that few CRN domains induce necrosis when expressed in planta and that one cell death inducing effector, enhances P. capsici virulence on Nicotiana benthamiana. These results suggest that the CRN protein family form an important class of intracellular effectors that target the host nucleus during infection. These results combined with domain expansion in hemi-biotrophic and necrotrophic pathogens, suggests specific contributions to pathogen lifestyles. This work will bolster CRN identification efforts in other sequenced oomycete species and set the stage for future functional studies towards understanding CRN effector functions.

Sustained subconjunctival protein delivery using a thermosetting gel delivery system.

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Rieke, Erin R; Amaral, Juan; Becerra, S Patricia; Lutz, Robert J

2010-02-01

An effective treatment modality for posterior eye diseases would provide prolonged delivery of therapeutic agents, including macromolecules, to eye tissues using a safe and minimally invasive method. The goal of this study was to assess the ability of a thermosetting gel to deliver a fluorescently labeled protein, Alexa 647 ovalbumin, to the choroid and retina of rats following a single subconjunctival injection of the gel. Additional experiments were performed to compare in vitro to in vivo ovalbumin release rates from the gel. The ovalbumin content of the eye tissues was monitored by spectrophotometric assays of tissue extracts of Alexa 647 ovalbumin from dissected sclera, choroid, and retina at time points ranging from 2 h to 14 days. At the same time points, fluorescence microscopy images of tissue samples were also obtained. Measurement of intact ovalbumin was verified by LDS-PAGE analysis of the tissue extract solutions. In vitro release of Alexa 488 ovalbumin into 37 degrees C PBS solutions from ovalbumin-loaded gel pellets was also monitored over time by spectrophotometric assay. In vivo ovalbumin release rates were determined by measurement of residual ovalbumin extracted from gel pellets removed from rat eyes at various time intervals. Our results indicate that ovalbumin concentrations can be maintained at measurable levels in the sclera, choroid, and retina of rats for up to 14 days using the thermosetting gel delivery system. The concentration of ovalbumin exhibited a gradient that decreased from sclera to choroid and to retina. The in vitro release rate profiles were similar to the in vivo release profiles. Our findings suggest that the thermosetting gel system may be a feasible method for safe and convenient sustained delivery of proteins to choroidal and retinal tissue in the posterior segments of the eye.

Intrathecal delivery of protein therapeutics to the brain: a critical reassessment.

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Calias, Pericles; Banks, William A; Begley, David; Scarpa, Maurizio; Dickson, Patricia

2014-11-01

Disorders of the central nervous system (CNS), including stroke, neurodegenerative diseases, and brain tumors, are the world's leading causes of disability. Delivery of drugs to the CNS is complicated by the blood-brain barriers that protect the brain from the unregulated leakage and entry of substances, including proteins, from the blood. Yet proteins represent one of the most promising classes of therapeutics for the treatment of CNS diseases. Many strategies for overcoming these obstacles are in development, but the relatively straightforward approach of bypassing these barriers through direct intrathecal administration has been largely overlooked. Originally discounted because of its lack of usefulness for delivering small, lipid-soluble drugs to the brain, the intrathecal route has emerged as a useful, in some cases perhaps the ideal, route of administration for certain therapeutic protein and targeted disease combinations. Here, we review blood-brain barrier functions and cerebrospinal fluid dynamics and their relevance to drug delivery via the intrathecal route, discuss animal and human studies that have investigated intrathecal delivery of protein therapeutics, and outline several characteristics of protein therapeutics that can allow them to be successfully delivered intrathecally. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular design and nanoparticle-mediated intracellular delivery of functional proteins to target cellular pathways

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Shah, Dhiral Ashwin

Intracellular delivery of specific proteins and peptides represents a novel method to influence stem cells for gain-of-function and loss-of-function. Signaling control is vital in stem cells, wherein intricate control of and interplay among critical pathways directs the fate of these cells into either self-renewal or differentiation. The most common route to manipulate cellular function involves the introduction of genetic material such as full-length genes and shRNA into the cell to generate (or prevent formation of) the target protein, and thereby ultimately alter cell function. However, viral-mediated gene delivery may result in relatively slow expression of proteins and prevalence of oncogene insertion into the cell, which can alter cell function in an unpredictable fashion, and non-viral delivery may lead to low efficiency of genetic delivery. For example, the latter case plagues the generation of induced pluripotent stem cells (iPSCs) and hinders their use for in vivo applications. Alternatively, introducing proteins into cells that specifically recognize and influence target proteins, can result in immediate deactivation or activation of key signaling pathways within the cell. In this work, we demonstrate the cellular delivery of functional proteins attached to hydrophobically modified silica (SiNP) nanoparticles to manipulate specifically targeted cell signaling proteins. In the Wnt signaling pathway, we have targeted the phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta) by designing a chimeric protein and delivering it in neural stem cells. Confocal imaging indicates that the SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, indicated by the elevation of beta-catenin levels, and increased transcription of Wnt target genes, such as c-MYC. The results presented here suggest that

Photo-synthesis of protein-based nanoparticles and the application in drug delivery

International Nuclear Information System (INIS)

Xie, Jinbing; Wang, Hongyang; Cao, Yi; Qin, Meng; Wang, Wei

2015-01-01

Recently, protein-based nanoparticles as drug delivery systems have attracted great interests due to the excellent behavior of high biocompatibility and biodegradability, and low toxicity. However, the synthesis techniques are generally costly, chemical reagents introduced, and especially present difficulties in producing homogeneous monodispersed nanoparticles. Here, we introduce a novel physical method to synthesize protein nanoparticles which can be accomplished under physiological condition only through ultraviolet (UV) illumination. By accurately adjusting the intensity and illumination time of UV light, disulfide bonds in proteins can be selectively reduced and the subsequent self-assembly process can be well controlled. Importantly, the co-assembly can also be dominated when the proteins mixed with either anti-cancer drugs, siRNA, or active targeting molecules. Both in vitro and in vivo experiments indicate that our synthesized protein–drug nanoparticles (drug-loading content and encapsulation efficiency being ca. 8.2% and 70%, respectively) not only possess the capability of traditional drug delivery systems (DDS), but also have a greater drug delivery efficiency to the tumor sites and a better inhibition of tumor growth (only 35% of volume comparing to the natural growing state), indicating it being a novel drug delivery system in tumor therapy

Mechanism of host substrate acetylation by a YopJ family effector.

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Zhang, Zhi-Min; Ma, Ka-Wai; Gao, Linfeng; Hu, Zhenquan; Schwizer, Simon; Ma, Wenbo; Song, Jikui

2017-07-24

The Yersinia outer protein J (YopJ) family of bacterial effectors depends on a novel acetyltransferase domain to acetylate signalling proteins from plant and animal hosts. However, the underlying mechanism is unclear. Here, we report the crystal structures of PopP2, a YopJ effector produced by the plant pathogen Ralstonia solanacearum, in complex with inositol hexaphosphate (InsP 6 ), acetyl-coenzyme A (AcCoA) and/or substrate Resistance to Ralstonia solanacearum 1 (RRS1-R) WRKY . PopP2 recognizes the WRKYGQK motif of RRS1-R WRKY to position a targeted lysine in the active site for acetylation. Importantly, the PopP2-RRS1-R WRKY association is allosterically regulated by InsP 6 binding, suggesting a previously unidentified role of the eukaryote-specific cofactor in substrate interaction. Furthermore, we provide evidence for the reaction intermediate of PopP2-mediated acetylation, an acetyl-cysteine covalent adduct, lending direct support to the 'ping-pong'-like catalytic mechanism proposed for YopJ effectors. Our study provides critical mechanistic insights into the virulence activity of YopJ class of acetyltransferases.

Bioresponsive nanoparticles based on poly(amidoamine)s for protein delivery

NARCIS (Netherlands)

Coué, G.M.J.P.C.

2011-01-01

This study describes the design and development of multifuctional poly(amidoamine)s (PAAs) capable to form self-assembled nanocomplexes with peptides and proteins, as functional bioresponsive vectors for protein delivery to targeting cells in vitro and in vivo. The representative examples of this

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

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A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or an...

Mathematical model of the binding of allosteric effectors to the Escherichia coli PII signal transduction protein GlnB.

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da Rocha, Ricardo Alves; Weschenfelder, Thiago André; de Castilhos, Fernanda; de Souza, Emanuel Maltempi; Huergo, Luciano Fernandes; Mitchell, David Alexander

2013-04-16

PII proteins are important regulators of nitrogen metabolism in a wide variety of organisms: the binding of the allosteric effectors ATP, ADP, and 2-oxoglutarate (2-OG) to PII proteins affects their ability to interact with target proteins. We modeled the simultaneous binding of ATP, ADP, and 2-OG to one PII protein, namely GlnB of Escherichia coli, using a modeling approach that allows the prediction of the proportions of individual binding states. Four models with different binding rules were compared. We selected one of these models (that assumes that the binding of the first nucleotide to GlnB makes it harder for subsequent nucleotides to bind) and used it to explore how physiological concentrations of ATP, ADP, and 2-OG would affect the proportions of those states of GlnB that interact with the target proteins ATase and NtrB. Our simulations indicate that GlnB can, as suggested by previous researchers, act as a sensor of both 2-OG and the ATP:ADP ratio. We conclude that our modeling approach will be an important tool in future studies concerning the PII binding states and their interactions with target proteins.

Adsorption and Desorption of Bioactive Proteins on Hydroxyapatite for Protein Delivery Systems

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Chie Kojima

2012-01-01

Full Text Available Hydroxyapatite (HA is a precursor of bone and has been studied as a biomaterial. We attempted HA to apply to protein delivery systems. In this study, the association and dissociation properties of two types of bioactive proteins, cytochrom c and insulin, to HA were investigated. Cytochrom c was less associated with HA than insulin, which was easily released from it. However, the release of insulin from HA was slow. Insulin was released from HA at pH 7.4 more rapidly than at pH 3. The association and dissociation properties might be influenced by the size, solubility and net charge of protein. HA is a potential protein carrier with controlled release.

Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells.

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Kwon, Kwang-Chul; Daniell, Henry

2016-08-01

Plants cells are now approved by the FDA for cost-effective production of protein drugs (PDs) in large-scale current Good Manufacturing Practice (cGMP) hydroponic growth facilities. In lyophilized plant cells, PDs are stable at ambient temperature for several years, maintaining their folding and efficacy. Upon oral delivery, PDs bioencapsulated in plant cells are protected in the stomach from acids and enzymes but are subsequently released into the gut lumen by microbes that digest the plant cell wall. The large mucosal area of the human intestine offers an ideal system for oral drug delivery. When tags (receptor-binding proteins or cell-penetrating peptides) are fused to PDs, they efficiently cross the intestinal epithelium and are delivered to the circulatory or immune system. Unique tags to deliver PDs to human immune or nonimmune cells have been developed recently. After crossing the epithelium, ubiquitous proteases cleave off tags at engineered sites. PDs are also delivered to the brain or retina by crossing the blood-brain or retinal barriers. This review highlights recent advances in PD delivery to treat Alzheimer's disease, diabetes, hypertension, Gaucher's or ocular diseases, as well as the development of affordable drugs by eliminating prohibitively expensive purification, cold chain and sterile delivery.

Bacterial Effectors and Their Functions in the Ubiquitin-Proteasome System: Insight from the Modes of Substrate Recognition

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Minsoo Kim

2014-08-01

Full Text Available Protein ubiquitination plays indispensable roles in the regulation of cell homeostasis and pathogenesis of neoplastic, infectious, and neurodegenerative diseases. Given the importance of this modification, it is to be expected that several pathogenic bacteria have developed the ability to utilize the host ubiquitin system for their own benefit. Modulation of the host ubiquitin system by bacterial effector proteins inhibits innate immune responses and hijacks central signaling pathways. Bacterial effectors mimic enzymes of the host ubiquitin system, but may or may not be structurally similar to the mammalian enzymes. Other effectors bind and modify components of the host ubiquitin system, and some are themselves subject to ubiquitination. This review will describe recent findings, based on structural analyses, regarding how pathogens use post-translational modifications of proteins to establish an infection.

Bacterial effectors and their functions in the ubiquitin-proteasome system: insight from the modes of substrate recognition.

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Kim, Minsoo; Otsubo, Ryota; Morikawa, Hanako; Nishide, Akira; Takagi, Kenji; Sasakawa, Chihiro; Mizushima, Tsunehiro

2014-08-18

Protein ubiquitination plays indispensable roles in the regulation of cell homeostasis and pathogenesis of neoplastic, infectious, and neurodegenerative diseases. Given the importance of this modification, it is to be expected that several pathogenic bacteria have developed the ability to utilize the host ubiquitin system for their own benefit. Modulation of the host ubiquitin system by bacterial effector proteins inhibits innate immune responses and hijacks central signaling pathways. Bacterial effectors mimic enzymes of the host ubiquitin system, but may or may not be structurally similar to the mammalian enzymes. Other effectors bind and modify components of the host ubiquitin system, and some are themselves subject to ubiquitination. This review will describe recent findings, based on structural analyses, regarding how pathogens use post-translational modifications of proteins to establish an infection.

Structure and evolution of barley powdery mildew effector candidates

DEFF Research Database (Denmark)

Pedersen, Carsten; Themaat, Emiel Ver Loren van; McGuffin, Liam J.

2012-01-01

Protein effectors of pathogenicity are instrumental in modulating host immunity and disease resistance. The powdery mildew pathogen of grasses Blumeria graminis causes one of the most important diseases of cereal crops. B. graminis is an obligate biotrophic pathogen and as such has an absolute...

Differential modulation of plant immune responses by diverse members of the Pseudomonas savastanoi pv. savastanoi HopAF type III effector family.

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Castañeda-Ojeda, M Pilar; López-Solanilla, Emilia; Ramos, Cayo

2017-06-01

The Pseudomonas savastanoi pv. savastanoi NCPPB 3335 type III secretion system (T3SS) effector repertoire includes 33 candidates, seven of which translocate into host cells and interfere with plant defences. The present study was performed to investigate the co-existence of both plasmid- and chromosomal-encoded members of the HopAF effector family, HopAF1-1 and HopAF1-2, respectively, in the genome of NCPPB 3335. Here, we show that the HopAF1 paralogues are widely distributed in the Pseudomonas syringae complex, where HopAF1-1 is most similar to the homologues encoded by other P. syringae pathovars infecting woody hosts that belong to phylogroups 1 and 3. We show that the expression of both HopAF1-1 and HopAF-2 is transcriptionally dependent on HrpL and demonstrate their delivery into Nicotiana tabacum leaves. Although the heterologous delivery of either HopAF1-1 or HopAF1-2 significantly suppressed the production of defence-associated reactive oxygen species levels, only HopAF1-2 reduced the levels of callose deposition. Moreover, the expression of HopAF1-2 by functionally effectorless P. syringae pv. tomato DC3000D28E completely inhibited the hypersensitive response in tobacco and significantly increased the competitiveness of the strain in Nicotiana benthamiana. Despite their functional differences, subcellular localization studies reveal that green fluorescent protein (GFP) fusions to either HopAF1-1 or HopAF1-2 are targeted to the plasma membrane when they are expressed in plant cells, a process that is completely dependent on the integrity of their N-myristoylation motif. Our results further support the notion that highly similar T3SS effectors might differentially interact with diverse plant targets, even when they co-localize in the same cell compartment. © 2016 BSPP AND JOHN WILEY & SONS LTD.

On prilled Nanotubes-in-Microgel Oral Systems for protein delivery.

Science.gov (United States)

de Kruif, Jan Kendall; Ledergerber, Gisela; Garofalo, Carla; Fasler-Kan, Elizaveta; Kuentz, Martin

2016-04-01

Newly discovered active macromolecules are highly promising for therapy, but poor bioavailability hinders their oral use. Microencapsulation approaches, such as protein prilling into microspheres, may enable protection from gastrointestinal (GI) enzymatic degradation. This would increase bioavailability mainly for local delivery to GI lumen or mucosa. This work's purpose was to design a novel architecture, namely a Nanotubes-in-Microgel Oral System, by prilling for protein delivery. Halloysite nanotubes (HNT) were selected as orally acceptable clay particles and their lumen was enlarged by alkaline etching. This chemical modification increased the luminal volume to a mean of 216.3 μL g(-1) (+40.8%). After loading albumin as model drug, the HNT were entrapped in microgels by prilling. The formation of Nanoparticles-in-Microsphere Oral System (NiMOS) yielded entrapment efficiencies up to 63.2%. NiMOS shape was spherical to toroidal, with a diameter smaller than 320 μm. Release profiles depended largely on the employed system and HNT type. Protein stability was determined throughout prilling and after in vitro enzymatic degradation. Prilling did not harm protein structure, and NiMOS demonstrated higher enzymatic protection than pure nanotubes or microgels, since up to 82% of BSA remained unscathed after in vitro digestion. Therefore, prilled NiMOS was shown to be a promising and flexible multi-compartment system for oral (local) macromolecular delivery. Copyright © 2016 Elsevier B.V. All rights reserved.

A Sample Delivery System for Planetary Missions

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National Aeronautics and Space Administration — The project will develop, test and characterize the performance of a prototype /sample delivery system (SDS) implemented as an end effector on a robotic arm capable...

Introduction to current and future protein therapeutics: a protein engineering perspective.

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Carter, Paul J

2011-05-15

Protein therapeutics and its enabling sister discipline, protein engineering, have emerged since the early 1980s. The first protein therapeutics were recombinant versions of natural proteins. Proteins purposefully modified to increase their clinical potential soon followed with enhancements derived from protein or glycoengineering, Fc fusion or conjugation to polyethylene glycol. Antibody-based drugs subsequently arose as the largest and fastest growing class of protein therapeutics. The rationale for developing better protein therapeutics with enhanced efficacy, greater safety, reduced immunogenicity or improved delivery comes from the convergence of clinical, scientific, technological and commercial drivers that have identified unmet needs and provided strategies to address them. Future protein drugs seem likely to be more extensively engineered to improve their performance, e.g., antibodies and Fc fusion proteins with enhanced effector functions or extended half-life. Two old concepts for improving antibodies, namely antibody-drug conjugates and bispecific antibodies, have advanced to the cusp of clinical success. As for newer protein therapeutic platform technologies, several engineered protein scaffolds are in early clinical development and offer differences and some potential advantages over antibodies. Copyright © 2011 Elsevier Inc. All rights reserved.

Preparation and characterization of alginate microspheres for sustained protein delivery within tissue scaffolds

International Nuclear Information System (INIS)

Zhai Peng; Chen, X B; Schreyer, David J

2013-01-01

Tissue engineering scaffolds are designed not only to provide structural support for the repair of damaged tissue, but can also serve the function of bioactive protein delivery. Here we present a study on the preparation and characterization of protein-loaded microspheres, either alone or incorporated into mock tissue scaffolds, for sustained protein delivery. Alginate microspheres were prepared by a novel, small-scale water-in-oil emulsion technique and loaded with fluorescently labeled immunoglobulin G (IgG). Microsphere size appears to be influenced by the magnitude and distribution of force generated by mechanical stirring during emulsion. Protein release studies show that sustained IgG release from microspheres could be achieved and that application of a secondary coating of chitosan could further slow the rate of protein release. Preservation of bioactivity of released IgG protein was confirmed using an immunohistochemical assay. When IgG-loaded microspheres were incorporated into mock scaffolds, initial protein release was diminished and the overall time course of release was extended. The present study demonstrates that protein-loaded microspheres can be prepared with a controlled release profile and preserved biological activity, and can be incorporated into scaffolds to achieve sustained and prolonged protein delivery in a tissue engineering application. (paper)

A Fungal Effector With Host Nuclear Localization and DNA-Binding Properties Is Required for Maize Anthracnose Development.

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Vargas, Walter A; Sanz-Martín, José M; Rech, Gabriel E; Armijos-Jaramillo, Vinicio D; Rivera, Lina P; Echeverria, María Mercedes; Díaz-Mínguez, José M; Thon, Michael R; Sukno, Serenella A

2016-02-01

Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host's nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host's nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.

The pore-forming bacterial effector, VopQ, halts autophagic turnover.

Science.gov (United States)

Sreelatha, Anju; Orth, Kim; Starai, Vincent J

2013-12-01

Vibrio parahemolyticus Type III effector VopQ is both necessary and sufficient to induce autophagy within one hour of infection. We demonstrated that VopQ interacts with the Vo domain of the conserved vacuolar H(+)-ATPase. Membrane-associated VopQ subsequently forms pores in the membranes of acidic compartments, resulting in immediate release of protons without concomitant release of lumenal protein contents. These studies show how a bacterial pathogen can compromise host ion potentials using a gated pore-forming effector to equilibrate levels of small molecules found in endolysosomal compartments and disrupt cellular processes such as autophagy.

Interaction of barley powdery mildew effector candidate CSEP0055 with the defence protein PR17c

DEFF Research Database (Denmark)

Zhang, Wenjing; Pedersen, Carsten; Kwaaitaal, Mark Adrianus Cornelis J

2012-01-01

A large number of effector candidates have been identified recently in powdery mildew fungi. However, their roles and how they perform their functions remain unresolved. In this study, we made use of host-induced gene silencing and confirmed that the secreted barley powdery mildew effector candid...

The effector repertoire of Fusarium oxysporum determines the tomato xylem proteome composition following infection

NARCIS (Netherlands)

Gawehns, Fleur; Ma, Lisong; Bruning, Oskar; Houterman, Petra M.; Boeren, Sjef; Cornelissen, B.J.C.; Rep, Martijn; Takken, Frank L.W.

2015-01-01

Plant pathogens secrete small proteins, of which some are effectors that promote infection. During colonization of the tomato xylem vessels the fungus Fusarium oxysporum f.sp. lycopersici (Fol) secretes small proteins that are referred to as SIX (Secreted In Xylem) proteins. Of these, Six1

Targeted Delivery of Protein Drugs by Nanocarriers

Directory of Open Access Journals (Sweden)

Antonella Battisti

2010-03-01

Full Text Available Recent advances in biotechnology demonstrate that peptides and proteins are the basis of a new generation of drugs. However, the transportation of protein drugs in the body is limited by their high molecular weight, which prevents the crossing of tissue barriers, and by their short lifetime due to immuno response and enzymatic degradation. Moreover, the ability to selectively deliver drugs to target organs, tissues or cells is a major challenge in the treatment of several human diseases, including cancer. Indeed, targeted delivery can be much more efficient than systemic application, while improving bioavailability and limiting undesirable side effects. This review describes how the use of targeted nanocarriers such as nanoparticles and liposomes can improve the pharmacokinetic properties of protein drugs, thus increasing their safety and maximizing the therapeutic effect.

Yeast as a Heterologous Model System to Uncover Type III Effector Function.

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Crina Popa

2016-02-01

Full Text Available Type III effectors (T3E are key virulence proteins that are injected by bacterial pathogens inside the cells of their host to subvert cellular processes and contribute to disease. The budding yeast Saccharomyces cerevisiae represents an important heterologous system for the functional characterisation of T3E proteins in a eukaryotic environment. Importantly, yeast contains eukaryotic processes with low redundancy and are devoid of immunity mechanisms that counteract T3Es and mask their function. Expression in yeast of effectors from both plant and animal pathogens that perturb conserved cellular processes often resulted in robust phenotypes that were exploited to elucidate effector functions, biochemical properties, and host targets. The genetic tractability of yeast and its amenability for high-throughput functional studies contributed to the success of this system that, in recent years, has been used to study over 100 effectors. Here, we provide a critical view on this body of work and describe advantages and limitations inherent to the use of yeast in T3E research. "Favourite" targets of T3Es in yeast are cytoskeleton components and small GTPases of the Rho family. We describe how mitogen-activated protein kinase (MAPK signalling, vesicle trafficking, membrane structures, and programmed cell death are also often altered by T3Es in yeast and how this reflects their function in the natural host. We describe how effector structure-function studies and analysis of candidate targeted processes or pathways can be carried out in yeast. We critically analyse technologies that have been used in yeast to assign biochemical functions to T3Es, including transcriptomics and proteomics, as well as suppressor, gain-of-function, or synthetic lethality screens. We also describe how yeast can be used to select for molecules that block T3E function in search of new antibacterial drugs with medical applications. Finally, we provide our opinion on the limitations

Updates on smart polymeric carrier systems for protein delivery.

Science.gov (United States)

El-Sherbiny, Ibrahim; Khalil, Islam; Ali, Isra; Yacoub, Magdi

2017-10-01

Smart materials are those materials that are responsive to chemical (organic molecules, chemical agents or specific agents), biochemical (protein, enzymes, growth factors, substrates or ligands), physical (electric field, magnetic field, temperature, pH, ionic strength or radiation) or mechanical (pressure or mechanical stress) signals. These responsive materials interact with the stimuli by changing their properties or conformational structures in a predictable manner. Recently, smart polymers have been utilized in various biomedical applications. Particularly, they have been used as a platform to synthesize stimuli-responsive systems that could deliver therapeutics to a specific site for a specific period with minimal adverse effects. For instance, stimuli-responsive polymers-based systems have been recently reported to deliver different bioactive molecules such as carbohydrates (heparin), chemotherapeutic agents (doxorubicin), small organic molecules (anti-coagulants), nucleic acids (siRNA), and proteins (growth factors and hormones). Protein therapeutics played a fundamental role in treatment of various chronic and some autoimmune diseases. For instance insulin has been used in treatment of diabetes. However, being a protein in nature, insulin delivery is limited by its instability, short half-life, and easy denaturation when administered orally. To overcome these challenges, and as highlighted in this review article, much research efforts have been recently devoted to design and develop convenient smart controlled nanosystems for protein therapeutics delivery.

Timing of in utero malaria exposure influences fetal CD4 T cell regulatory versus effector differentiation

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Mary Prahl

2016-10-01

Full Text Available Abstract Background In malaria-endemic areas, the first exposure to malaria antigens often occurs in utero when the fetal immune system is poised towards the development of tolerance. Children exposed to placental malaria have an increased risk of clinical malaria in the first few years of life compared to unexposed children. Recent work has suggested the potential of pregnancy-associated malaria to induce immune tolerance in children living in malaria-endemic areas. A study was completed to evaluate the effect of malaria exposure during pregnancy on fetal immune tolerance and effector responses. Methods Using cord blood samples from a cohort of mother-infant pairs followed from early in pregnancy until delivery, flow cytometry analysis was completed to assess the relationship between pregnancy-associated malaria and fetal cord blood CD4 and dendritic cell phenotypes. Results Cord blood FoxP3+ Treg counts were higher in infants born to mothers with Plasmodium parasitaemia early in pregnancy (12–20 weeks of gestation; p = 0.048, but there was no association between Treg counts and the presence of parasites in the placenta at the time of delivery (by loop-mediated isothermal amplification (LAMP; p = 0.810. In contrast, higher frequencies of activated CD4 T cells (CD25+FoxP3−CD127+ were observed in the cord blood of neonates with active placental Plasmodium infection at the time of delivery (p = 0.035. This population exhibited evidence of effector memory differentiation, suggesting priming of effector T cells in utero. Lastly, myeloid dendritic cells were higher in the cord blood of infants with histopathologic evidence of placental malaria (p < 0.0001. Conclusion Together, these data indicate that in utero exposure to malaria drives expansion of both regulatory and effector T cells in the fetus, and that the timing of this exposure has a pivotal role in determining the polarization of the fetal immune response.

Elastin-like-polypeptide based fusion proteins for osteogenic factor delivery in bone healing.

Science.gov (United States)

McCarthy, Bryce; Yuan, Yuan; Koria, Piyush

2016-07-08

Modern treatments of bone injuries and diseases are becoming increasingly dependent on the usage of growth factors to stimulate bone growth. Bone morphogenetic protein-2 (BMP-2), a potent osteogenic inductive protein, exhibits promising results in treatment models, but recently has had its practical efficacy questioned due to the lack of local retention, ectopic bone formation, and potentially lethal inflammation. Where a new delivery technique of the BMP-2 is necessary, here we demonstrate the viability of an elastin-like peptide (ELP) fusion protein containing BMP-2 for delivery of the BMP-2. This fusion protein retains the performance characteristics of both the BMP-2 and ELP. The fusion protein was found to induce osteogenic differentiation of mesenchymal stem cells as evidenced by the production of alkaline phosphatase and extracellular calcium deposits in response to treatment by the fusion protein. Retention of the ELPs inverse phase transition property has allowed for expression of the fusion protein within a bacterial host (such as Escherichia coli) and easy and rapid purification using inverse transition cycling. The fusion protein formed self-aggregating nanoparticles at human-body temperature. The data collected suggests the viability of these fusion protein nanoparticles as a dosage-efficient and location-precise noncytotoxic delivery vehicle for BMP-2 in bone treatment. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1029-1037, 2016. © 2016 American Institute of Chemical Engineers.

Co-ordinate regulation of distinct host cell signalling pathways by multifunctional enteropathogenic Escherichia coli effector molecules.

Science.gov (United States)

Kenny, Brendan; Ellis, Sarah; Leard, Alan D; Warawa, Jonathan; Mellor, Harry; Jepson, Mark A

2002-05-01

Enteropathogenic Escherichia coli (EPEC) is a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. A/E pathogens encode a type III secretion system to transfer effector proteins into host cells. The EPEC Tir effector protein acts as a receptor for the bacterial surface protein intimin and is involved in the formation of Cdc42-independent, actin-rich pedestal structures beneath the adhered bacteria. In this paper, we demonstrate that EPEC binding to HeLa cells also induces Tir-independent, cytoskeletal rearrangement evidenced by the early, transient formation of filopodia-like structures at sites of infection. Filopodia formation is dependent on expression of the EPEC Map effector molecule - a protein that targets mitochondria and induces their dysfunction. We show that Map-induced filopodia formation is independent of mitochondrial targeting and is abolished by cellular expression of the Cdc42 inhibitory WASP-CRIB domain, demonstrating that Map has at least two distinct functions in host cells. The transient nature of the filopodia is related to an ability of EPEC to downregulate Map-induced cell signalling that, like pedestal formation, was dependent on both Tir and intimin proteins. The ability of Tir to downregulate filopodia was impaired by disrupting a putative GTPase-activating protein (GAP) motif, suggesting that Tir may possess such a function, with its interaction with intimin triggering this activity. Furthermore, we also found that Map-induced cell signalling inhibits pedestal formation, revealing that the cellular effects of Tir and Map must be co-ordinately regulated during infection. Possible implications of the multifunctional nature of EPEC effector molecules in pathogenesis are discussed.

Pseudomonas syringae pv. Tomato DC3000 Type III secretion effector polymutants reveal an interplay between hopAD1 and AvrPtoB

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The model pathogen Pseudomonas syringae pv. tomato DC3000 suppresses the two-tiered innate immune system of plants by injecting a complex repertoire of effector proteins into host cells via the type III secretion system. The model effector AvrPtoB has multiple domains and plant protein interactors i...

An overview on the delivery of antitumor drug doxorubicin by carrier proteins.

Science.gov (United States)

Agudelo, D; Bérubé, G; Tajmir-Riahi, H A

2016-07-01

Serum proteins play an increasing role as drug carriers in the clinical settings. In this review, we have compared the binding modalities of anticancer drug doxorubicin (DOX) to three model carrier proteins, human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (β-LG) in order to determine the potential application of these model proteins in DOX delivery. Molecular modeling studies showed stronger binding of DOX with HSA than BSA and β-LG with the free binding energies of -10.75 (DOX-HSA), -9.31 (DOX-BSA) and -8.12kcal/mol (DOX-β-LG). Extensive H-boding network stabilizes DOX-protein conjugation and played a major role in drug-protein complex formation. DOX complexation induced major alterations of HSA and BSA conformations, while did not alter β-LG secondary structure. The literature review shows that these proteins can potentially be used for delivery of DOX in vitro and in vivo. Copyright © 2016 Elsevier B.V. All rights reserved.

Recombinant Amphiphilic Protein Micelles for Drug Delivery

OpenAIRE

Kim, Wookhyun; Xiao, Jiantao; Chaikof, Elliot L.

2011-01-01

Amphiphilic block polypeptides can self-assemble into a range of nanostructures in solution, including micelles and vesicles. Our group has recently described the capacity of recombinant amphiphilic diblock copolypeptides to form highly stable micelles. In this report, we demonstrate the utility of protein nanoparticles to serve as a vehicle for controlled drug delivery. Drug-loaded micelles were produced by encapsulating dipyridamole as a model hydrophobic drug with anti-inflammatory activit...

Structure of the effector-binding domain of the arabinose repressor AraR from Bacillus subtilis

Energy Technology Data Exchange (ETDEWEB)

Procházková, Kateřina; Čermáková, Kateřina [Academy of Sciences of the Czech Republic, Flemingovo nam. 2, Prague 6 (Czech Republic); Pachl, Petr; Sieglová, Irena [Academy of Sciences of the Czech Republic, Flemingovo nam. 2, Prague 6 (Czech Republic); Academy of Sciences of the Czech Republic, Videnska 1083, Prague 4 (Czech Republic); Fábry, Milan [Academy of Sciences of the Czech Republic, Videnska 1083, Prague 4 (Czech Republic); Otwinowski, Zbyszek [UT Southwestern Medical Center, Dallas, Texas (United States); Řezáčová, Pavlína, E-mail: rezacova@uochb.cas.cz [Academy of Sciences of the Czech Republic, Flemingovo nam. 2, Prague 6 (Czech Republic); Academy of Sciences of the Czech Republic, Videnska 1083, Prague 4 (Czech Republic)

2012-02-01

The crystal structure of the effector-binding domain of the transcriptional repressor AraR from B. subtilis in complex with the effector molecule (l-arabinose) was determined at 2.2 Å resolution. A detailed analysis of the crystal identified a dimer organization that is distinctive from that of other members of the GalR/LacI family. In Bacillus subtilis, the arabinose repressor AraR negatively controls the expression of genes in the metabolic pathway of arabinose-containing polysaccharides. The protein is composed of two domains of different phylogenetic origin and function: an N-terminal DNA-binding domain belonging to the GntR family and a C-terminal effector-binding domain that shows similarity to members of the GalR/LacI family. The crystal structure of the C-terminal effector-binding domain of AraR in complex with the effector l-arabinose has been determined at 2.2 Å resolution. The l-arabinose binding affinity was characterized by isothermal titration calorimetry and differential scanning fluorimetry; the K{sub d} value was 8.4 ± 0.4 µM. The effect of l-arabinose on the protein oligomeric state was investigated in solution and detailed analysis of the crystal identified a dimer organization which is distinctive from that of other members of the GalR/LacI family.

Plant protein-based hydrophobic fine and ultrafine carrier particles in drug delivery systems.

Science.gov (United States)

Malekzad, Hedieh; Mirshekari, Hamed; Sahandi Zangabad, Parham; Moosavi Basri, S M; Baniasadi, Fazel; Sharifi Aghdam, Maryam; Karimi, Mahdi; Hamblin, Michael R

2018-02-01

For thousands of years, plants and their products have been used as the mainstay of medicinal therapy. In recent years, besides attempts to isolate the active ingredients of medicinal plants, other new applications of plant products, such as their use to prepare drug delivery vehicles, have been discovered. Nanobiotechnology is a branch of pharmacology that can provide new approaches for drug delivery by the preparation of biocompatible carrier nanoparticles (NPs). In this article, we review recent studies with four important plant proteins that have been used as carriers for targeted delivery of drugs and genes. Zein is a water-insoluble protein from maize; Gliadin is a 70% alcohol-soluble protein from wheat and corn; legumin is a casein-like protein from leguminous seeds such as peas; lectins are glycoproteins naturally occurring in many plants that recognize specific carbohydrate residues. NPs formed from these proteins show good biocompatibility, possess the ability to enhance solubility, and provide sustained release of drugs and reduce their toxicity and side effects. The effects of preparation methods on the size and loading capacity of these NPs are also described in this review.

Cytokine Secreting Microparticles Engineer the Fate and the Effector Functions of T-Cells.

Science.gov (United States)

Majedi, Fatemeh S; Hasani-Sadrabadi, Mohammad Mahdi; Kidani, Yoko; Thauland, Timothy J; Moshaverinia, Alireza; Butte, Manish J; Bensinger, Steven J; Bouchard, Louis-S

2018-02-01

T-cell immunotherapy is a promising approach for cancer, infection, and autoimmune diseases. However, significant challenges hamper its therapeutic potential, including insufficient activation, delivery, and clonal expansion of T-cells into the tumor environment. To facilitate T-cell activation and differentiation in vitro, core-shell microparticles are developed for sustained delivery of cytokines. These particles are enriched by heparin to enable a steady release of interleukin-2 (IL-2), the major T-cell growth factor, over 10+ d. The controlled delivery of cytokines is used to steer lineage specification of cultured T-cells. This approach enables differentiation of T-cells into central memory and effector memory subsets. It is shown that the sustained release of stromal cell-derived factor 1α could accelerate T-cell migration. It is demonstrated that CD4+ T-cells could be induced to high concentrations of regulatory T-cells through controlled release of IL-2 and transforming growth factor beta. It is found that CD8+ T-cells that received IL-2 from microparticles are more likely to gain effector functions as compared with traditional administration of IL-2. Culture of T-cells within 3D scaffolds that contain IL-2-secreting microparticles enhances proliferation as compared with traditional, 2D approaches. This yield a new method to control the fate of T-cells and ultimately to new strategies for immune therapy. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functionalized linear poly(amidoamine)s are efficient vectors for intracellular protein delivery

NARCIS (Netherlands)

Coué, G.M.J.P.C.; Engbersen, Johannes F.J.

2011-01-01

An effective intracellular protein delivery system was developed based on functionalized linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Three differently functionalized PAAs were synthesized, two of these having repetitive disulfide

Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells.

Science.gov (United States)

Röder, Ruth; Helma, Jonas; Preiß, Tobias; Rädler, Joachim O; Leonhardt, Heinrich; Wagner, Ernst

2017-01-01

Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells. Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies. Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.

Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation.

Directory of Open Access Journals (Sweden)

Roger W Kramer

2007-02-01

Full Text Available Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.

The effector SPRYSEC-19 of Globodera rostochiensis suppresses CC-NB-LRR-mediated disease resistance in plants.

Science.gov (United States)

Postma, Wiebe J; Slootweg, Erik J; Rehman, Sajid; Finkers-Tomczak, Anna; Tytgat, Tom O G; van Gelderen, Kasper; Lozano-Torres, Jose L; Roosien, Jan; Pomp, Rikus; van Schaik, Casper; Bakker, Jaap; Goverse, Aska; Smant, Geert

2012-10-01

The potato cyst nematode Globodera rostochiensis invades roots of host plants where it transforms cells near the vascular cylinder into a permanent feeding site. The host cell modifications are most likely induced by a complex mixture of proteins in the stylet secretions of the nematodes. Resistance to nematodes conferred by nucleotide-binding-leucine-rich repeat (NB-LRR) proteins usually results in a programmed cell death in and around the feeding site, and is most likely triggered by the recognition of effectors in stylet secretions. However, the actual role of these secretions in the activation and suppression of effector-triggered immunity is largely unknown. Here we demonstrate that the effector SPRYSEC-19 of G. rostochiensis physically associates in planta with the LRR domain of a member of the SW5 resistance gene cluster in tomato (Lycopersicon esculentum). Unexpectedly, this interaction did not trigger defense-related programmed cell death and resistance to G. rostochiensis. By contrast, agroinfiltration assays showed that the coexpression of SPRYSEC-19 in leaves of Nicotiana benthamiana suppresses programmed cell death mediated by several coiled-coil (CC)-NB-LRR immune receptors. Furthermore, SPRYSEC-19 abrogated resistance to Potato virus X mediated by the CC-NB-LRR resistance protein Rx1, and resistance to Verticillium dahliae mediated by an unidentified resistance in potato (Solanum tuberosum). The suppression of cell death and disease resistance did not require a physical association of SPRYSEC-19 and the LRR domains of the CC-NB-LRR resistance proteins. Altogether, our data demonstrated that potato cyst nematodes secrete effectors that enable the suppression of programmed cell death and disease resistance mediated by several CC-NB-LRR proteins in plants.

In Planta Functional Analysis and Subcellular Localization of the Oomycete Pathogen Plasmopara viticola Candidate RXLR Effector Repertoire

Directory of Open Access Journals (Sweden)

Yunxiao Liu

2018-04-01

Full Text Available Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent Plasmopara viticola is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes (PvRXLRs were cloned from the P. viticola isolate “JL-7-2” genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous Agrobacterium-mediated Nicotiana benthamiana expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83 could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable N-terminal transit peptide and underwent processing in planta. Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria.

Local Delivery Is Critical for Monocyte Chemotactic Protein-1 Mediated Site-Specific Murine Aneurysm Healing.

Science.gov (United States)

Hourani, Siham; Motwani, Kartik; Wajima, Daisuke; Fazal, Hanain; Jones, Chad H; Doré, Sylvain; Hosaka, Koji; Hoh, Brian L

2018-01-01

Local delivery of monocyte chemotactic protein-1 (MCP-1/CCL2) via our drug-eluting coil has been shown to promote intrasaccular aneurysm healing via an inflammatory pathway. In this study, we validate the importance of local MCP-1 in murine aneurysm healing. Whether systemic, rather than local, delivery of MCP-1 can direct site-specific aneurysm healing has significant translational implications. If systemic MCP-1 is effective, then MCP-1 could be administered as a pill rather than by endovascular procedure. Furthermore, we confirm that MCP-1 is the primary effector in our MCP-1 eluting coil-mediated murine aneurysm healing model. We compare aneurysm healing with repeated intraperitoneal MCP-1 versus vehicle injection, in animals with control poly(lactic-co-glycolic) acid (PLGA)-coated coils. We demonstrate elimination of the MCP-1-associated tissue-healing response by knockout of MCP-1 or CCR2 (MCP-1 receptor) and by selectively inhibiting MCP-1 or CCR2. Using immunofluorescent probing, we explore the cell populations found in healed aneurysm tissue following each intervention. Systemically administered MCP-1 with PLGA coil control does not produce comparable aneurysm healing, as seen with MCP-1 eluting coils. MCP-1-directed aneurysm healing is eliminated by selective inhibition of MCP-1 or CCR2 and in MCP-1-deficient or CCR2-deficient mice. No difference was detected in M2 macrophage and myofibroblast/smooth muscle cell staining with systemic MCP-1 versus vehicle in aneurysm wall, but a significant increase in these cell types was observed with MCP-1 eluting coil implant and attenuated by MCP-1/CCR2 blockade or deficiency. We show that systemic MCP-1 concurrent with PLGA-coated platinum coil implant is not sufficient to produce site-specific aneurysm healing. MCP-1 is a critical, not merely complementary, actor in the aneurysm healing pathway.

Intracellular localisation of proteins to specific cellular areas by nanocapsule mediated delivery.

Science.gov (United States)

Wang, Huabin; Chen, Ligang; Sun, Xianchao; Fu, Ailing

2017-09-01

Nanocapsules are promising carriers with great potential for intracellular protein transport. Although many studies have intended to improve cell uptake efficacy, there is an increasing interest in understanding of subcellular distribution of cargoes inside cells, which is essential for purposeful delivery of biomolecules into specific sites within cells. Herein, we interrogate the intracellular localisation of exogenous proteins, including fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) and green fluorescent protein (GFP), mediated by specially designed nanocapsules. The results show that the designed nanocapsules can deliver the two types of fluorescent proteins into different cellular destinations (cytosol, nucleus or the whole cell), depending on the composition of nanocapsules. Meanwhile, several impact factors that influence the distribution of proteins in cells have also been investigated, and the results suggest that the localisation of capsule-mediated proteins in cells is strongly affected by the surface properties of nanocapsules, the types of stabilisers and proteins, and environmental temperatures. The rational control of intracellular localised delivery of exogenous proteins as we demonstrated in this study might open new avenues to obtain desired magnitude of drug effects for modulating cell activity.

The Bacterial Effector HopX1 Targets JAZ Transcriptional Repressors to Activate Jasmonate Signaling and Promote Infection in Arabidopsis

Science.gov (United States)

Gimenez-Ibanez, Selena; Boter, Marta; Fernández-Barbero, Gemma; Chini, Andrea; Rathjen, John P.; Solano, Roberto

2014-01-01

Pathogenicity of Pseudomonas syringae is dependent on a type III secretion system, which secretes a suite of virulence effector proteins into the host cytoplasm, and the production of a number of toxins such as coronatine (COR), which is a mimic of the plant hormone jasmonate-isoleuce (JA-Ile). Inside the plant cell, effectors target host molecules to subvert the host cell physiology and disrupt defenses. However, despite the fact that elucidating effector action is essential to understanding bacterial pathogenesis, the molecular function and host targets of the vast majority of effectors remain largely unknown. Here, we found that effector HopX1 from Pseudomonas syringae pv. tabaci (Pta) 11528, a strain that does not produce COR, interacts with and promotes the degradation of JAZ proteins, a key family of JA-repressors. We show that hopX1 encodes a cysteine protease, activity that is required for degradation of JAZs by HopX1. HopX1 associates with JAZ proteins through its central ZIM domain and degradation occurs in a COI1-independent manner. Moreover, ectopic expression of HopX1 in Arabidopsis induces the expression of JA-dependent genes, represses salicylic acid (SA)-induced markers, and complements the growth of a COR-deficient P. syringae pv. tomato (Pto) DC3000 strain during natural bacterial infections. Furthermore, HopX1 promoted susceptibility when delivered by the natural type III secretion system, to a similar extent as the addition of COR, and this effect was dependent on its catalytic activity. Altogether, our results indicate that JAZ proteins are direct targets of bacterial effectors to promote activation of JA-induced defenses and susceptibility in Arabidopsis. HopX1 illustrates a paradigm of an alternative evolutionary solution to COR with similar physiological outcome. PMID:24558350

Structural basis for sequence-specific recognition of DNA by TAL effectors

KAUST Repository

Deng, Dong

2012-01-05

TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved amino acids and targets a specific base pair by using two hypervariable residues [known as repeat variable diresidues (RVDs)] at positions 12 and 13. Here, we report the crystal structures of an 11.5-repeat TAL effector in both DNA-free and DNA-bound states. Each TAL repeat comprises two helices connected by a short RVD-containing loop. The 11.5 repeats form a right-handed, superhelical structure that tracks along the sense strand of DNA duplex, with RVDs contacting the major groove. The 12th residue stabilizes the RVD loop, whereas the 13th residue makes a base-specific contact. Understanding DNA recognition by TAL effectors may facilitate rational design of DNA-binding proteins with biotechnological applications.

Bioreducible poly(amidoamine)s with charge-reversel properties for intracellular protein delivery

NARCIS (Netherlands)

Coué, G.M.J.P.C.; Engbersen, Johannes F.J.; Hennink, W.E.; Engbersen, J.F.J.

2010-01-01

An effective intracellular protein delivery system was developed using bioreducible disulfide-containing poly(amidoamine)s with negatively charged citraconic side groups that can give charge-reversal upon pH decrease. These water-soluble and linear polymers efficiently self-assemble with proteins

Overview on zein protein: a promising pharmaceutical excipient in drug delivery systems and tissue engineering.

Science.gov (United States)

Labib, Gihan

2018-01-01

Natural pharmaceutical excipients have been applied extensively in the past decades owing to their safety and biocompatibility. Zein, a natural protein of plant origin offers great benefit over other synthetic polymers used in controlled drug and biomedical delivery systems. It was used in a variety of medical fields including pharmaceutical and biomedical drug targeting, vaccine, tissue engineering, and gene delivery. Being biodegradable and biocompatible, the current review focuses on the history and the medical application of zein as an attractive still promising biopolymer. Areas covered: The current review gives a broadscope on zein as a still promising protein excipient in different fields. Zein- based drug and biomedical delivery systems are discussed with special focus on current and potential application in controlled drug delivery systems, and tissue engineering. Expert opinion: Zein as a protein of natural origin can still be considered a promising polymer in the field of drug delivery systems as well as in tissue engineering. Although different researchers spotted light on zein application in different industrial fields extensively, the feasibility of its use in the field of drug delivery replenished by investigators in recent years has not yet been fully approached.

Aerosol delivery of Akt controls protein translation in the lungs of dual luciferase reporter mice.

Science.gov (United States)

Tehrani, A M; Hwang, S-K; Kim, T-H; Cho, C-S; Hua, J; Nah, W-S; Kwon, J-T; Kim, J-S; Chang, S-H; Yu, K-N; Park, S-J; Bhandari, D R; Lee, K-H; An, G-H; Beck, G R; Cho, M-H

2007-03-01

Lung cancer has emerged as a leading cause of cancer death in the world; however, most of the current conventional therapies are not sufficiently effective in altering the progression of disease. Therefore, development of novel treatment approaches is needed. Although several genes and methods have been used for cancer gene therapy, a number of problems such as specificity, efficacy and toxicity reduce their application. This has led to re-emergence of aerosol gene delivery as a noninvasive method for lung cancer treatment. In this study, nano-sized glucosylated polyethyleneimine (GPEI) was used as a gene delivery carrier to investigate the effects of Akt wild type (WT) and kinase deficient (KD) on Akt-related signaling pathways and protein translation in the lungs of CMV- LucR-cMyc-IRES-LucF dual reporter mice. These mice are a powerful tool for the discrimination between cap-dependent/-independent protein translation. Aerosols containing self-assembled nano-sized GPEI/Akt WT or GPEI/Akt KD were delivered into the lungs of reporter mice through nose-only-inhalation-chamber with the aid of nebulizer. Aerosol delivery of Akt WT caused the increase of protein expression levels of Akt-related signals, whereas aerosol delivery of Akt KD did not. Furthermore, dual luciferase activity assay showed that aerosol delivery of Akt WT enhanced cap-dependent protein translation, whereas a reduction in cap-dependent protein translation by Akt KD was observed. Our results clearly showed that targeting Akt may be a good strategy for prevention as well as treatment of lung cancer. These studies suggest that our aerosol delivery is compatible for in vivo gene delivery which could be used as a noninvasive gene therapy in the future.

Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

Science.gov (United States)

Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

2015-09-01

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional Analysis of Barley Powdery Mildew Effector Candidates and Identification of their Barley Targets

DEFF Research Database (Denmark)

Ahmed, Ali Abdurehim

The genome of barley powdery mildew fungus (Blumeria graminis f. sp. hordei, Bgh) encodes around 500 Candidate Secreted Effector Proteins (CSEPs), which are believed to be delivered to the barley cells either to interfere with plant defence and/or promote nutrient uptake. So far, little is known...... about the function of many CSEPs in virulence and the identities of their host targets. In this PhD study, we investigated the function of nine CSEPs and found that CSEP0081, CSEP0105, CSEP0162 and CSEP0254 act as effectors by promoting the Bgh infection success. Independent silencing of these CSEPs...... proteins (sHsps), Hsp16.9 and Hsp17.5, were identified as interactors for both CSEP0105 and CSEP0162. These interactions were confirmed in planta by BiFC and co-localization studies. Small heat shock proteins are highly conserved ATP-independent chaperones that protect the cell from stress-induced protein...

Promotion of the transdermal delivery of protein drugs by N-trimethyl chitosan nanoparticles combined with polypropylene electret.

Science.gov (United States)

Tu, Ye; Wang, Xinxia; Lu, Ying; Zhang, He; Yu, Yuan; Chen, Yan; Liu, Junjie; Sun, Zhiguo; Cui, Lili; Gao, Jing; Zhong, Yanqiang

We recently reported that electret, which was prepared by a corona charging system with polypropylene film, could enhance the transdermal delivery of several drugs of low molecular weight. The aim of this study was to investigate whether electret could enhance the transdermal delivery of protein drugs by N -trimethyl chitosan nanoparticles (TMC NPs) prepared by an ionic gelation method. A series of experiments were performed, including in vitro skin permeation assays and anti-inflammatory effects, to evaluate the transdermal delivery of protein drugs by TMC NPs in the presence of electret. The results showed that in the presence of electret, the transdermal delivery of protein drugs in TMC NPs was significantly enhanced, as demonstrated by in vitro permeation studies and confocal laser scanning microscopy. Notably, superoxide dismutase-loaded TMC NPs combined with electret exhibited the best inhibitory effect on the edema of the mouse ear. TMC NPs combined with electret represent a novel platform for the transdermal delivery of protein drugs.

Defective Proteasome Delivery of Polyubiquitinated Proteins by Ubiquilin-2 Proteins Containing ALS Mutations.

Directory of Open Access Journals (Sweden)

Lydia Chang

Full Text Available Ubiquilin proteins facilitate delivery of ubiquitinated proteins to the proteasome for degradation. Interest in the proteins has been heightened by the discovery that gene mutations in UBQLN2 cause dominant inheritance of amyotrophic lateral sclerosis (ALS. However, the mechanisms by which the mutations cause ALS are not known. Here we report on the underlying defect of ubiquilin-2 proteins containing ALS-linked mutations in affecting proteasome-mediated degradation. We found that overexpression of ubiquilin-2 proteins containing any one of five different ALS mutations slow degradation of Myc, a prototypic proteasome substrate. Examination of coprecipitating proteins indicated that the mutant proteins are generally capable of binding polyubiquitinated proteins, but defective in binding the proteasome. GST-pulldown studies revealed that many of the mutants bind weaker to the S5a subunit of the proteasome, compared with wild type (WT ubiquilin-2 protein. The results suggest the mutant proteins are unable to deliver their captured cargo to the proteasome for degradation, which presumably leads to toxicity. Quantification of cell death is consistent with this idea. Measurement of protein turnover further indicated the mutant proteins have longer half-lives than WT ubiquilin-2. Our studies provide novel insight into the mechanism by which ALS-linked mutations in UBQLN2 interfere with protein degradation.

Hybrid protein-inorganic nanoparticles: From tumor-targeted drug delivery to cancer imaging.

Science.gov (United States)

Elzoghby, Ahmed O; Hemasa, Ayman L; Freag, May S

2016-12-10

Recently, a great interest has been paid to the development of hybrid protein-inorganic nanoparticles (NPs) for drug delivery and cancer diagnostics in order to combine the merits of both inorganic and protein nanocarriers. This review primarily discusses the most outstanding advances in the applications of the hybrids of naturally-occurring proteins with iron oxide, gadolinium, gold, silica, calcium phosphate NPs, carbon nanotubes, and quantum dots in drug delivery and cancer imaging. Various strategies that have been utilized for the preparation of protein-functionalized inorganic NPs and the mechanisms involved in the drug loading process are discussed. How can the protein functionalization overcome the limitations of colloidal stability, poor dispersibility and toxicity associated with inorganic NPs is also investigated. Moreover, issues relating to the influence of protein hybridization on the cellular uptake, tumor targeting efficiency, systemic circulation, mucosal penetration and skin permeation of inorganic NPs are highlighted. A special emphasis is devoted to the novel approaches utilizing the protein-inorganic nanohybrids in combined cancer therapy, tumor imaging, and theranostic applications as well as stimuli-responsive drug release from the nanohybrids. Copyright © 2016 Elsevier B.V. All rights reserved.

Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity*

Science.gov (United States)

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J.; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-01

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. PMID:25422319

Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity.

Science.gov (United States)

Sato, Tatsuhiro; Akasu, Hitomi; Shimono, Wataru; Matsu, Chisa; Fujiwara, Yuki; Shibagaki, Yoshio; Heard, Jeffrey J; Tamanoi, Fuyuhiko; Hattori, Seisuke

2015-01-09

Rheb small GTPases, which consist of Rheb1 and Rheb2 (also known as RhebL1) in mammalian cells, are unique members of the Ras superfamily and play central roles in regulating protein synthesis and cell growth by activating mTOR. To gain further insight into the function of Rheb, we carried out a search for Rheb-binding proteins and found that Rheb binds to CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), a multifunctional enzyme required for the de novo synthesis of pyrimidine nucleotides. CAD binding is more pronounced with Rheb2 than with Rheb1. Rheb binds CAD in a GTP- and effector domain-dependent manner. The region of CAD where Rheb binds is located at the C-terminal region of the carbamoyl-phosphate synthetase domain and not in the dihydroorotase and aspartate transcarbamoylase domains. Rheb stimulated carbamoyl-phosphate synthetase activity of CAD in vitro. In addition, an elevated level of intracellular UTP pyrimidine nucleotide was observed in Tsc2-deficient cells, which was attenuated by knocking down of Rheb. Immunostaining analysis showed that expression of Rheb leads to increased accumulation of CAD on lysosomes. Both a farnesyltransferase inhibitor that blocks membrane association of Rheb and knockdown of Rheb mislocalized CAD. These results establish CAD as a downstream effector of Rheb and suggest a possible role of Rheb in regulating de novo pyrimidine nucleotide synthesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection.

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Qiao, Yongli; Shi, Jinxia; Zhai, Yi; Hou, Yingnan; Ma, Wenbo

2015-05-05

A broad range of parasites rely on the functions of effector proteins to subvert host immune response and facilitate disease development. The notorious Phytophthora pathogens evolved effectors with RNA silencing suppression activity to promote infection in plant hosts. Here we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) can bind to an evolutionarily conserved nuclear protein containing the aspartate-glutamate-alanine-histidine-box RNA helicase domain in plants. This protein, designated PSR1-Interacting Protein 1 (PINP1), regulates the accumulation of both microRNAs and endogenous small interfering RNAs in Arabidopsis. A null mutation of PINP1 causes embryonic lethality, and silencing of PINP1 leads to developmental defects and hypersusceptibility to Phytophthora infection. These phenotypes are reminiscent of transgenic plants expressing PSR1, supporting PINP1 as a direct virulence target of PSR1. We further demonstrate that the localization of the Dicer-like 1 protein complex is impaired in the nucleus of PINP1-silenced or PSR1-expressing cells, indicating that PINP1 may facilitate small RNA processing by affecting the assembly of dicing complexes. A similar function of PINP1 homologous genes in development and immunity was also observed in Nicotiana benthamiana. These findings highlight PINP1 as a previously unidentified component of RNA silencing that regulates distinct classes of small RNAs in plants. Importantly, Phytophthora has evolved effectors to target PINP1 in order to promote infection.

A Legionella Effector Disrupts Host Cytoskeletal Structure by Cleaving Actin.

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Yao Liu

2017-01-01

Full Text Available Legionella pneumophila, the etiological agent of Legionnaires' disease, replicates intracellularly in protozoan and human hosts. Successful colonization and replication of this pathogen in host cells requires the Dot/Icm type IVB secretion system, which translocates approximately 300 effector proteins into the host cell to modulate various cellular processes. In this study, we identified RavK as a Dot/Icm substrate that targets the host cytoskeleton and reduces actin filament abundance in mammalian cells upon ectopic expression. RavK harbors an H95EXXH99 motif associated with diverse metalloproteases, which is essential for the inhibition of yeast growth and for the induction of cell rounding in HEK293T cells. We demonstrate that the actin protein itself is the cellular target of RavK and that this effector cleaves actin at a site between residues Thr351 and Phe352. Importantly, RavK-mediated actin cleavage also occurs during L. pneumophila infection. Cleavage by RavK abolishes the ability of actin to form polymers. Furthermore, an F352A mutation renders actin resistant to RavK-mediated cleavage; expression of the mutant in mammalian cells suppresses the cell rounding phenotype caused by RavK, further establishing that actin is the physiological substrate of RavK. Thus, L. pneumophila exploits components of the host cytoskeleton by multiple effectors with distinct mechanisms, highlighting the importance of modulating cellular processes governed by the actin cytoskeleton in the intracellular life cycle of this pathogen.

Role of Rab family GTPases and their effectors in melanosomal logistics.

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Ohbayashi, Norihiko; Fukuda, Mitsunori

2012-04-01

Rab GTPases constitute a family of small GTPases that regulate a variety of membrane trafficking events in all eukaryotic cells by recruiting their specific effector molecules. Recent accumulating evidence indicates that members of the mammalian Rab small GTPase family are involved in certain physiological and pathological processes. In particular, functional impairments of specific Rab proteins, e.g. Rab38 and Rab27A, their regulators or their effectors cause pigmentation disorders in humans and coat colour variations in mice because such impairments cause defects in melanosomal logistics, i.e. defects in melanosome biogenesis and transport. Genetic and biochemical analyses of the gene products responsible for mammalian pigmentation disorders in the past decade have revealed that Rab-mediated endosomal transport systems and melanosome transport systems play crucial roles in the efficient darkening of mammalian hair and skin. In this article, we review current knowledge regarding melanosomal logistics, with particular focus on the roles of Rab small GTPases and their effectors.

Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence.

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Meghraoui, Alaeddine; Schiavolin, Lionel; Allaoui, Abdelmounaaïm

2014-07-01

Infection of colonic epithelial cells by Shigella is associated with the type III secretion system, which serves as a molecular syringe to inject effectors into host cells. This system includes an extracellular needle used as a conduit for secreted proteins. Two of these proteins, IpaB and IpaD, dock at the needle tip to control secretion and are also involved in the insertion of a translocation pore into host cell membrane allowing effector delivery. To better understand the function of IpaD, we substituted thirteen residues conserved among homologous proteins in other bacterial species. Generated variants were tested for their ability to surface expose IpaB and IpaD, to control secretion, to insert the translocation pore, and to invade host cells. In addition to a first group of seven ipaD variants that behaved similarly to the wild-type strain, we identified a second group with mutations V314D and I319D that deregulated secretion of all effectors, but remained fully invasive. Moreover, we identified a third group with mutations Y153A, T161D, Q165L and Y276A, that exhibited increased levels of translocators secretion, pore formation, and cell entry. Altogether, our results offer a better understanding of the role of IpaD in the control of Shigella virulence. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Hydrodynamic Delivery of Cre Protein to Lineage-Mark or Time-Stamp Mouse Hepatocytes In situ

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Sonsteng, Katherine M.; Prigge, Justin R.; Talago, Emily A.; June, Ronald K.; Schmidt, Edward E.

2014-01-01

Cre-responsive fluorescent marker alleles are powerful tools for cell lineage tracing in mice; however their utility is limited by regulation of Cre activity. When targeting hepatocytes, hydrodynamic delivery of a Cre-expression plasmid can convert Cre-responsive alleles without inducing the intracellular or systemic antiviral responses often associated with viral-derived Cre-expression vectors. In this method, rapid high-volume intravenous inoculation induces hepatocyte-targeted uptake of extracellular molecules. Here we tested whether hydrodynamic delivery of Cre protein or Cre fused to the HIV-TAT cell-penetrating peptide could convert Cre-responsive reporters in hepatocytes of mice. Hydrodynamic delivery of 2 nmol of either Cre or TAT-Cre protein converted the reporter allele in 5 to 20% of hepatocytes. Neither protein gave detectable Cre activity in endothelia, non-liver organs, or non-hepatocyte cells in liver. Using mice homozygous for a Cre-responsive marker that directs red- (Cre-naïve) or green- (Cre-converted) fluorescent proteins to the nucleus, we assessed sub-saturation Cre-activity. One month after hydrodynamic inoculation with Cre protein, 58% of hepatocyte nuclei that were green were also red, indicating that less than half of the hepatocytes that had obtained enough Cre to convert one marker allele to green were able to convert all alleles. For comparison, one month after hydrodynamic delivery of a Cre-expression plasmid with a weak promoter, only 26% of the green nuclei were also red. Our results show that hydrodynamic delivery of Cre protein allows rapid allelic conversion in hepatocytes, but Cre-activity is sub-saturating so many cells will not convert multiple Cre-responsive alleles. PMID:24626158

Mechanism of IRSp53 inhibition and combinatorial activation by Cdc42 and downstream effectors.

Science.gov (United States)

Kast, David J; Yang, Changsong; Disanza, Andrea; Boczkowska, Malgorzata; Madasu, Yadaiah; Scita, Giorgio; Svitkina, Tatyana; Dominguez, Roberto

2014-04-01

The Rho family GTPase effector IRSp53 has essential roles in filopodia formation and neuronal development, but its regulatory mechanism is poorly understood. IRSp53 contains a membrane-binding BAR domain followed by an unconventional CRIB motif that overlaps with a proline-rich region (CRIB-PR) and an SH3 domain that recruits actin cytoskeleton effectors. Using a fluorescence reporter assay, we show that human IRSp53 adopts a closed inactive conformation that opens synergistically with the binding of human Cdc42 to the CRIB-PR and effector proteins, such as the tumor-promoting factor Eps8, to the SH3 domain. The crystal structure of Cdc42 bound to the CRIB-PR reveals a new mode of effector binding to Rho family GTPases. Structure-inspired mutations disrupt autoinhibition and Cdc42 binding in vitro and decouple Cdc42- and IRSp53-dependent filopodia formation in cells. The data support a combinatorial mechanism of IRSp53 activation.

Near-Infrared Light Activation of Proteins Inside Living Cells Enabled by Carbon Nanotube-Mediated Intracellular Delivery.

Science.gov (United States)

Li, He; Fan, Xinqi; Chen, Xing

2016-02-01

Light-responsive proteins have been delivered into the cells for controlling intracellular events with high spatial and temporal resolution. However, the choice of wavelength is limited to the UV and visible range; activation of proteins inside the cells using near-infrared (NIR) light, which has better tissue penetration and biocompatibility, remains elusive. Here, we report the development of a single-walled carbon nanotube (SWCNT)-based bifunctional system that enables protein intracellular delivery, followed by NIR activation of the delivered proteins inside the cells. Proteins of interest are conjugated onto SWCNTs via a streptavidin-desthiobiotin (SA-DTB) linkage, where the protein activity is blocked. SWCNTs serve as both a nanocarrier for carrying proteins into the cells and subsequently a NIR sensitizer to photothermally cleave the linkage and release the proteins. The released proteins become active and exert their functions inside the cells. We demonstrated this strategy by intracellular delivery and NIR-triggered nuclear translocation of enhanced green fluorescent protein, and by intracellular delivery and NIR-activation of a therapeutic protein, saporin, in living cells. Furthermore, we showed that proteins conjugated onto SWCNTs via the SA-DTB linkage could be delivered to the tumors, and optically released and activated by using NIR light in living mice.

Oral delivery of bioencapsulated proteins across blood-brain and blood-retinal barriers.

Science.gov (United States)

Kohli, Neha; Westerveld, Donevan R; Ayache, Alexandra C; Verma, Amrisha; Shil, Pollob; Prasad, Tuhina; Zhu, Ping; Chan, Sic L; Li, Qiuhong; Daniell, Henry

2014-03-01

Delivering neurotherapeutics to target brain-associated diseases is a major challenge. Therefore, we investigated oral delivery of green fluorescence protein (GFP) or myelin basic protein (MBP) fused with the transmucosal carrier cholera toxin B subunit (CTB), expressed in chloroplasts (bioencapsulated within plant cells) to the brain and retinae of triple transgenic Alzheimer's disease (3×TgAD) mice, across the blood-brain barriers (BBB) and blood-retinal barriers (BRB). Human neuroblastoma cells internalized GFP when incubated with CTB-GFP but not with GFP alone. Oral delivery of CTB-MBP in healthy and 3×TgAD mice shows increased MBP levels in different regions of the brain, crossing intact BBB. Thioflavin S-stained amyloid plaque intensity was reduced up to 60% by CTB-MBP incubation with human AD and 3×TgAD mice brain sections ex vivo. Amyloid loads were reduced in vivo by 70% in hippocampus and cortex brain regions of 3×TgAD mice fed with bioencapsulated CTB-MBP, along with reduction in the ratio of insoluble amyloid β 42 (Aβ42) to soluble fractions. CTB-MBP oral delivery reduced Aβ42 accumulation in retinae and prevented loss of retinal ganglion cells in 3×TgAD mice. Lyophilization of leaves increased CTB-MBP concentration by 17-fold and stabilized it during long-term storage in capsules, facilitating low-cost oral delivery of therapeutic proteins across the BBB and BRB.

The novel cyst nematode effector protein 19C07 interacts with the Arabidopsis auxin influx transporter LAX3 to control feeding site development.

Science.gov (United States)

Lee, Chris; Chronis, Demosthenis; Kenning, Charlotte; Peret, Benjamin; Hewezi, Tarek; Davis, Eric L; Baum, Thomas J; Hussey, Richard; Bennett, Malcolm; Mitchum, Melissa G

2011-02-01

Plant-parasitic cyst nematodes penetrate plant roots and transform cells near the vasculature into specialized feeding sites called syncytia. Syncytia form by incorporating neighboring cells into a single fused cell by cell wall dissolution. This process is initiated via injection of esophageal gland cell effector proteins from the nematode stylet into the host cell. Once inside the cell, these proteins may interact with host proteins that regulate the phytohormone auxin, as cellular concentrations of auxin increase in developing syncytia. Soybean cyst nematode (Heterodera glycines) Hg19C07 is a novel effector protein expressed specifically in the dorsal gland cell during nematode parasitism. Here, we describe its ortholog in the beet cyst nematode (Heterodera schachtii), Hs19C07. We demonstrate that Hs19C07 interacts with the Arabidopsis (Arabidopsis thaliana) auxin influx transporter LAX3. LAX3 is expressed in cells overlying lateral root primordia, providing auxin signaling that triggers the expression of cell wall-modifying enzymes, allowing lateral roots to emerge. We found that LAX3 and polygalacturonase, a LAX3-induced cell wall-modifying enzyme, are expressed in the developing syncytium and in cells to be incorporated into the syncytium. We observed no decrease in H. schachtii infectivity in aux1 and lax3 single mutants. However, a decrease was observed in both the aux1lax3 double mutant and the aux1lax1lax2lax3 quadruple mutant. In addition, ectopic expression of 19C07 was found to speed up lateral root emergence. We propose that Hs19C07 most likely increases LAX3-mediated auxin influx and may provide a mechanism for cyst nematodes to modulate auxin flow into root cells, stimulating cell wall hydrolysis for syncytium development.

Transient exposure to proteins SOX2, Oct-4, and NANOG immortalizes exhausted tumor-infiltrating CTLs

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Bhadurihauck, Anjuli; Li, Lei [Department of Animal and Avian Sciences, University of Maryland, College Park, 20742, MD (United States); Li, Qianqian; Wang, Jianjun [Department of Biochemistry and Molecular Biology, Wayne State University, Detroit, 48201 (United States); Xiao, Zhengguo, E-mail: xiao0028@umd.edu [Department of Animal and Avian Sciences, University of Maryland, College Park, 20742, MD (United States)

2016-05-13

Adoptive cell transfer therapy (ACT) is one of the most promising immunotherapies against cancer, using tumor-infiltrating lymphocytes (TILs) expanded in vitro. Tumor-infiltrating cytotoxic T lymphocytes (TICTLs) play a prominent role in cancer control. TILs terminally differentiate in response to immunosuppressive environments within tumors, and thus are slow to expand and challenging to maintain both in vitro and in patients. To reverse this exhaustion, we utilize a nuclear protein delivery system that exposes TICTLs to the SOX2, Oct-4, and NANOG (SON) proteins. Unlike activated naïve CTLs (effector CTLs), TICTLs respond favorably to SON treatment, exhibiting steady proliferation and extended survivability independent of cytokine and antigen stimulation. Though TICTLs treated with SON (STICTLs) still express T cell receptors as well as other critical downstream components, they are unresponsive to antigen challenge, suggesting that SON treatment regresses TICTLs into a state similar to that of an early double negative T cell. Our findings indicate the TICTL response to SON proteins is unique when compared to effector CTLs, suggesting TICTLs may be sensitive to regulation by other lineage-specific transcription factors and opening a promising new avenue into cancer immunotherapy. To our knowledge, this is the first report on lineage reprogramming of TILs using protein stem cell transcription factors delivered directly to the nucleus. -- Highlights: •TICTLs are sensitive to reprogramming by proteins of stem cell transcription factors, but effector CTLs were not. •TICTLs are regressed back to an early double negative T cell stage. •TCR signaling is deregulated by these transcription factors.

Delivery of Therapeutic Proteins via Extracellular Vesicles: Review and Potential Treatments for Parkinson's Disease, Glioma, and Schwannoma.

Science.gov (United States)

Hall, Justin; Prabhakar, Shilpa; Balaj, Leonora; Lai, Charles P; Cerione, Richard A; Breakefield, Xandra O

2016-04-01

Extracellular vesicles present an attractive delivery vehicle for therapeutic proteins. They intrinsically contain many proteins which can provide information to other cells. Advantages include reduced immune reactivity, especially if derived from the same host, stability in biologic fluids, and ability to target uptake. Those from mesenchymal stem cells appear to be intrinsically therapeutic, while those from cancer cells promote tumor progression. Therapeutic proteins can be loaded into vesicles by overexpression in the donor cell, with oligomerization and membrane sequences increasing their loading. Examples of protein delivery for therapeutic benefit in pre-clinical models include delivery of: catalase for Parkinson's disease to reduce oxidative stress and thus help neurons to survive; prodrug activating enzymes which can convert a prodrug which crosses the blood-brain barrier into a toxic chemotherapeutic drug for schwannomas and gliomas; and the apoptosis-inducing enzyme, caspase-1 under a Schwann cell specific promoter for schwannoma. This therapeutic delivery strategy is novel and being explored for a number of diseases.

Recent advances in topical delivery of proteins and peptides mediated by soft matter nanocarriers.

Science.gov (United States)

Witting, Madeleine; Obst, Katja; Friess, Wolfgang; Hedtrich, Sarah

2015-11-01

Proteins and peptides are increasingly important therapeutics for the treatment of severe and complex diseases like cancer or autoimmune diseases due to their high specificity and potency. Their unique structure and labile physicochemical properties, however, require special attention in the production and formulation process as well as during administration. Aside from conventional systemic injections, the topical application of proteins and peptides is an appealing alternative due to its non-invasive nature and thus high acceptance by patients. For this approach, soft matter nanocarriers are interesting delivery systems which offer beneficial properties such as high biocompatibility, easiness of modifications, as well as targeted drug delivery and release. This review aims to highlight and discuss technological developments in the field of soft matter nanocarriers for the delivery of proteins and peptides via the skin, the eye, the nose, and the lung, and to provide insights in advantages, limitations, and practicability of recent advances. Copyright © 2015 Elsevier Inc. All rights reserved.

Bioreducible poly(amidoamine)s as carriers for intracellular protein delivery to intestinal cells

NARCIS (Netherlands)

Cohen, S.; Coué, G.M.J.P.C.; Beno, D.; Korenstein, R.; Engbersen, Johannes F.J.

2012-01-01

An effective intracellular protein delivery system was developed based on linear poly(amidoamine)s (PAAs) that form self-assembled cationic nanocomplexes with oppositely charged proteins. Two differently functionalized PAAs were synthesized by Michael-type polyaddition of 4-amino-1-butanol (ABOL) to

Orally active-targeted drug delivery systems for proteins and peptides.

Science.gov (United States)

Li, Xiuying; Yu, Miaorong; Fan, Weiwei; Gan, Yong; Hovgaard, Lars; Yang, Mingshi

2014-09-01

In the past decade, extensive efforts have been devoted to designing 'active targeted' drug delivery systems (ATDDS) to improve oral absorption of proteins and peptides. Such ATDDS enhance cellular internalization and permeability of proteins and peptides via molecular recognition processes such as ligand-receptor or antigen-antibody interaction, and thus enhance drug absorption. This review focuses on recent advances with orally ATDDS, including ligand-protein conjugates, recombinant ligand-protein fusion proteins and ligand-modified carriers. In addition to traditional intestinal active transport systems of substrates and their corresponding receptors, transporters and carriers, new targets such as intercellular adhesion molecule-1 and β-integrin are also discussed. ATDDS can improve oral absorption of proteins and peptides. However, currently, no clinical studies on ATDDS for proteins and peptides are underway, perhaps due to the complexity and limited knowledge of transport mechanisms. Therefore, more research is warranted to optimize ATDDS efficiency.

A review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules.

Science.gov (United States)

Choonara, Bibi F; Choonara, Yahya E; Kumar, Pradeep; Bijukumar, Divya; du Toit, Lisa C; Pillay, Viness

2014-11-15

The oral delivery of proteins and peptides is a dynamic research field despite the numerous challenges limiting their effective delivery. Successful oral delivery of proteins and peptides requires the accomplishment of three key tasks: protection of the macromolecules from degradation in the gastrointestinal tract (GIT), permeation through the intestinal barrier and absorption of molecules into the systemic circulation. Currently, no clinically useful oral formulations have been developed but several attempts have been made to overcome the challenges of low oral bioavailability resulting from poor absorption, poor permeation and enzymatic degradation of the proteins and peptides in the GIT. Present strategies attempt to provide structural protection of the proteins and peptides and improved absorption through the use of enzyme inhibitors, absorption enhancers, novel polymeric delivery systems and chemical modification. However, each of these technologies has their limitations despite showing positive results. This review attempts to discuss the physical and chemical barriers of the GIT with particular emphasis on the current approaches employed to overcome these barriers, including the evaluation of other non-parenteral routes of protein and peptide delivery. In addition, this review assimilates oral formulation strategies under development and within the clinical trial stage in relation to their benefits and drawbacks with regard to facilitating optimal protection and absorption of proteins and peptides, as well as pertinent future challenges and opportunities governing oral drug delivery. Copyright © 2014 Elsevier Inc. All rights reserved.

Convergent and Divergent Signaling in PAMP-Triggered Immunity and Effector-Triggered Immunity.

Science.gov (United States)

Peng, Yujun; van Wersch, Rowan; Zhang, Yuelin

2018-04-01

Plants use diverse immune receptors to sense pathogen attacks. Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors localized on the plasma membrane leads to PAMP-triggered immunity (PTI). Detection of pathogen effectors by intracellular or plasma membrane-localized immune receptors results in effector-triggered immunity (ETI). Despite the large variations in the magnitude and duration of immune responses triggered by different PAMPs or pathogen effectors during PTI and ETI, plasma membrane-localized immune receptors activate similar downstream molecular events such as mitogen-activated protein kinase activation, oxidative burst, ion influx, and increased biosynthesis of plant defense hormones, indicating that defense signals initiated at the plasma membrane converge at later points. On the other hand, activation of ETI by immune receptors localized to the nucleus appears to be more directly associated with transcriptional regulation of defense gene expression. Here, we review recent progress in signal transductions downstream of different groups of plant immune receptors, highlighting the converging and diverging molecular events.

Global study of holistic morphological effectors in the budding yeast Saccharomyces cerevisiae.

Science.gov (United States)

Suzuki, Godai; Wang, Yang; Kubo, Karen; Hirata, Eri; Ohnuki, Shinsuke; Ohya, Yoshikazu

2018-02-20

The size of the phenotypic effect of a gene has been thoroughly investigated in terms of fitness and specific morphological traits in the budding yeast Saccharomyces cerevisiae, but little is known about gross morphological abnormalities. We identified 1126 holistic morphological effectors that cause severe gross morphological abnormality when deleted, and 2241 specific morphological effectors with weak holistic effects but distinctive effects on yeast morphology. Holistic effectors fell into many gene function categories and acted as network hubs, affecting a large number of morphological traits, interacting with a large number of genes, and facilitating high protein expression. Holistic morphological abnormality was useful for estimating the importance of a gene to morphology. The contribution of gene importance to fitness and morphology could be used to efficiently classify genes into functional groups. Holistic morphological abnormality can be used as a reproducible and reliable gene feature for high-dimensional morphological phenotyping. It can be used in many functional genomic applications.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector

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Pratibha Sharma

2017-06-01

Full Text Available Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs technology to interrupt type IV secretion system (T4SS effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1, which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.

Diversifying Selection in the Wheat Stem Rust Fungus Acts Predominantly on Pathogen-Associated Gene Families and Reveals Candidate Effectors

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Jana eSperschneider

2014-09-01

Full Text Available Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus, Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with target host defence proteins. Previous efforts to understand virulence largely relied on the prediction of secreted, small and cysteine-rich proteins as candidate effectors and thus delivered an overwhelming number of candidates. Here, we implement an alternative analysis strategy that uses the signal of adaptive evolution as a line of evidence for effector function, combined with comparative information and expression data. We demonstrate that in planta up-regulated genes that are rapidly evolving are found almost exclusively in pathogen-associated gene families, affirming the impact of host-pathogen co-evolution on genome structure and the adaptive diversification of specialised gene families. In particular, we predict 42 effector candidates that are conserved only across pathogens, induced during infection and rapidly evolving. One of our top candidates has recently been shown to induce genotype-specific hypersensitive cell death in wheat. This shows that comparative genomics incorporating the evolutionary signal of adaptation is powerful for predicting effector candidates for laboratory verification. Our system can be applied to a wide range of pathogens and will give insight into host-pathogen dynamics, ultimately leading to progress in strategies for disease control.

Activation and polar sequestration of PopA, a c-di-GMP effector protein involved in Caulobacter crescentus cell cycle control

DEFF Research Database (Denmark)

Ozaki, Shogo; Schalch-Moser, Annina; Zumthor, Ludwig

2014-01-01

that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co-option as c-di-GMP effector protein. While the C-terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N-terminal receiver domain, functional adaptation has reversed signal......A to the cell pole in response to c-di-GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S-phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit...

Protein stability in pulmonary drug delivery via nebulization.

Science.gov (United States)

Hertel, Sebastian P; Winter, Gerhard; Friess, Wolfgang

2015-10-01

Protein inhalation is a delivery route which offers high potential for direct local lung application of proteins. Liquid formulations are usually available in early stages of biopharmaceutical development and nebulizers are the device of choice for atomization avoiding additional process steps like drying and enabling fast progression to clinical trials. While some proteins were proven to remain stable throughout aerosolization e.g. DNase, many biopharmaceuticals are more susceptible towards the stresses encountered during nebulization. The main reason for protein instability is unfolding and aggregation at the air-liquid interface, a problem which is of particular challenge in the case of ultrasound and jet nebulizers due to recirculation of much of the generated droplets. Surfactants are an important formulation component to protect the sensitive biomolecules. A second important challenge is warming of ultrasound and vibrating mesh devices, which can be overcome by overfilling, precooled solutions or cooling of the reservoir. Ultimately, formulation development has to go hand in hand with device evaluation. Copyright © 2014 Elsevier B.V. All rights reserved.

T3SEdb: data warehousing of virulence effectors secreted by the bacterial Type III Secretion System.

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Tay, Daniel Ming Ming; Govindarajan, Kunde Ramamoorthy; Khan, Asif M; Ong, Terenze Yao Rui; Samad, Hanif M; Soh, Wei Wei; Tong, Minyan; Zhang, Fan; Tan, Tin Wee

2010-10-15

Effectors of Type III Secretion System (T3SS) play a pivotal role in establishing and maintaining pathogenicity in the host and therefore the identification of these effectors is important in understanding virulence. However, the effectors display high level of sequence diversity, therefore making the identification a difficult process. There is a need to collate and annotate existing effector sequences in public databases to enable systematic analyses of these sequences for development of models for screening and selection of putative novel effectors from bacterial genomes that can be validated by a smaller number of key experiments. Herein, we present T3SEdb http://effectors.bic.nus.edu.sg/T3SEdb, a specialized database of annotated T3SS effector (T3SE) sequences containing 1089 records from 46 bacterial species compiled from the literature and public protein databases. Procedures have been defined for i) comprehensive annotation of experimental status of effectors, ii) submission and curation review of records by users of the database, and iii) the regular update of T3SEdb existing and new records. Keyword fielded and sequence searches (BLAST, regular expression) are supported for both experimentally verified and hypothetical T3SEs. More than 171 clusters of T3SEs were detected based on sequence identity comparisons (intra-cluster difference up to ~60%). Owing to this high level of sequence diversity of T3SEs, the T3SEdb provides a large number of experimentally known effector sequences with wide species representation for creation of effector predictors. We created a reliable effector prediction tool, integrated into the database, to demonstrate the application of the database for such endeavours. T3SEdb is the first specialised database reported for T3SS effectors, enriched with manual annotations that facilitated systematic construction of a reliable prediction model for identification of novel effectors. The T3SEdb represents a platform for inclusion of

T3SEdb: data warehousing of virulence effectors secreted by the bacterial Type III Secretion System

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Tong Minyan

2010-10-01

Full Text Available Abstract Background Effectors of Type III Secretion System (T3SS play a pivotal role in establishing and maintaining pathogenicity in the host and therefore the identification of these effectors is important in understanding virulence. However, the effectors display high level of sequence diversity, therefore making the identification a difficult process. There is a need to collate and annotate existing effector sequences in public databases to enable systematic analyses of these sequences for development of models for screening and selection of putative novel effectors from bacterial genomes that can be validated by a smaller number of key experiments. Results Herein, we present T3SEdb http://effectors.bic.nus.edu.sg/T3SEdb, a specialized database of annotated T3SS effector (T3SE sequences containing 1089 records from 46 bacterial species compiled from the literature and public protein databases. Procedures have been defined for i comprehensive annotation of experimental status of effectors, ii submission and curation review of records by users of the database, and iii the regular update of T3SEdb existing and new records. Keyword fielded and sequence searches (BLAST, regular expression are supported for both experimentally verified and hypothetical T3SEs. More than 171 clusters of T3SEs were detected based on sequence identity comparisons (intra-cluster difference up to ~60%. Owing to this high level of sequence diversity of T3SEs, the T3SEdb provides a large number of experimentally known effector sequences with wide species representation for creation of effector predictors. We created a reliable effector prediction tool, integrated into the database, to demonstrate the application of the database for such endeavours. Conclusions T3SEdb is the first specialised database reported for T3SS effectors, enriched with manual annotations that facilitated systematic construction of a reliable prediction model for identification of novel effectors

Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system.

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Limaye, Arati; Koya, Vijay; Samsam, Mohtashem; Daniell, Henry

2006-05-01

Oral delivery of biopharmaceutical proteins expressed in plant cells should reduce their cost of production, purification, processing, cold storage, transportation, and delivery. However, poor intestinal absorption of intact proteins is a major challenge. To overcome this limitation, we investigate here the concept of receptor-mediated oral delivery of chloroplast-expressed foreign proteins. Therefore, the transmucosal carrier cholera toxin B-subunit and green fluorescent protein (CTB-GFP), separated by a furin cleavage site, was expressed via the tobacco chloroplast genome. Polymerase chain reaction (PCR) and Southern blot analyses confirmed site-specific transgene integration and homoplasmy. Immunoblot analysis and ELISA confirmed expression of monomeric and pentameric forms of CTB-GFP, up to 21.3% of total soluble proteins. An in vitro furin cleavage assay confirmed integrity of the engineered furin cleavage site, and a GM1 binding assay confirmed the functionality of CTB-GFP pentamers. Following oral administration of CTB-GFP expressing leaf material to mice, GFP was observed in the mice intestinal mucosa, liver, and spleen in fluorescence and immunohistochemical studies, while CTB remained in the intestinal cell. This report of receptor-mediated oral delivery of a foreign protein into the circulatory system opens the door for low-cost production and delivery of human therapeutic proteins.

Autoreactive T effector memory differentiation mirrors β-cell function in type 1 diabetes.

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Yeo, Lorraine; Woodwyk, Alyssa; Sood, Sanjana; Lorenc, Anna; Eichmann, Martin; Pujol-Autonell, Irma; Melchiotti, Rossella; Skowera, Ania; Fidanis, Efthymios; Dolton, Garry M; Tungatt, Katie; Sewell, Andrew K; Heck, Susanne; Saxena, Alka; Beam, Craig A; Peakman, Mark

2018-05-31

In type 1 diabetes, cytotoxic CD8 T cells with specificity for β-cell autoantigens are found in the pancreatic islets where they are implicated in the destruction of insulin-secreting β cells. In contrast, the disease relevance of β-cell-reactive CD8 T cells that are detectable in the circulation, and their relationship to β-cell function, are not known. Here, we tracked multiple, circulating β-cell-reactive CD8 T cell subsets and measured β-cell function longitudinally for two years, starting immediately after diagnosis of type 1 diabetes. We found that change in β-cell-specific effector memory CD8 T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8 T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer specific protein 37 and CD16, and reduced expression of CD28) compared with their CD57-negative counterparts, and network association modelling indicated that the dynamics of β-cell-reactive CD57+ effector memory CD8 T cell subsets were strongly linked. Thus, coordinated changes in circulating β-cell-specific CD8 T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.

Intracellular Protein Delivery and Gene Transfection by Electroporation Using a Microneedle Electrode Array

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Choi, Seong-O; Kim, Yeu-Chun; Lee, Jeong Woo; Park, Jung-Hwan

2012-01-01

The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, we present intracellular delivery of molecules and transfection with plasmid DNA by electroporation using a novel microneedle electrode array designed for targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50 % of cells and transfection of 12 % of cells with a gene for green fluorescent protein is demonstrated at high cell viability. We conclude that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA and other biopharmaceuticals. PMID:22328093

Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene.

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Raul A Cernadas

2014-02-01

Full Text Available Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo, which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting.

Influence of Crude Protein Intake on the Duration of Delivery and Litter Size in Sows

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D. Tydlitát

2008-01-01

Full Text Available The aim of the study was to evaluate the influence of different intakes of crude protein during the period from 94 to 100 days of pregnancy to the parturition, lengths of pregnancy and delivery, number and birth weights of piglets and concentrations of progesterone, 17-β estradiol and cortisol on days 100, 110 and 114 of pregnancy in sows. Daily feed intake of the sow represented 2.5 kg of complete mixtures containing 13% (group A, n = 23, 15% (group B, n = 52, 18% (group C, n = 10 and 21% (group D, n = 10 of crude protein. Lengths of pregnancy in experimental groups were not significantly different. The mean durations of delivery synchronously increased with the intake of crude protein; significant difference was found between groups A (4.5 h and D (8.6 h (p p < 0.05. The average birth weights of piglets did not differ between experimental groups. No statistical differences in hormone concentrations were found between experimental groups. High intake of crude protein in sows before parturition prolonged delivery and increased the number of stillborn piglets.

Interleukin-10 induction is an important virulence function of the Yersinia pseudotuberculosis type III effector YopM.

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McPhee, Joseph B; Mena, Patricio; Zhang, Yue; Bliska, James B

2012-07-01

Pathogenic Yersinia species modulate host immune responses through the activity of a plasmid-encoded type III secretion system and its associated effector proteins. One effector, YopM, is a leucine-rich-repeat-containing protein that is important for virulence in murine models of Yersinia infection. Although the mechanism by which YopM promotes virulence is unknown, we previously demonstrated that YopM was required for the induction of high levels of the immunosuppressive cytokine interleukin-10 (IL-10) in sera of C57BL/6J mice infected with Yersinia pseudotuberculosis. To determine if IL-10 production is important for the virulence function of YopM, C57BL/6J or congenic IL-10⁻/⁻ mice were infected intravenously with wild-type or yopM mutant Y. pseudotuberculosis strains. Analysis of cytokine levels in serum and bacterial colonization in the spleen and liver showed that YopM is required for IL-10 induction in C57BL/6J mice infected with either the IP32953 or the 32777 strain of Y. pseudotuberculosis, demonstrating that the phenotype is conserved in the species. In single-strain infections, the ability of the 32777ΔyopM mutant to colonize the liver was significantly increased by the delivery of exogenous IL-10 to C57BL/6J mice. In mixed infections, the competitive advantage of a yopM⁺ 32777 strain over an isogenic yopM mutant to colonize spleen and liver, as observed for C57BL/6J mice, was significantly reduced in IL-10⁻/⁻ animals. Thus, by experimentally controlling IL-10 levels in a mouse infection model, we obtained evidence that the induction of this cytokine is an important mechanism by which YopM contributes to Y. pseudotuberculosis virulence.

Addressing the Immunogenicity of the Cargo and of the Targeting Antibodies with a Focus on Deimmunized Bacterial Toxins and on Antibody-Targeted Human Effector Proteins

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Grinberg, Yehudit; Benhar, Itai

2017-01-01

Third-generation immunotoxins are composed of a human, or humanized, targeting moiety, usually a monoclonal antibody or an antibody fragment, and a non-human effector molecule. Due to the non-human origin of the cytotoxic domain, these molecules stimulate potent anti-drug immune responses, which limit treatment options. Efforts are made to deimmunize such immunotoxins or to combine treatment with immunosuppression. An alternative approach is using the so-called ?human cytotoxic fusion protein...

Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan--a review.

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George, Meera; Abraham, T Emilia

2006-08-10

The protein pharmaceutical market is rapidly growing, since it is gaining support from the recombinant DNA technology. To deliver these drugs via the oral route, the most preferred route, is the toughest challenge. In the design of oral delivery of peptide or protein drugs, pH sensitive hydrogels like alginate and chitosan have attracted increasing attention, since most of the synthetic polymers are immunogenic and the incorporation of proteins in to these polymers require harsh environment which may denature and inactivate the desired protein. Alginate is a water-soluble linear polysaccharide composed of alternating blocks of 1-4 linked alpha-L-guluronic and beta-D-mannuronic acid residues where as chitosan is a co polymer of D-glucosamine and N-acetyl glucosamine. The incorporation of protein into these two matrices can be done under relatively mild environment and hence the chances of protein denaturation are minimal. The limitations of these polymers, like drug leaching during preparation can be overcome by different techniques which increase their encapsulation efficiency. Alginate, being an anionic polymer with carboxyl end groups, is a good mucoadhesive agent. The pore size of alginate gel microbeads has been shown to be between 5 and 200 nm and coated beads and microspheres are found to be better oral delivery vehicles. Cross-linked alginate has more capacity to retain the entrapped drugs and mixing of alginate with other polymers such as neutral gums, pectin, chitosan, and eudragit have been found to solve the problem of drug leaching. Chitosan has only limited ability for controlling the release of encapsulated compound due to its hydrophilic nature and easy solubility in acidic medium. By simple covalent modifications of the polymer, its physicochemical properties can be changed and can be made suitable for the peroral drug delivery purpose. Ionic interactions between positively charged amino groups in chitosan and the negatively charged mucus gel layer

Effector gene birth in plant parasitic nematodes: Neofunctionalization of a housekeeping glutathione synthetase gene

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Lilley, Catherine J.; Maqbool, Abbas; Wu, Duqing; Yusup, Hazijah B.; Jones, Laura M.; Birch, Paul R. J.; Urwin, Peter E.

2018-01-01

Plant pathogens and parasites are a major threat to global food security. Plant parasitism has arisen four times independently within the phylum Nematoda, resulting in at least one parasite of every major food crop in the world. Some species within the most economically important order (Tylenchida) secrete proteins termed effectors into their host during infection to re-programme host development and immunity. The precise detail of how nematodes evolve new effectors is not clear. Here we reconstruct the evolutionary history of a novel effector gene family. We show that during the evolution of plant parasitism in the Tylenchida, the housekeeping glutathione synthetase (GS) gene was extensively replicated. New GS paralogues acquired multiple dorsal gland promoter elements, altered spatial expression to the secretory dorsal gland, altered temporal expression to primarily parasitic stages, and gained a signal peptide for secretion. The gene products are delivered into the host plant cell during infection, giving rise to “GS-like effectors”. Remarkably, by solving the structure of GS-like effectors we show that during this process they have also diversified in biochemical activity, and likely represent the founding members of a novel class of GS-like enzyme. Our results demonstrate the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors, and the foundation to uncover a novel enzymatic function. PMID:29641602

pH-sensitive degradable nanoparticles for highly efficient intracellular delivery of exogenous protein

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Xu D

2013-09-01

Full Text Available Dan Xu,1 Fei Wu,1 Yinghui Chen,2,* Liangming Wei,3,* Weien Yuan1,* 1School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 2Department of Neurology, Jinshan Hospital, Fudan University, Shanghai, 3Key Laboratory for Thin Film and Microfabrication of the Ministry of Education, Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, Shanghai, People's Republic of China*These authors contributed equally to this workBackground: Encapsulating exogenous proteins into a nanosized particulate system for delivery into cells is a great challenge. To address this issue, we developed a novel nanoparticle delivery method that differs from the nanoparticles reported to date because its core was composed of cross-linked dextran glassy nanoparticles which had pH in endosome-responsive environment and the protein was loaded in the core of cross-linked dextran glassy nanoparticles.Methods: In this study, dextran in a poly(ethylene glycol aqueous two-phase system created a different chemical environment in which proteins were encapsulated very efficiently (84.3% and 89.6% for enhanced green fluorescent protein and bovine serum albumin, respectively by thermodynamically favored partition. The structures of the nanoparticles were confirmed by confocal laser scanning microscopy and scanning electron microscopy.Results: The nanoparticles had a normal size distribution and a mean diameter of 186 nm. MTT assays showed that the nanoparticles were nontoxic up to a concentration of 2000 µg/mL in human hepatocarcinoma cell line SMMC-7721, HeLa, and BRL-3A cells. Of note, confocal laser scanning microscopy studies showed that nanoparticles loaded with fluorescein isothiocyanate-bovine serum albumin were efficiently delivered and released proteins into the cytoplasm of HeLa cells. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that nanoparticles with a functional protein (apoptin efficiently induced

Inclusion bodies as potential vehicles for recombinant protein delivery into epithelial cells

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2012-01-01

Background We present the potential of inclusion bodies (IBs) as a protein delivery method for polymeric filamentous proteins. We used as cell factory a strain of E. coli, a conventional host organism, and keratin 14 (K14) as an example of a complex protein. Keratins build the intermediate filament cytoskeleton of all epithelial cells. In order to build filaments, monomeric K14 needs first to dimerize with its binding partner (keratin 5, K5), which is then followed by heterodimer assembly into filaments. Results K14 IBs were electroporated into SW13 cells grown in culture together with a “reporter” plasmid containing EYFP labeled keratin 5 (K5) cDNA. As SW13 cells do not normally express keratins, and keratin filaments are built exclusively of keratin heterodimers (i.e. K5/K14), the short filamentous structures we obtained in this study can only be the result of: a) if both IBs and plasmid DNA are transfected simultaneously into the cell(s); b) once inside the cells, K14 protein is being released from IBs; c) released K14 is functional, able to form heterodimers with EYFP-K5. Conclusions Soluble IBs may be also developed for complex cytoskeletal proteins and used as nanoparticles for their delivery into epithelial cells. PMID:22624805

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors*

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Aurass, Philipp; Gerlach, Thomas; Becher, Dörte; Voigt, Birgit; Karste, Susanne; Bernhardt, Jörg; Riedel, Katharina; Hecker, Michael; Flieger, Antje

2016-01-01

Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phase-specific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests

Meta-analytic approach to the accurate prediction of secreted virulence effectors in gram-negative bacteria

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Sato Yoshiharu

2011-11-01

Full Text Available Abstract Background Many pathogens use a type III secretion system to translocate virulence proteins (called effectors in order to adapt to the host environment. To date, many prediction tools for effector identification have been developed. However, these tools are insufficiently accurate for producing a list of putative effectors that can be applied directly for labor-intensive experimental verification. This also suggests that important features of effectors have yet to be fully characterized. Results In this study, we have constructed an accurate approach to predicting secreted virulence effectors from Gram-negative bacteria. This consists of a support vector machine-based discriminant analysis followed by a simple criteria-based filtering. The accuracy was assessed by estimating the average number of true positives in the top-20 ranking in the genome-wide screening. In the validation, 10 sets of 20 training and 20 testing examples were randomly selected from 40 known effectors of Salmonella enterica serovar Typhimurium LT2. On average, the SVM portion of our system predicted 9.7 true positives from 20 testing examples in the top-20 of the prediction. Removal of the N-terminal instability, codon adaptation index and ProtParam indices decreased the score to 7.6, 8.9 and 7.9, respectively. These discrimination features suggested that the following characteristics of effectors had been uncovered: unstable N-terminus, non-optimal codon usage, hydrophilic, and less aliphathic. The secondary filtering process represented by coexpression analysis and domain distribution analysis further refined the average true positive counts to 12.3. We further confirmed that our system can correctly predict known effectors of P. syringae DC3000, strongly indicating its feasibility. Conclusions We have successfully developed an accurate prediction system for screening effectors on a genome-wide scale. We confirmed the accuracy of our system by external validation

Evidence for acquisition of virulence effectors in pathogenic chytrids

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Summers Kyle

2011-07-01

Full Text Available Abstract Background The decline in amphibian populations across the world is frequently linked to the infection of the chytrid fungus Batrachochytrium dendrobatidis (Bd. This is particularly perplexing because Bd was only recently discovered in 1999 and no chytrid fungus had previously been identified as a vertebrate pathogen. Results In this study, we show that two large families of known virulence effector genes, crinkler (CRN proteins and serine peptidases, were acquired by Bd from oomycete pathogens and bacteria, respectively. These two families have been duplicated after their acquisition by Bd. Additional selection analyses indicate that both families evolved under strong positive selection, suggesting that they are involved in the adaptation of Bd to its hosts. Conclusions We propose that the acquisition of virulence effectors, in combination with habitat disruption and climate change, may have driven the Bd epidemics and the decline in amphibian populations. This finding provides a starting point for biochemical investigations of chytridiomycosis.

Duplications and losses in gene families of rust pathogens highlight putative effectors.

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Pendleton, Amanda L; Smith, Katherine E; Feau, Nicolas; Martin, Francis M; Grigoriev, Igor V; Hamelin, Richard; Nelson, C Dana; Burleigh, J Gordon; Davis, John M

2014-01-01

Rust fungi are a group of fungal pathogens that cause some of the world's most destructive diseases of trees and crops. A shared characteristic among rust fungi is obligate biotrophy, the inability to complete a lifecycle without a host. This dependence on a host species likely affects patterns of gene expansion, contraction, and innovation within rust pathogen genomes. The establishment of disease by biotrophic pathogens is reliant upon effector proteins that are encoded in the fungal genome and secreted from the pathogen into the host's cell apoplast or within the cells. This study uses a comparative genomic approach to elucidate putative effectors and determine their evolutionary histories. We used OrthoMCL to identify nearly 20,000 gene families in proteomes of 16 diverse fungal species, which include 15 basidiomycetes and one ascomycete. We inferred patterns of duplication and loss for each gene family and identified families with distinctive patterns of expansion/contraction associated with the evolution of rust fungal genomes. To recognize potential contributors for the unique features of rust pathogens, we identified families harboring secreted proteins that: (i) arose or expanded in rust pathogens relative to other fungi, or (ii) contracted or were lost in rust fungal genomes. While the origin of rust fungi appears to be associated with considerable gene loss, there are many gene duplications associated with each sampled rust fungal genome. We also highlight two putative effector gene families that have expanded in Cqf that we hypothesize have roles in pathogenicity.

The Effector SPRYSEC-19 of Globodera rostochiensis Suppresses CC-NB-LRR-Mediated Disease Resistance in Plants1[C][W][OA

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Postma, Wiebe J.; Slootweg, Erik J.; Rehman, Sajid; Finkers-Tomczak, Anna; Tytgat, Tom O.G.; van Gelderen, Kasper; Lozano-Torres, Jose L.; Roosien, Jan; Pomp, Rikus; van Schaik, Casper; Bakker, Jaap; Goverse, Aska; Smant, Geert

2012-01-01

The potato cyst nematode Globodera rostochiensis invades roots of host plants where it transforms cells near the vascular cylinder into a permanent feeding site. The host cell modifications are most likely induced by a complex mixture of proteins in the stylet secretions of the nematodes. Resistance to nematodes conferred by nucleotide-binding-leucine-rich repeat (NB-LRR) proteins usually results in a programmed cell death in and around the feeding site, and is most likely triggered by the recognition of effectors in stylet secretions. However, the actual role of these secretions in the activation and suppression of effector-triggered immunity is largely unknown. Here we demonstrate that the effector SPRYSEC-19 of G. rostochiensis physically associates in planta with the LRR domain of a member of the SW5 resistance gene cluster in tomato (Lycopersicon esculentum). Unexpectedly, this interaction did not trigger defense-related programmed cell death and resistance to G. rostochiensis. By contrast, agroinfiltration assays showed that the coexpression of SPRYSEC-19 in leaves of Nicotiana benthamiana suppresses programmed cell death mediated by several coiled-coil (CC)-NB-LRR immune receptors. Furthermore, SPRYSEC-19 abrogated resistance to Potato virus X mediated by the CC-NB-LRR resistance protein Rx1, and resistance to Verticillium dahliae mediated by an unidentified resistance in potato (Solanum tuberosum). The suppression of cell death and disease resistance did not require a physical association of SPRYSEC-19 and the LRR domains of the CC-NB-LRR resistance proteins. Altogether, our data demonstrated that potato cyst nematodes secrete effectors that enable the suppression of programmed cell death and disease resistance mediated by several CC-NB-LRR proteins in plants. PMID:22904163

Method for Targeted Therapeutic Delivery of Proteins into Cells | NCI Technology Transfer Center | TTC

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The Protein Expression Laboratory at the National Cancer Institute in Frederick, MD is seeking statements of capability or interest from parties interested in collaborative research to further develop a platform technology for the targeted intra-cellular delivery of proteins using virus-like particles (VLPs).

Thermo-responsive human α-elastin self-assembled nanoparticles for protein delivery.

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Kim, Jae Dong; Jung, Youn Jae; Woo, Chang Hee; Choi, Young Chan; Choi, Ji Suk; Cho, Yong Woo

2017-01-01

Self-assembled nanoparticles based on PEGylated human α-elastin were prepared as a potential vehicle for sustained protein delivery. The α-elastin was extracted from human adipose tissue and modified with methoxypolyethyleneglycol (mPEG) to control particle size and enhance the colloidal stability. The PEGylated human α-elastin showed sol-to-particle transition with a lower critical solution temperature (LCST) of 25°C-40°C in aqueous media. The PEGylated human α-elastin nanoparticles (PhENPs) showed a narrow size distribution with an average diameter of 330±33nm and were able to encapsulate significant amounts of insulin and bovine serum albumin (BSA) upon simple mixing at low temperature in water and subsequent heating to physiological temperature. The release profiles of insulin and BSA showed sustained release for 72h. Overall, the thermo-responsive self-assembled PhENPs provide a useful tool for a range of protein delivery and tissue engineering applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Systematic Identification of Intracellular-Translocated Candidate Effectors in Edwardsiella piscicida

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Lingzhi Zhang

2018-02-01

Full Text Available Many bacterial pathogens inject effectors directly into host cells to target a variety of host cellular processes and promote bacterial dissemination and survival. Identifying the bacterial effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Edwardsiella piscicida is a pathogen with a wide host range, and very few of its effectors have been identified to date. Here, based on the genes significantly regulated by macrophage infection, we identified 25 intracellular translocation-positive candidate effectors, including all five previously reported effectors, namely EseG, EseJ, EseH, EseK, and EvpP. A subsequent secretion analysis revealed diverse secretion patterns for the 25 effector candidates, suggesting that multiple transport pathways were involved in the internalization of these candidate effectors. Further, we identified two novel type VI secretion system (T6SS putative effectors and three outer membrane vesicles (OMV-dependent putative effectors among the candidate effectors described above, and further analyzed their contribution to bacterial virulence in a zebrafish model. This work demonstrates an effective approach for screening bacterial effectors and expands the effectors repertoire in E. piscicida.

Cell-penetrating DNA-binding protein as a safe and efficient naked DNA delivery carrier in vitro and in vivo

International Nuclear Information System (INIS)

Kim, Eun-Sung; Yang, Seung-Woo; Hong, Dong-Ki; Kim, Woo-Taek; Kim, Ho-Guen; Lee, Sang-Kyou

2010-01-01

Non-viral gene delivery is a safe and suitable alternative to viral vector-mediated delivery to overcome the immunogenicity and tumorigenesis associated with viral vectors. Using the novel, human-origin Hph-1 protein transduction domain that can facilitate the transduction of protein into cells, we developed a new strategy to deliver naked DNA in vitro and in vivo. The new DNA delivery system contains Hph-1-GAL4 DNA-binding domain (DBD) fusion protein and enhanced green fluorescent protein (EGFP) reporter plasmid that includes the five repeats of GAL4 upstream activating sequence (UAS). Hph-1-GAL4-DBD protein formed complex with plasmid DNA through the specific interaction between GAL4-DBD and UAS, and delivered into the cells via the Hph-1-PTD. The pEGFP DNA was successfully delivered by the Hph-1-GAL4 system, and the EGFP was effectively expressed in mammalian cells such as HeLa and Jurkat, as well as in Bright Yellow-2 (BY-2) plant cells. When 10 μg of pEGFP DNA was intranasally administered to mice using Hph-1-GAL4 protein, a high level of EGFP expression was detected throughout the lung tissue for 7 days. These results suggest that an Hph-1-PTD-mediated DNA delivery strategy may be an useful non-viral DNA delivery system for gene therapy and DNA vaccines.

Cell-penetrating DNA-binding protein as a safe and efficient naked DNA delivery carrier in vitro and in vivo

Energy Technology Data Exchange (ETDEWEB)

Kim, Eun-Sung; Yang, Seung-Woo [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Hong, Dong-Ki; Kim, Woo-Taek [Department of Biology, Yonsei University, Seoul 120-749 (Korea, Republic of); Kim, Ho-Guen [Department of Pathology, Yonsei Medical School, Seoul 120-752 (Korea, Republic of); Lee, Sang-Kyou, E-mail: sjrlee@yonsei.ac.kr [Department of Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

2010-01-29

Non-viral gene delivery is a safe and suitable alternative to viral vector-mediated delivery to overcome the immunogenicity and tumorigenesis associated with viral vectors. Using the novel, human-origin Hph-1 protein transduction domain that can facilitate the transduction of protein into cells, we developed a new strategy to deliver naked DNA in vitro and in vivo. The new DNA delivery system contains Hph-1-GAL4 DNA-binding domain (DBD) fusion protein and enhanced green fluorescent protein (EGFP) reporter plasmid that includes the five repeats of GAL4 upstream activating sequence (UAS). Hph-1-GAL4-DBD protein formed complex with plasmid DNA through the specific interaction between GAL4-DBD and UAS, and delivered into the cells via the Hph-1-PTD. The pEGFP DNA was successfully delivered by the Hph-1-GAL4 system, and the EGFP was effectively expressed in mammalian cells such as HeLa and Jurkat, as well as in Bright Yellow-2 (BY-2) plant cells. When 10 {mu}g of pEGFP DNA was intranasally administered to mice using Hph-1-GAL4 protein, a high level of EGFP expression was detected throughout the lung tissue for 7 days. These results suggest that an Hph-1-PTD-mediated DNA delivery strategy may be an useful non-viral DNA delivery system for gene therapy and DNA vaccines.

Multidisciplinary perspectives for Alzheimer's and Parkinson's diseases: hydrogels for protein delivery and cell-based drug delivery as therapeutic strategies.

Science.gov (United States)

Giordano, Carmen; Albani, Diego; Gloria, Antonio; Tunesi, Marta; Batelli, Sara; Russo, Teresa; Forloni, Gianluigi; Ambrosio, Luigi; Cigada, Alberto

2009-12-01

This review presents two intriguing multidisciplinary strategies that might make the difference in the treatment of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The first proposed strategy is based on the controlled delivery of recombinant proteins known to play a key role in these neurodegenerative disorders that are released in situ by optimized polymer-based systems. The second strategy is the use of engineered cells, encapsulated and delivered in situ by suitable polymer-based systems, that act as drug reservoirs and allow the delivery of selected molecules to be used in the treatment of Alzheimer's and Parkinson's diseases. In both these scenarios, the design and development of optimized polymer-based drug delivery and cell housing systems for central nervous system applications represent a key requirement. Materials science provides suitable hydrogel-based tools to be optimized together with suitably designed recombinant proteins or drug delivering-cells that, once in situ, can provide an effective treatment for these neurodegenerative disorders. In this scenario, only interdisciplinary research that fully integrates biology, biochemistry, medicine and materials science can provide a springboard for the development of suitable therapeutic tools, not only for the treatment of Alzheimer's and Parkinson's diseases but also, prospectively, for a wide range of severe neurodegenerative disorders.

Rab proteins: The key regulators of intracellular vesicle transport

International Nuclear Information System (INIS)

Bhuin, Tanmay; Roy, Jagat Kumar

2014-01-01

Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future

Rab proteins: The key regulators of intracellular vesicle transport

Energy Technology Data Exchange (ETDEWEB)

Bhuin, Tanmay [Cell and Developmental Biology Unit, Department of Zoology, The University of Burdwan, Golapbag 713104 (India); Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in [Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005 (India)

2014-10-15

Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

The interplay between a Phytophthora RXLR effector and an Arabidopsis lectin receptor kinase

NARCIS (Netherlands)

Bouwmeester, K.

2010-01-01

Phytophthora infestans – the causal agent of potato late blight – secretes a plethora of effector proteins to facilitate plant infection. The central subject of this thesis is ipiO, one of the first cloned Phytophthora genes with a putative function in pathogenicity as was anticipated based on its

Development of amphiphilic gamma-PGA-nanoparticle based tumor vaccine: potential of the nanoparticulate cytosolic protein delivery carrier.

Science.gov (United States)

Yoshikawa, Tomoaki; Okada, Naoki; Oda, Atsushi; Matsuo, Kazuhiko; Matsuo, Keisuke; Mukai, Yohei; Yoshioka, Yasuo; Akagi, Takami; Akashi, Mitsuru; Nakagawa, Shinsaku

2008-02-08

Nanoscopic therapeutic systems that incorporate biomacromolecules, such as protein and peptides, are emerging as the next generation of nanomedicine aimed at improving the therapeutic efficacy of biomacromolecular drugs. In this study, we report that poly(gamma-glutamic acid)-based nanoparticles (gamma-PGA NPs) are excellent protein delivery carriers for tumor vaccines that delivered antigenic proteins to antigen-presenting cells and elicited potent immune responses. Importantly, gamma-PGA NPs efficiently delivered entrapped antigenic proteins through cytosolic translocation from the endosomes, which is a key process of gamma-PGA NP-mediated anti-tumor immune responses. Our findings suggest that the gamma-PGA NP system is suitable for the intracellular delivery of protein-based drugs as well as tumor vaccines.

Effector-Triggered Self-Replication in Coupled Subsystems.

Science.gov (United States)

Komáromy, Dávid; Tezcan, Meniz; Schaeffer, Gaël; Marić, Ivana; Otto, Sijbren

2017-11-13

In living systems processes like genome duplication and cell division are carefully synchronized through subsystem coupling. If we are to create life de novo, similar control over essential processes such as self-replication need to be developed. Here we report that coupling two dynamic combinatorial subsystems, featuring two separate building blocks, enables effector-mediated control over self-replication. The subsystem based on the first building block shows only self-replication, whereas that based on the second one is solely responsive toward a specific external effector molecule. Mixing the subsystems arrests replication until the effector molecule is added, resulting in the formation of a host-effector complex and the liberation of the building block that subsequently engages in self-replication. The onset, rate and extent of self-replication is controlled by the amount of effector present. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative analysis of the predicted secretomes of Rosaceae scab pathogens Venturia inaequalis and V. pirina reveals expanded effector families and putative determinants of host range.

Science.gov (United States)

Deng, Cecilia H; Plummer, Kim M; Jones, Darcy A B; Mesarich, Carl H; Shiller, Jason; Taranto, Adam P; Robinson, Andrew J; Kastner, Patrick; Hall, Nathan E; Templeton, Matthew D; Bowen, Joanna K

2017-05-02

Fungal plant pathogens belonging to the genus Venturia cause damaging scab diseases of members of the Rosaceae. In terms of economic impact, the most important of these are V. inaequalis, which infects apple, and V. pirina, which is a pathogen of European pear. Given that Venturia fungi colonise the sub-cuticular space without penetrating plant cells, it is assumed that effectors that contribute to virulence and determination of host range will be secreted into this plant-pathogen interface. Thus the predicted secretomes of a range of isolates of Venturia with distinct host-ranges were interrogated to reveal putative proteins involved in virulence and pathogenicity. Genomes of Venturia pirina (one European pear scab isolate) and Venturia inaequalis (three apple scab, and one loquat scab, isolates) were sequenced and the predicted secretomes of each isolate identified. RNA-Seq was conducted on the apple-specific V. inaequalis isolate Vi1 (in vitro and infected apple leaves) to highlight virulence and pathogenicity components of the secretome. Genes encoding over 600 small secreted proteins (candidate effectors) were identified, most of which are novel to Venturia, with expansion of putative effector families a feature of the genus. Numerous genes with similarity to Leptosphaeria maculans AvrLm6 and the Verticillium spp. Ave1 were identified. Candidates for avirulence effectors with cognate resistance genes involved in race-cultivar specificity were identified, as were putative proteins involved in host-species determination. Candidate effectors were found, on average, to be in regions of relatively low gene-density and in closer proximity to repeats (e.g. transposable elements), compared with core eukaryotic genes. Comparative secretomics has revealed candidate effectors from Venturia fungal plant pathogens that attack pome fruit. Effectors that are putative determinants of host range were identified; both those that may be involved in race-cultivar and host

PEG-Immobilized Keratin for Protein Drug Sequestration and pH-Mediated Delivery

Directory of Open Access Journals (Sweden)

Roche C. de Guzman

2016-01-01

Full Text Available Protein drugs like growth factors are promising therapeutics for damaged-tissue repair. Their local delivery often requires biomaterial carriers for achieving the therapeutic dose range while extending efficacy. In this study, polyethylene glycol (PEG and keratin were crosslinked and used as sponge-like scaffolds (KTN-PEG to absorb test proteins with different isoelectric points (pI: albumin (~5, hemoglobin (~7, and lysozyme (~11. The protein release kinetics was influenced by charge at physiological pH 7.4. The keratin network, with pI 5.3, electrostatically attracted lysozyme and repulsed albumin generating the release rate profile: albumin > hemoglobin > lysozyme. However, under acidic conditions (pH 4, all proteins including keratins were positively charged and consequently intermolecular repulsion altered the release hierarchy, now determined by size (MW diffusion: lysozyme (14 kDa > hemoglobin (64 kDa > albumin (66 kDa. Vascular endothelial growth factor C (VEGF-C, with properties comparable to lysozyme, was absorbed into the KTN-PEG scaffold. Endothelial cells cultured on this substrate had significantly larger numbers than on scaffolds without VEGF-C suggesting that the ionically bound and retained growth factor at neutral pH indirectly increased acute cell attachment and viability. PEG and keratin based sequestrations of proteins with basic pIs are therefore a feasible strategy with potential applications for selective biologics delivery.

Controlling chitosan-based encapsulation for protein and vaccine delivery

Science.gov (United States)

Koppolu, Bhanu prasanth; Smith, Sean G.; Ravindranathan, Sruthi; Jayanthi, Srinivas; Kumar, Thallapuranam K.S.; Zaharoff, David A.

2014-01-01

Chitosan-based nano/microencapsulation is under increasing investigation for the delivery of drugs, biologics and vaccines. Despite widespread interest, the literature lacks a defined methodology to control chitosan particle size and drug/protein release kinetics. In this study, the effects of precipitation-coacervation formulation parameters on chitosan particle size, protein encapsulation efficiency and protein release were investigated. Chitosan particle sizes, which ranged from 300 nm to 3 μm, were influenced by chitosan concentration, chitosan molecular weight and addition rate of precipitant salt. The composition of precipitant salt played a significant role in particle formation with upper Hofmeister series salts containing strongly hydrated anions yielding particles with a low polydispersity index (PDI) while weaker anions resulted in aggregated particles with high PDIs. Sonication power had minimal effect on mean particle size, however, it significantly reduced polydispersity. Protein loading efficiencies in chitosan nano/microparticles, which ranged from 14.3% to 99.2%, was inversely related to the hydration strength of precipitant salts, protein molecular weight and directly related to the concentration and molecular weight of chitosan. Protein release rates increased with particle size and were generally inversely related to protein molecular weight. This study demonstrates that chitosan nano/microparticles with high protein loading efficiencies can be engineered with well-defined sizes and controllable release kinetics through manipulation of specific formulation parameters. PMID:24560459

Addressing challenges of heterogeneous tumor treatment through bispecific protein-mediated pretargeted drug delivery.

Science.gov (United States)

Yang, Qi; Parker, Christina L; McCallen, Justin D; Lai, Samuel K

2015-12-28

Tumors are frequently characterized by genomically and phenotypically distinct cancer cell subpopulations within the same tumor or between tumor lesions, a phenomenon termed tumor heterogeneity. These diverse cancer cell populations pose a major challenge to targeted delivery of diagnostic and/or therapeutic agents, as the conventional approach of conjugating individual ligands to nanoparticles is often unable to facilitate intracellular delivery to the full spectrum of cancer cells present in a given tumor lesion or patient. As a result, many cancers are only partially suppressed, leading to eventual tumor regrowth and/or the development of drug-resistant tumors. Pretargeting (multistep targeting) approaches involving the administration of 1) a cocktail of bispecific proteins that can collectively bind to the entirety of a mixed tumor population followed by 2) nanoparticles containing therapeutic and/or diagnostic agents that can bind to the bispecific proteins accumulated on the surface of target cells offer the potential to overcome many of the challenges associated with drug delivery to heterogeneous tumors. Despite its considerable success in improving the efficacy of radioimmunotherapy, the pretargeting strategy remains underexplored for a majority of nanoparticle therapeutic applications, especially for targeted delivery to heterogeneous tumors. In this review, we will present concepts in tumor heterogeneity, the shortcomings of conventional targeted systems, lessons learned from pretargeted radioimmunotherapy, and important considerations for harnessing the pretargeting strategy to improve nanoparticle delivery to heterogeneous tumors. Copyright © 2015 Elsevier B.V. All rights reserved.

The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes.

Science.gov (United States)

Marwaha, Rituraj; Arya, Subhash B; Jagga, Divya; Kaur, Harmeet; Tuli, Amit; Sharma, Mahak

2017-04-03

Endocytic, autophagic, and phagocytic vesicles move on microtubule tracks to fuse with lysosomes. Small GTPases, such as Rab7 and Arl8b, recruit their downstream effectors to mediate this transport and fusion. However, the potential cross talk between these two GTPases is unclear. Here, we show that the Rab7 effector PLEKHM1 simultaneously binds Rab7 and Arl8b, bringing about clustering and fusion of late endosomes and lysosomes. We show that the N-terminal RUN domain of PLEKHM1 is necessary and sufficient for interaction with Arl8b and its subsequent localization to lysosomes. Notably, we also demonstrate that Arl8b mediates recruitment of HOPS complex to PLEKHM1-positive vesicle contact sites. Consequently, Arl8b binding to PLEKHM1 is required for its function in delivery and, therefore, degradation of endocytic and autophagic cargo in lysosomes. Finally, we also show that PLEKHM1 competes with SKIP for Arl8b binding, which dictates lysosome positioning. These findings suggest that Arl8b, along with its effectors, orchestrates lysosomal transport and fusion. © 2017 Marwaha et al.

An effector of the Irish potato famine pathogen antagonizes a host autophagy cargo receptor

Science.gov (United States)

Dagdas, Yasin F; Belhaj, Khaoula; Maqbool, Abbas; Chaparro-Garcia, Angela; Pandey, Pooja; Petre, Benjamin; Tabassum, Nadra; Cruz-Mireles, Neftaly; Hughes, Richard K; Sklenar, Jan; Win, Joe; Menke, Frank; Findlay, Kim; Banfield, Mark J; Kamoun, Sophien; Bozkurt, Tolga O

2016-01-01

Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy-related processes is unknown. Here, we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autophagy cargo receptor Joka2 out of ATG8CL complexes and interferes with Joka2's positive effect on pathogen defense. Thus, a plant pathogen effector has evolved to antagonize a host autophagy cargo receptor to counteract host defenses. DOI: http://dx.doi.org/10.7554/eLife.10856.001 PMID:26765567

Heterologous protein secretion in Lactococcus lactis: a novel antigen delivery system

Directory of Open Access Journals (Sweden)

Langella P.

1999-01-01

Full Text Available Lactic acid bacteria (LAB are Gram-positive bacteria and are generally regarded as safe (GRAS organisms. Therefore, LAB could be used for heterologous protein secretion and they are good potential candidates as antigen delivery vehicles. To develop such live vaccines, a better control of protein secretion is required. We developed an efficient secretion system in the model LAB, Lactococcus lactis. Staphylococcal nuclease (Nuc was used as the reporter protein. We first observed that the quantity of secreted Nuc correlated with the copy number of the cloning vector. The nuc gene was cloned on a high-copy number cloning vector and no perturbation of the metabolism of the secreting strain was observed. Replacement of nuc native promoter by a strong lactococcal one led to a significant increase of nuc expression. Secretion efficiency (SE of Nuc in L. lactis was low, i.e., only 60% of the synthesized Nuc was secreted. Insertion of a synthetic propeptide between the signal peptide and the mature moiety of Nuc increased the SE of Nuc. On the basis of these results, we developed a secretion system and we applied it to the construction of an L. lactis strain which secretes a bovine coronavirus (BCV epitope-protein fusion (BCV-Nuc. BCV-Nuc was recognized by both anti-BCV and anti-Nuc antibodies. Secretion of this antigenic fusion is the first step towards the development of a novel antigen delivery system based on LAB-secreting strains.

In Vitro and In Vivo Investigation of the Potential of Amorphous Microporous Silica as a Protein Delivery Vehicle

Directory of Open Access Journals (Sweden)

Amol Chaudhari

2013-01-01

Full Text Available Delivering growth factors (GFs at bone/implant interface needs to be optimized to achieve faster osseointegration. Amorphous microporous silica (AMS has a potential to be used as a carrier and delivery platform for GFs. In this work, adsorption (loading and release (delivery mechanism of a model protein, bovine serum albumin (BSA, from AMS was investigated in vitro as well as in vivo. In general, strong BSA adsorption to AMS was observed. The interaction was stronger at lower pH owing to favorable electrostatic interaction. In vitro evaluation of BSA release revealed a peculiar release profile, involving a burst release followed by a 6 h period without appreciable BSA release and a further slower release later. Experimental data supporting this observation are discussed. Apart from understanding protein/biomaterial (BSA/AMS interaction, determination of in vivo protein release is an essential aspect of the evaluation of a protein delivery system. In this regard micropositron emission tomography (μ-PET was used in an exploratory experiment to determine in vivo BSA release profile from AMS. Results suggest stronger in vivo retention of BSA when adsorbed on AMS. This study highlights the possible use of AMS as a controlled protein delivery platform which may facilitate osseointegration.

Increase in DNA vaccine efficacy by virosome delivery and co-expression of a cytolytic protein.

Science.gov (United States)

Gargett, Tessa; Grubor-Bauk, Branka; Miller, Darren; Garrod, Tamsin; Yu, Stanley; Wesselingh, Steve; Suhrbier, Andreas; Gowans, Eric J

2014-06-01

The potential of DNA vaccines has not been realised due to suboptimal delivery, poor antigen expression and the lack of localised inflammation, essential for antigen presentation and an effective immune response to the immunogen. Initially, we examined the delivery of a DNA vaccine encoding a model antigen, luciferase (LUC), to the respiratory tract of mice by encapsulation in a virosome. Virosomes that incorporated influenza virus haemagglutinin effectively delivered DNA to cells in the mouse respiratory tract and resulted in antigen expression and systemic and mucosal immune responses to the immunogen after an intranasal (IN) prime/intradermal (ID) boost regimen, whereas a multidose ID regimen only generated systemic immunity. We also examined systemic immune responses to LUC after ID vaccination with a DNA vaccine, which also encoded one of the several cytolytic or toxic proteins. Although the herpes simplex virus thymidine kinase, in the presence of the prodrug, ganciclovir, resulted in cell death, this failed to increase the humoral or cell-mediated immune responses. In contrast, the co-expression of LUC with the rotavirus non-structural protein 4 (NSP4) protein or a mutant form of mouse perforin, proteins which are directly cytolytic, resulted in increased LUC-specific humoral and cell-mediated immunity. On the other hand, co-expression of LUC with diphtheria toxin subunit A or overexpression of perforin or NSP4 resulted in a lower level of immunity. In summary, the efficacy of DNA vaccines can be improved by targeted IN delivery of DNA or by the induction of cell death in vaccine-targeted cells after ID delivery.

Duplications and losses in gene families of rust pathogens highlight putative effectors

Directory of Open Access Journals (Sweden)

Amanda L. Pendleton

2014-06-01

Full Text Available Rust fungi are a group of fungal pathogens that cause some of the world’s most destructive diseases of trees and crops. A shared characteristic among rust fungi is obligate biotrophy, the inability to complete a lifecycle without a host. This dependence on a host species likely affects patterns of gene expansion, contraction, and innovation within rust pathogen genomes. The establishment of disease by biotrophic pathogens is reliant upon effector proteins that are encoded in the fungal genome and secreted from the pathogen into the host’s cell apoplast or within the cells. This study uses a comparative genomic approach to elucidate putative effectors and determine their evolutionary histories. We used OrthoMCL to identify nearly 20,000 gene families in proteomes of sixteen diverse fungal species, which include fifteen basidiomycetes and one ascomycete. We inferred patterns of duplication and loss for each gene family and identified families with distinctive patterns of expansion/contraction associated with the evolution of rust fungal genomes. To recognize potential contributors for the unique features of rust pathogens, we identified families harboring secreted proteins that: i arose or expanded in rust pathogens relative to other fungi, or ii contracted or were lost in rust fungal genomes. While the origin of rust fungi appears to be associated with considerable gene loss, there are many gene duplications associated with each sampled rust fungal genome. We also highlight two putative effector gene families that have expanded in Cqf that we hypothesize have roles in pathogenicity.

Poly(lactic-co-glycolic acid) devices: Production and applications for sustained protein delivery.

Science.gov (United States)

Lee, Parker W; Pokorski, Jonathan K

2018-03-13

Injectable or implantable poly(lactic-co-glycolic acid) (PLGA) devices for the sustained delivery of proteins have been widely studied and utilized to overcome the necessity of repeated administrations for therapeutic proteins due to poor pharmacokinetic profiles of macromolecular therapies. These devices can come in the form of microparticles, implants, or patches depending on the disease state and route of administration. Furthermore, the release rate can be tuned from weeks to months by controlling the polymer composition, geometry of the device, or introducing additives during device fabrication. Slow-release devices have become a very powerful tool for modern medicine. Production of these devices has initially focused on emulsion-based methods, relying on phase separation to encapsulate proteins within polymeric microparticles. Process parameters and the effect of additives have been thoroughly researched to ensure protein stability during device manufacturing and to control the release profile. Continuous fluidic production methods have also been utilized to create protein-laden PLGA devices through spray drying and electrospray production. Thermal processing of PLGA with solid proteins is an emerging production method that allows for continuous, high-throughput manufacturing of PLGA/protein devices. Overall, polymeric materials for protein delivery remain an emerging field of research for the creation of single administration treatments for a wide variety of disease. This review describes, in detail, methods to make PLGA devices, comparing traditional emulsion-based methods to emerging methods to fabricate protein-laden devices. This article is categorized under: Biology-Inspired Nanomaterials > Protein and Virus-Based Structures Implantable Materials and Surgical Technologies > Nanomaterials and Implants Biology-Inspired Nanomaterials > Peptide-Based Structures. © 2018 Wiley Periodicals, Inc.

Biodegradable Magnetic Silica@Iron Oxide Nanovectors with Ultra-Large Mesopores for High Protein Loading, Magnetothermal Release, and Delivery

KAUST Repository

Omar, Haneen

2016-11-29

The delivery of large cargos of diameter above 15 nm for biomedical applications has proved challenging since it requires biocompatible, stably-loaded, and biodegradable nanomaterials. In this study, we describe the design of biodegradable silica-iron oxide hybrid nanovectors with large mesopores for large protein delivery in cancer cells. The mesopores of the nanomaterials spanned from 20 to 60 nm in diameter and post-functionalization allowed the electrostatic immobilization of large proteins (e.g. mTFP-Ferritin, ~ 534 kDa). Half of the content of the nanovectors was based with iron oxide nanophases which allowed the rapid biodegradation of the carrier in fetal bovine serum and a magnetic responsiveness. The nanovectors released large protein cargos in aqueous solution under acidic pH or magnetic stimuli. The delivery of large proteins was then autonomously achieved in cancer cells via the silica-iron oxide nanovectors, which is thus a promising for biomedical applications.

Specific Hypersensitive Response–Associated Recognition of New Apoplastic Effectors from Cladosporium fulvum in Wild Tomato

NARCIS (Netherlands)

Mesarich, Carl H.; Ӧkmen, Bilal; Rovenich, Hanna; Griffiths, Scott A.; Wang, Changchun; Karimi Jashni, Mansoor; Mihajlovski, Aleksandar; Collemare, Jérôme; Hunziker, Lukas; Deng, Cecilia H.; Burgt, Van Der Ate; Beenen, Henriek G.; Templeton, Matthew D.; Bradshaw, Rosie E.; Wit, De Pierre J.G.M.

2018-01-01

Tomato leaf mold disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes

Role of Soluble Innate Effector Molecules in Pulmonary Defense against Fungal Pathogens

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Soledad R. Ordonez

2017-10-01

Full Text Available Fungal infections of the lung are life-threatening but rarely occur in healthy, immunocompetent individuals, indicating efficient clearance by pulmonary defense mechanisms. Upon inhalation, fungi will first encounter the airway surface liquid which contains several soluble effector molecules that form the first barrier of defense against fungal infections. These include host defense peptides, like LL-37 and defensins that can neutralize fungi by direct killing of the pathogen, and collectins, such as surfactant protein A and D, that can aggregate fungi and stimulate phagocytosis. In addition, these molecules have immunomodulatory activities which can aid in fungal clearance from the lung. However, existing observations are based on in vitro studies which do not reflect the complexity of the lung and its airway surface liquid. Ionic strength, pH, and the presence of mucus can have strong detrimental effects on antifungal activity, while the potential synergistic interplay between soluble effector molecules is largely unknown. In this review, we describe the current knowledge on soluble effector molecules that contribute to antifungal activity, the importance of environmental factors and discuss the future directions required to understand the innate antifungal defense in the lung.

Role of Soluble Innate Effector Molecules in Pulmonary Defense against Fungal Pathogens

Science.gov (United States)

Ordonez, Soledad R.; Veldhuizen, Edwin J. A.; van Eijk, Martin; Haagsman, Henk P.

2017-01-01

Fungal infections of the lung are life-threatening but rarely occur in healthy, immunocompetent individuals, indicating efficient clearance by pulmonary defense mechanisms. Upon inhalation, fungi will first encounter the airway surface liquid which contains several soluble effector molecules that form the first barrier of defense against fungal infections. These include host defense peptides, like LL-37 and defensins that can neutralize fungi by direct killing of the pathogen, and collectins, such as surfactant protein A and D, that can aggregate fungi and stimulate phagocytosis. In addition, these molecules have immunomodulatory activities which can aid in fungal clearance from the lung. However, existing observations are based on in vitro studies which do not reflect the complexity of the lung and its airway surface liquid. Ionic strength, pH, and the presence of mucus can have strong detrimental effects on antifungal activity, while the potential synergistic interplay between soluble effector molecules is largely unknown. In this review, we describe the current knowledge on soluble effector molecules that contribute to antifungal activity, the importance of environmental factors and discuss the future directions required to understand the innate antifungal defense in the lung. PMID:29163395

Identification and characterization of a 29-kilodalton protein from Mycobacterium tuberculosis culture filtrate recognized by mouse memory effector cells

DEFF Research Database (Denmark)

Rosenkrands, I; Rasmussen, P.B.; Carnio, M

1998-01-01

Culture filtrate proteins from Mycobacterium tuberculosis induce protective immunity in various animal models of tuberculosis. Two molecular mass regions (6 to 10 kDa and 24 to 36 kDa) of short-term culture filtrate are preferentially recognized by Th1 cells in animal models as well as by patients...... the antigen 85 complex was selected. The 29-kDa antigen (CFP29) was purified from M. tuberculosis short-term culture filtrate by thiophilic adsorption chromatography, anion-exchange chromatography, and gel filtration, In its native form, CFP29 forms a polymer with a high molecular mass. CFP29 was mapped......, and they both elicited the release of high levels of gamma interferon from mouse memory effector cells isolated during the recall of protective immunity to tuberculosis. Interspecies analysis by immunoblotting and PCR demonstrated that CFP29 is widely distributed in mycobacterial species....

Tissue specific heterogeneity in effector immune cell response

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Saba eTufail

2013-08-01

Full Text Available Post pathogen invasion, migration of effector T-cell subsets to specific tissue locations is of prime importance for generation of robust immune response. Effector T cells are imprinted with distinct ‘homing codes’ (adhesion molecules and chemokine receptors during activation which regulate their targeted trafficking to specific tissues. Internal cues in the lymph node microenvironment along with external stimuli from food (vitamin A and sunlight (vitamin D3 prime dendritic cells, imprinting them to play centrestage in the induction of tissue tropism in effector T cells. B cells as well, in a manner similar to effector T cells, exhibit tissue tropic migration. In this review, we have focused on the factors regulating the generation and migration of effector T cells to various tissues alongwith giving an overview of tissue tropism in B cells.

Delivery of peptide and protein drugs over the blood-brain barrier.

Science.gov (United States)

Brasnjevic, Ivona; Steinbusch, Harry W M; Schmitz, Christoph; Martinez-Martinez, Pilar

2009-04-01

Peptide and protein (P/P) drugs have been identified as showing great promises for the treatment of various neurodegenerative diseases. A major challenge in this regard, however, is the delivery of P/P drugs over the blood-brain barrier (BBB). Intense research over the last 25 years has enabled a better understanding of the cellular and molecular transport mechanisms at the BBB, and several strategies for enhanced P/P drug delivery over the BBB have been developed and tested in preclinical and clinical-experimental research. Among them, technology-based approaches (comprising functionalized nanocarriers and liposomes) and pharmacological strategies (such as the use of carrier systems and chimeric peptide technology) appear to be the most promising ones. This review combines a comprehensive overview on the current understanding of the transport mechanisms at the BBB with promising selected strategies published so far that can be applied to facilitate enhanced P/P drug delivery over the BBB.

New players in the same old game: a system level in silico study to predict type III secretion system and effector proteins in bacterial genomes reveals common themes in T3SS mediated pathogenesis.

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Sadarangani, Vineet; Datta, Sunando; Arunachalam, Manonmani

2013-07-26

Type III secretion system (T3SS) plays an important role in virulence or symbiosis of many pathogenic or symbiotic bacteria [CHM 2:291-294, 2007; Physiology (Bethesda) 20:326-339, 2005]. T3SS acts like a tunnel between a bacterium and its host through which the bacterium injects 'effector' proteins into the latter [Nature 444:567-573, 2006; COSB 18:258-266, 2008]. The effectors spatially and temporally modify the host signalling pathways [FEMS Microbiol Rev 35:1100-1125, 2011; Cell Host Microbe5:571-579, 2009]. In spite its crucial role in host-pathogen interaction, the study of T3SS and the associated effectors has been limited to a few bacteria [Cell Microbiol 13:1858-1869, 2011; Nat Rev Microbiol 6:11-16, 2008; Mol Microbiol 80:1420-1438, 2011]. Before one set out to perform systematic experimental studies on an unknown set of bacteria it would be beneficial to identify the potential candidates by developing an in silico screening algorithm. A system level study would also be advantageous over traditional laboratory methods to extract an overriding theme for host-pathogen interaction, if any, from the vast resources of data generated by sequencing multiple bacterial genomes. We have developed an in silico protocol in which the most conserved set of T3SS proteins was used as the query against the entire bacterial database with increasingly stringent search parameters. It enabled us to identify several uncharacterized T3SS positive bacteria. We adopted a similar strategy to predict the presence of the already known effectors in the newly identified T3SS positive bacteria. The huge resources of biochemical data [FEMS Microbiol Rev 35:1100-1125, 2011; Cell Host Microbe 5:571-579, 2009; BMC Bioinformatics 7(11):S4, 2010] on the T3SS effectors enabled us to search for the common theme in T3SS mediated pathogenesis. We identified few cellular signalling networks in the host, which are manipulated by most of the T3SS containing pathogens. We went on to look for

Effector-Triggered Immunity Determines Host Genotype-Specific Incompatibility in Legume-Rhizobium Symbiosis.

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Yasuda, Michiko; Miwa, Hiroki; Masuda, Sachiko; Takebayashi, Yumiko; Sakakibara, Hitoshi; Okazaki, Shin

2016-08-01

Symbiosis between legumes and rhizobia leads to the formation of N2-fixing root nodules. In soybean, several host genes, referred to as Rj genes, control nodulation. Soybean cultivars carrying the Rj4 gene restrict nodulation by specific rhizobia such as Bradyrhizobium elkanii We previously reported that the restriction of nodulation was caused by B. elkanii possessing a functional type III secretion system (T3SS), which is known for its delivery of virulence factors by pathogenic bacteria. In the present study, we investigated the molecular basis for the T3SS-dependent nodulation restriction in Rj4 soybean. Inoculation tests revealed that soybean cultivar BARC-2 (Rj4/Rj4) restricted nodulation by B. elkanii USDA61, whereas its nearly isogenic line BARC-3 (rj4/rj4) formed nitrogen-fixing nodules with the same strain. Root-hair curling and infection threads were not observed in the roots of BARC-2 inoculated with USDA61, indicating that Rj4 blocked B. elkanii infection in the early stages. Accumulation of H2O2 and salicylic acid (SA) was observed in the roots of BARC-2 inoculated with USDA61. Transcriptome analyses revealed that inoculation of USDA61, but not its T3SS mutant in BARC-2, induced defense-related genes, including those coding for hypersensitive-induced responsive protein, which act in effector-triggered immunity (ETI) in Arabidopsis. These findings suggest that B. elkanii T3SS triggers the SA-mediated ETI-type response in Rj4 soybean, which consequently blocks symbiotic interactions. This study revealed a common molecular mechanism underlying both plant-pathogen and plant-symbiont interactions, and suggests that establishment of a root nodule symbiosis requires the evasion or suppression of plant immune responses triggered by rhizobial effectors. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Special-purpose multifingered robotic end-effectors

International Nuclear Information System (INIS)

Crowder, R.M.

1990-01-01

A number of advanced multifingered robotic end-effectors have been developed recently in which the finger joints are powered from external actuators. Although this gives dexterous performance, there are considerable problems with power transmission, due to the use of flexible tendons between the external actuators and the individual finger joints. If a multifingered robotic end-effector is to be operated in a confined space, local actuation of the fingers needs to be fully considered, even if there is a reduction in hand dexterity over that of an externally mounted actuator system. The University of Southampton has developed a number of end-effectors that incorporate integral finger actuators and mechanisms, two examples of which are discussed in this paper

Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

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Elena Elizabeth Bagley

2014-06-01

Full Text Available Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1 currents in periaqueductal gray (PAG neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1

Identification and characterization of LysM effectors in Penicillium expansum.

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Levin, Elena; Ballester, Ana Rosa; Raphael, Ginat; Feigenberg, Oleg; Liu, Yongsheng; Norelli, John; Gonzalez-Candelas, Luis; Ma, Jing; Dardick, Christopher; Wisniewski, Michael; Droby, Samir

2017-01-01

P. expansum is regarded as one of the most important postharvest rots of apple fruit and is also of great concern to fruit processing industries. Elucidating the pathogenicity mechanism of this pathogen is of utmost importance for the development of effective and safe management strategies. Although, many studies on modification of the host environment by the pathogen were done, its interactions with fruit during the early stages of infection and the virulence factors that mediate pathogenicity have not been fully defined. Effectors carrying LysM domain have been identified in numerous pathogenic fungi and their role in the first stages of infection has been established. In this study, we identified 18 LysM genes in the P. expansum genome. Amino acid sequence analysis indicated that P. expansum LysM proteins belong to a clade of fungal-specific LysM. Eleven of the discovered LysM genes were found to have secretory pathway signal peptide, among them, 4 (PeLysM1 PeLysM2, PeLysM3 and PeLysM4) were found to be highly expressed during the infection and development of decay of apple fruit. Effect of targeted deletion of the four putative PeLysM effectors on the growth and pathogenicity was studied. Possible interactions of PeLysM with host proteins was investigated using the yeast-two-hybrid system.

Identification and characterization of LysM effectors in Penicillium expansum.

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Elena Levin

Full Text Available P. expansum is regarded as one of the most important postharvest rots of apple fruit and is also of great concern to fruit processing industries. Elucidating the pathogenicity mechanism of this pathogen is of utmost importance for the development of effective and safe management strategies. Although, many studies on modification of the host environment by the pathogen were done, its interactions with fruit during the early stages of infection and the virulence factors that mediate pathogenicity have not been fully defined. Effectors carrying LysM domain have been identified in numerous pathogenic fungi and their role in the first stages of infection has been established. In this study, we identified 18 LysM genes in the P. expansum genome. Amino acid sequence analysis indicated that P. expansum LysM proteins belong to a clade of fungal-specific LysM. Eleven of the discovered LysM genes were found to have secretory pathway signal peptide, among them, 4 (PeLysM1 PeLysM2, PeLysM3 and PeLysM4 were found to be highly expressed during the infection and development of decay of apple fruit. Effect of targeted deletion of the four putative PeLysM effectors on the growth and pathogenicity was studied. Possible interactions of PeLysM with host proteins was investigated using the yeast-two-hybrid system.

MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects.

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Naor, Adit; Panas, Michael W; Marino, Nicole; Coffey, Michael J; Tonkin, Christopher J; Boothroyd, John C

2018-04-03

The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma , we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔ myr1 , and RHΔ asp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, "hidden" responses arising in RHΔ myr1 - but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite's ability to co-opt host cell functions. IMPORTANCE Toxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1

Integrating Protein Engineering and Bioorthogonal Click Conjugation for Extracellular Vesicle Modulation and Intracellular Delivery.

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Ming Wang

Full Text Available Exosomes are small, cell-secreted vesicles that transfer proteins and genetic information between cells. This intercellular transmission regulates many physiological and pathological processes. Therefore, exosomes have emerged as novel biomarkers for disease diagnosis and as nanocarriers for drug delivery. Here, we report an easy-to-adapt and highly versatile methodology to modulate exosome composition and conjugate exosomes for intracellular delivery. Our strategy combines the metabolic labeling of newly synthesized proteins or glycan/glycoproteins of exosome-secreting cells with active azides and bioorthogonal click conjugation to modify and functionalize the exosomes. The azide-integrated can be conjugated to a variety of small molecules and proteins and can efficiently deliver conjugates into cells. The metabolic engineering of exosomes diversifies the chemistry of exosomes and expands the functions that can be introduced into exosomes, providing novel, powerful tools to study the roles of exosomes in biology and expand the biomedical potential of exosomes.

Hacker Within! Ehrlichia chaffeensis Effector Driven Phagocyte Reprogramming Strategy

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Taslima Taher Lina

2016-05-01

Full Text Available Ehrlichia chaffeensis is a small, gram negative, obligately intracellular bacterium that preferentially infects mononuclear phagocytes. It is the etiologic agent of human monocytotropic ehrlichiosis (HME, an emerging life-threatening tick-borne zoonosis. Mechanisms by which E. chaffeensis establishes intracellular infection, and avoids host defenses are not well understood, but involve functionally relevant host-pathogen interactions associated with tandem and ankyrin repeat effector proteins. In this review, we discuss the recent advances in our understanding of the molecular and cellular mechanisms that underlie Ehrlichia host cellular reprogramming strategies that enable intracellular survival.

The Salmonella type III effector SspH2 specifically exploits the NLR co-chaperone activity of SGT1 to subvert immunity.

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Amit P Bhavsar

Full Text Available To further its pathogenesis, S. Typhimurium delivers effector proteins into host cells, including the novel E3 ubiquitin ligase (NEL effector SspH2. Using model systems in a cross-kingdom approach we gained further insight into the molecular function of this effector. Here, we show that SspH2 modulates innate immunity in both mammalian and plant cells. In mammalian cell culture, SspH2 significantly enhanced Nod1-mediated IL-8 secretion when transiently expressed or bacterially delivered. In addition, SspH2 also enhanced an Rx-dependent hypersensitive response in planta. In both of these nucleotide-binding leucine rich repeat receptor (NLR model systems, SspH2-mediated phenotypes required its catalytic E3 ubiquitin ligase activity and interaction with the conserved host protein SGT1. SGT1 has an essential cell cycle function and an additional function as an NLR co-chaperone in animal and plant cells. Interaction between SspH2 and SGT1 was restricted to SGT1 proteins that have NLR co-chaperone function and accordingly, SspH2 did not affect SGT1 cell cycle functions. Mechanistic studies revealed that SspH2 interacted with, and ubiquitinated Nod1 and could induce Nod1 activity in an agonist-independent manner if catalytically active. Interestingly, SspH2 in vitro ubiquitination activity and protein stability were enhanced by SGT1. Overall, this work adds to our understanding of the sophisticated mechanisms used by bacterial effectors to co-opt host pathways by demonstrating that SspH2 can subvert immune responses by selectively exploiting the functions of a conserved host co-chaperone.

Activating human genes with zinc finger proteins, transcription activator-like effectors and CRISPR/Cas9 for gene therapy and regenerative medicine.

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Gersbach, Charles A; Perez-Pinera, Pablo

2014-08-01

New technologies have recently been developed to control the expression of human genes in their native genomic context by engineering synthetic transcription factors that can be targeted to any DNA sequence. The ability to precisely regulate any gene as it occurs naturally in the genome provides a means to address a variety of diseases and disorders. This approach also circumvents some of the traditional challenges of gene therapy. In this editorial, we review the technologies that have enabled targeted human gene activation, including the engineering of transcription factors based on zinc finger proteins, transcription activator-like effectors and the CRISPR/Cas9 system. Additionally, we highlight examples in which these methods have been developed for therapeutic applications and discuss challenges and opportunities.

Comparative reactivity of human IgE to cynomolgus monkey and human effector cells and effects on IgE effector cell potency

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Saul, Louise; Saul, Louise; Josephs, Debra H; Josephs, Debra H; Cutler, Keith; Cutler, Keith; Bradwell, Andrew; Bradwell, Andrew; Karagiannis, Panagiotis; Karagiannis, Panagiotis; Selkirk, Chris; Selkirk, Chris; Gould, Hannah J; Gould, Hannah J; Jones, Paul; Jones, Paul; Spicer, James F; Spicer, James F; Karagiannis, Sophia N; Karagiannis, Sophia N

2014-01-01

Background: Due to genetic similarities with humans, primates of the macaque genus such as the cynomolgus monkey are often chosen as models for toxicology studies of antibody therapies. IgE therapeutics in development depend upon engagement with the FcεRI and FcεRII receptors on immune effector cells for their function. Only limited knowledge of the primate IgE immune system is available to inform the choice of models for mechanistic and safety evaluations.   Methods: The recognition of human IgE by peripheral blood lymphocytes from cynomolgus monkey and man was compared. We used effector cells from each species in ex vivo affinity, dose-response, antibody-receptor dissociation and potency assays. Results: We report cross-reactivity of human IgE Fc with cynomolgus monkey cells, and comparable binding kinetics to peripheral blood lymphocytes from both species. In competition and dissociation assays, however, human IgE dissociated faster from cynomolgus monkey compared with human effector cells. Differences in association and dissociation kinetics were reflected in effector cell potency assays of IgE-mediated target cell killing, with higher concentrations of human IgE needed to elicit effector response in the cynomolgus monkey system. Additionally, human IgE binding on immune effector cells yielded significantly different cytokine release profiles in each species. Conclusion: These data suggest that human IgE binds with different characteristics to human and cynomolgus monkey IgE effector cells. This is likely to affect the potency of IgE effector functions in these two species, and so has relevance for the selection of biologically-relevant model systems when designing pre-clinical toxicology and functional studies. PMID:24492303

Global impact of Salmonella type III secretion effector SteA on host cells

International Nuclear Information System (INIS)

Cardenal-Muñoz, Elena; Gutiérrez, Gabriel; Ramos-Morales, Francisco

2014-01-01

Highlights: • We analyzed HeLa cells transcriptome in response to Salmonella SteA. • Significant differential expression was detected for 58 human genes. • They are involved in ECM organization and regulation of some signaling pathways. • Cell death, cell adhesion and cell migration were decreased in SteA-expressing cells. • These results contribute to understand the role of SteA during infections. - Abstract: Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell–cell adhesion and migration

Global impact of Salmonella type III secretion effector SteA on host cells

Energy Technology Data Exchange (ETDEWEB)

Cardenal-Muñoz, Elena; Gutiérrez, Gabriel; Ramos-Morales, Francisco

2014-07-11

Highlights: • We analyzed HeLa cells transcriptome in response to Salmonella SteA. • Significant differential expression was detected for 58 human genes. • They are involved in ECM organization and regulation of some signaling pathways. • Cell death, cell adhesion and cell migration were decreased in SteA-expressing cells. • These results contribute to understand the role of SteA during infections. - Abstract: Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell–cell adhesion and migration.

BSA Nanoparticles for siRNA Delivery: Coating Effects on Nanoparticle Properties, Plasma Protein Adsorption, and In Vitro siRNA Delivery

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Haran Yogasundaram

2012-01-01

Full Text Available Developing vehicles for the delivery of therapeutic molecules, like siRNA, is an area of active research. Nanoparticles composed of bovine serum albumin, stabilized via the adsorption of poly-L-lysine (PLL, have been shown to be potentially inert drug-delivery vehicles. With the primary goal of reducing nonspecific protein adsorption, the effect of using comb-type structures of poly(ethylene glycol (1 kDa, PEG units conjugated to PLL (4.2 and 24 kDa on BSA-NP properties, apparent siRNA release rate, cell viability, and cell uptake were evaluated. PEGylated PLL coatings resulted in NPs with ζ-potentials close to neutral. Incubation with platelet-poor plasma showed the composition of the adsorbed proteome was similar for all systems. siRNA was effectively encapsulated and released in a sustained manner from all NPs. With 4.2 kDa PLL, cellular uptake was not affected by the presence of PEG, but PEG coating inhibited uptake with 24 kDa PLL NPs. Moreover, 24 kDa PLL systems were cytotoxic and this cytotoxicity was diminished upon PEG incorporation. The overall results identified a BSA-NP coating structure that provided effective siRNA encapsulation while reducing ζ-potential, protein adsorption, and cytotoxicity, necessary attributes for in vivo application of drug-delivery vehicles.

Matricellular proteins in drug delivery: Therapeutic targets, active agents, and therapeutic localization.

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Sawyer, Andrew J; Kyriakides, Themis R

2016-02-01

Extracellular matrix is composed of a complex array of molecules that together provide structural and functional support to cells. These properties are mainly mediated by the activity of collagenous and elastic fibers, proteoglycans, and proteins such as fibronectin and laminin. ECM composition is tissue-specific and could include matricellular proteins whose primary role is to modulate cell-matrix interactions. In adults, matricellular proteins are primarily expressed during injury, inflammation and disease. Particularly, they are closely associated with the progression and prognosis of cardiovascular and fibrotic diseases, and cancer. This review aims to provide an overview of the potential use of matricellular proteins in drug delivery including the generation of therapeutic agents based on the properties and structures of these proteins as well as their utility as biomarkers for specific diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural insight into gene transcriptional regulation and effector binding by the Lrp/AsnC family

NARCIS (Netherlands)

Thaw, P.; Sedelnikova, S.E.; Muranova, T.; Wiese, S.; Ayora, S.; Alonso, J.C.; Brinkman, A.B.; Akerboom, A.P.; Oost, van der J.; Rafferty, J.B.

2006-01-01

The Lrp/AsnC family of transcriptional regulatory proteins is found in both archaea and bacteria. Members of the family influence cellular metabolism in both a global (Lrp) and specific (AsnC) manner, often in response to exogenous amino acid effectors. In the present study we have determined both

Biodegradable "Smart" Polyphosphazenes with Intrinsic Multifunctionality as Intracellular Protein Delivery Vehicles.

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Martinez, Andre P; Qamar, Bareera; Fuerst, Thomas R; Muro, Silvia; Andrianov, Alexander K

2017-06-12

A series of biodegradable drug delivery polymers with intrinsic multifunctionality have been designed and synthesized utilizing a polyphosphazene macromolecular engineering approach. Novel water-soluble polymers, which contain carboxylic acid and pyrrolidone moieties attached to an inorganic phosphorus-nitrogen backbone, were characterized by a suite of physicochemical methods to confirm their structure, composition, and molecular sizes. All synthesized polyphosphazenes displayed composition-dependent hydrolytic degradability in aqueous solutions at neutral pH. Their formulations were stable at lower temperatures, potentially indicating adequate shelf life, but were characterized by accelerated degradation kinetics at elevated temperatures, including 37 °C. It was found that synthesized polyphosphazenes are capable of environmentally triggered self-assembly to produce nanoparticles with narrow polydispersity in the size range of 150-700 nm. Protein loading capacity of copolymers has been validated via their ability to noncovalently bind avidin without altering biological functionality. Acid-induced membrane-disruptive activity of polyphosphazenes has been established with an onset corresponding to the endosomal pH range and being dependent on polymer composition. The synthesized polyphosphazenes facilitated cell-surface interactions followed by time-dependent, vesicular-mediated, and saturable internalization of a model protein cargo into cancer cells, demonstrating the potential for intracellular delivery.

Evaluation of Aerosol Delivery of Nanosuspension for Pre-clinical Pulmonary Drug Delivery

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Chiang Po-Chang

2009-01-01

Full Text Available Abstract Asthma and chronic obstructive pulmonary disease (COPD are pulmonary diseases that are characterized by inflammatory cell infiltration, cytokine production, and airway hyper-reactivity. Most of the effector cells responsible for these pathologies reside in the lungs. One of the most direct ways to deliver drugs to the target cells is via the trachea. In a pre-clinical setting, this can be achieved via intratracheal (IT, intranasal (IN, or aerosol delivery in the desired animal model. In this study, we pioneered the aerosol delivery of a nanosuspension formulation in a rodent model. The efficiency of different dosing techniques and formulations to target the lungs were compared, and fluticasone was used as the model compound. For the aerosol particle size determination, a ten-stage cascade impactor was used. The mass median aerodynamic diameter (MMAD was calculated based on the percent cumulative accumulation at each stage. Formulations with different particle size of fluticasone were made for evaluation. The compatibility of regular fluticasone suspension and nanosuspension for aerosol delivery was also investigated. The in vivo studies were conducted on mice with optimized setting. It was found that the aerosol delivery of fluticasone with nanosuspension was as efficient as intranasal (IN dosing, and was able to achieve dose dependent lung deposition.

Powdery mildew fungal effector candidates share N-terminal Y/F/WxC-motif

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Emmersen Jeppe

2010-05-01

Full Text Available Abstract Background Powdery mildew and rust fungi are widespread, serious pathogens that depend on developing haustoria in the living plant cells. Haustoria are separated from the host cytoplasm by a plant cell-derived extrahaustorial membrane. They secrete effector proteins, some of which are subsequently transferred across this membrane to the plant cell to suppress defense. Results In a cDNA library from barley epidermis containing powdery mildew haustoria, two-thirds of the sequenced ESTs were fungal and represented ~3,000 genes. Many of the most highly expressed genes encoded small proteins with N-terminal signal peptides. While these proteins are novel and poorly related, they do share a three-amino acid motif, which we named "Y/F/WxC", in the N-terminal of the mature proteins. The first amino acid of this motif is aromatic: tyrosine, phenylalanine or tryptophan, and the last is always cysteine. In total, we identified 107 such proteins, for which the ESTs represent 19% of the fungal clones in our library, suggesting fundamental roles in haustoria function. While overall sequence similarity between the powdery mildew Y/F/WxC-proteins is low, they do have a highly similar exon-intron structure, suggesting they have a common origin. Interestingly, searches of public fungal genome and EST databases revealed that haustoria-producing rust fungi also encode large numbers of novel, short proteins with signal peptides and the Y/F/WxC-motif. No significant numbers of such proteins were identified from genome and EST sequences from either fungi which do not produce haustoria or from haustoria-producing Oomycetes. Conclusion In total, we identified 107, 178 and 57 such Y/F/WxC-proteins from the barley powdery mildew, the wheat stem rust and the wheat leaf rust fungi, respectively. All together, our findings suggest the Y/F/WxC-proteins to be a new class of effectors from haustoria-producing pathogenic fungi.

Characterisation of cell death inducing Phytophthora capsici CRN effectors suggests diverse activities in the host nucleus

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Remco eStam

2013-10-01

Full Text Available Plant-Microbe interactions are complex associations that feature recognition of Pathogen Associated Molecular Patterns by the plant immune system and dampening of subsequent responses by pathogen encoded secreted effectors. With large effector repertoires now identified in a range of sequenced microbial genomes, much attention centres on understanding their roles in immunity or disease. These studies not only allow identification of pathogen virulence factors and strategies, they also provide an important molecular toolset suited for studying immunity in plants. The Phytophthora intracellular effector repertoire encodes a large class of proteins that translocate into host cells and exclusively target the host nucleus. Recent functional studies have implicated the CRN protein family as an important class of diverse effectors that target distinct subnuclear compartments and modify host cell signalling. Here, we characterised three necrosis inducing CRNs and show that there are differences in the levels of cell death. We show that only expression of CRN20_624 has an additive effect on PAMP induced cell death but not AVR3a induced ETI. Given their distinctive phenotypes, we assessed localisation of each CRN with a set of nuclear markers and found clear differences in CRN subnuclear distribution patterns. These assays also revealed that expression of CRN83_152 leads to a distinct change in nuclear chromatin organisation, suggesting a distinct series of events that leads to cell death upon over-expression. Taken together, our results suggest diverse functions carried by CRN C-termini, which can be exploited to identify novel processes that take place in the host nucleus and are required for immunity or susceptibility.

Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

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Liu, Minqiang; Li, Wu; Xiang, Xiaohong; Xie, Jianping

2015-07-01

Tuberculosis remains a serious human public health concern. The coevolution between its pathogen Mycobacterium tuberculosis and human host complicated the way to prevent and cure TB. Apoptosis plays subtle role in this interaction. The pathogen endeavors to manipulate the apoptosis via diverse effectors targeting key signaling nodes. In this paper, we summarized the effectors pathogen used to subvert the apoptosis, such as LpqH, ESAT-6/CFP-10, LAMs. The interplay between different forms of cell deaths, such as apoptosis, autophagy, necrosis, is also discussed with a focus on the modes of action of effectors, and implications for better TB control.

Golgi bypass for local delivery of axonal proteins, fact or fiction?

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González, Carolina; Cornejo, Víctor Hugo; Couve, Andrés

2018-04-06

Although translation of cytosolic proteins is well described in axons, much less is known about the synthesis, processing and trafficking of transmembrane and secreted proteins. A canonical rough endoplasmic reticulum or a stacked Golgi apparatus has not been detected in axons, generating doubts about the functionality of a local route. However, axons contain mRNAs for membrane and secreted proteins, translation factors, ribosomal components, smooth endoplasmic reticulum and post-endoplasmic reticulum elements that may contribute to local biosynthesis and plasma membrane delivery. Here we consider the evidence supporting a local secretory system in axons. We discuss exocytic elements and examples of autonomous axonal trafficking that impact development and maintenance. We also examine whether unconventional post-endoplasmic reticulum pathways may replace the canonical Golgi apparatus. Copyright © 2018. Published by Elsevier Ltd.

Controlled release and intracellular protein delivery from mesoporous silica nanoparticles.

Science.gov (United States)

Deodhar, Gauri V; Adams, Marisa L; Trewyn, Brian G

2017-01-01

Protein therapeutics are promising candidates for disease treatment due to their high specificity and minimal adverse side effects; however, targeted protein delivery to specific sites has proven challenging. Mesoporous silica nanoparticles (MSN) have demonstrated to be ideal candidates for this application, given their high loading capacity, biocompatibility, and ability to protect host molecules from degradation. These materials exhibit tunable pore sizes, shapes and volumes, and surfaces which can be easily functionalized. This serves to control the movement of molecules in and out of the pores, thus entrapping guest molecules until a specific stimulus triggers release. In this review, we will cover the benefits of using MSN as protein therapeutic carriers, demonstrating that there is great diversity in the ways MSN can be used to service proteins. Methods for controlling the physical dimensions of pores via synthetic conditions, applications of therapeutic protein loaded MSN materials in cancer therapies, delivering protein loaded MSN materials to plant cells using biolistic methods, and common stimuli-responsive functionalities will be discussed. New and exciting strategies for controlled release and manipulation of proteins are also covered in this review. While research in this area has advanced substantially, we conclude this review with future challenges to be tackled by the scientific community. Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Strategies for the photo-control of endogenous protein activity.

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Brechun, Katherine E; Arndt, Katja M; Woolley, G Andrew

2017-08-01

Photo-controlled or 'optogenetic' effectors interfacing with endogenous protein machinery allow the roles of endogenous proteins to be probed. There are two main approaches being used to develop optogenetic effectors: (i) caging strategies using photo-controlled conformational changes, and (ii) protein relocalization strategies using photo-controlled protein-protein interactions. Numerous specific examples of these approaches have been reported and efforts to develop general methods for photo-control of endogenous proteins are a current focus. The development of improved screening and selection methods for photo-switchable proteins would advance the field. Copyright © 2016 Elsevier Ltd. All rights reserved.

Actin re-organization induced by Chlamydia trachomatis serovar D--evidence for a critical role of the effector protein CT166 targeting Rac.

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Jessica Thalmann

Full Text Available The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs. CGTs contain an essential DXD-motif and mono-glucosylate GTP-binding proteins of the Rho/Ras families, the master regulators of the actin cytoskeleton. CT166 is preformed in elementary bodies of C. trachomatis D and is detected in the host-cell shortly after infection. Infection with high MOI of C. trachomatis serovar D containing the CT166 ORF induces actin re-organization resulting in cell rounding and a decreased cell diameter. A comparable phenotype was observed in HeLa cells treated with the Rho-GTPase-glucosylating Toxin B from Clostridium difficile (TcdB or HeLa cells ectopically expressing CT166. CT166 with a mutated DXD-motif (CT166-mut exhibited almost unchanged actin dynamics, suggesting that CT166-induced actin re-organization depends on the glucosyltransferase motif of CT166. The cytotoxic necrotizing factor 1 (CNF1 from E. coli deamidates and thereby activates Rho-GTPases and transiently protects them against TcdB-induced glucosylation. CNF1-treated cells were found to be protected from TcdB- and CT166-induced actin re-organization. CNF1 treatment as well as ectopic expression of non-glucosylable Rac1-G12V, but not RhoA-G14A, reverted CT166-induced actin re-organization, suggesting that CT166-induced actin re-organization depends on the glucosylation of Rac1. In accordance, over-expression of CT166-mut diminished TcdB induced cell rounding, suggesting shared substrates. Cell rounding induced by high MOI infection with C. trachomatis D was reduced in cells expressing CT166-mut or Rac1-G12V, and in CNF1 treated cells. These observations indicate that the cytopathic effect of C. trachomatis D is mediated by CT166 induced Rac1 glucosylation

Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Munc13-4 effector mechanism

International Nuclear Information System (INIS)

Saxena, Sunil K.; Horiuchi, Hisanori; Fukuda, Mitsunori

2006-01-01

Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays with two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins

Macrophages are critical effectors of antibody therapies for cancer.

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Weiskopf, Kipp; Weissman, Irving L

2015-01-01

Macrophages are innate immune cells that derive from circulating monocytes, reside in all tissues, and participate in many states of pathology. Macrophages play a dichotomous role in cancer, where they promote tumor growth but also serve as critical immune effectors of therapeutic antibodies. Macrophages express all classes of Fcγ receptors, and they have immense potential to destroy tumors via the process of antibody-dependent phagocytosis. A number of studies have demonstrated that macrophage phagocytosis is a major mechanism of action of many antibodies approved to treat cancer. Consequently, a number of approaches to augment macrophage responses to therapeutic antibodies are under investigation, including the exploration of new targets and development of antibodies with enhanced functions. For example, the interaction of CD47 with signal-regulatory protein α (SIRPα) serves as a myeloid-specific immune checkpoint that limits the response of macrophages to antibody therapies, and CD47-blocking agents overcome this barrier to augment phagocytosis. The response of macrophages to antibody therapies can also be enhanced with engineered Fc variants, bispecific antibodies, or antibody-drug conjugates. Macrophages have demonstrated success as effectors of cancer immunotherapy, and further investigation will unlock their full potential for the benefit of patients.

Transcriptional Dynamics Driving MAMP-Triggered Immunity and Pathogen Effector-Mediated Immunosuppression in Arabidopsis Leaves Following Infection with Pseudomonas syringae pv tomato DC3000.

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Lewis, Laura A; Polanski, Krzysztof; de Torres-Zabala, Marta; Jayaraman, Siddharth; Bowden, Laura; Moore, Jonathan; Penfold, Christopher A; Jenkins, Dafyd J; Hill, Claire; Baxter, Laura; Kulasekaran, Satish; Truman, William; Littlejohn, George; Prusinska, Justyna; Mead, Andrew; Steinbrenner, Jens; Hickman, Richard; Rand, David; Wild, David L; Ott, Sascha; Buchanan-Wollaston, Vicky; Smirnoff, Nick; Beynon, Jim; Denby, Katherine; Grant, Murray

2015-11-01

Transcriptional reprogramming is integral to effective plant defense. Pathogen effectors act transcriptionally and posttranscriptionally to suppress defense responses. A major challenge to understanding disease and defense responses is discriminating between transcriptional reprogramming associated with microbial-associated molecular pattern (MAMP)-triggered immunity (MTI) and that orchestrated by effectors. A high-resolution time course of genome-wide expression changes following challenge with Pseudomonas syringae pv tomato DC3000 and the nonpathogenic mutant strain DC3000hrpA- allowed us to establish causal links between the activities of pathogen effectors and suppression of MTI and infer with high confidence a range of processes specifically targeted by effectors. Analysis of this information-rich data set with a range of computational tools provided insights into the earliest transcriptional events triggered by effector delivery, regulatory mechanisms recruited, and biological processes targeted. We show that the majority of genes contributing to disease or defense are induced within 6 h postinfection, significantly before pathogen multiplication. Suppression of chloroplast-associated genes is a rapid MAMP-triggered defense response, and suppression of genes involved in chromatin assembly and induction of ubiquitin-related genes coincide with pathogen-induced abscisic acid accumulation. Specific combinations of promoter motifs are engaged in fine-tuning the MTI response and active transcriptional suppression at specific promoter configurations by P. syringae. © 2015 American Society of Plant Biologists. All rights reserved.

Protein cages and synthetic polymers: a fruitful symbiosis for drug delivery applications, bionanotechnology and materials science.

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Rother, Martin; Nussbaumer, Martin G; Renggli, Kasper; Bruns, Nico

2016-11-07

Protein cages are hollow protein nanoparticles, such as viral capsids, virus-like particles, ferritin, heat-shock proteins and chaperonins. They have well-defined capsule-like structures with a monodisperse size. Their protein subunits can be modified by genetic engineering at predetermined positions, allowing for example site-selective introduction of attachment points for functional groups, catalysts or targeting ligands on their outer surface, in their interior and between subunits. Therefore, protein cages have been extensively explored as functional entities in bionanotechnology, as drug-delivery or gene-delivery vehicles, as nanoreactors or as templates for the synthesis of organic and inorganic nanomaterials. The scope of functionalities and applications of protein cages can be significantly broadened if they are combined with synthetic polymers on their surface or within their interior. For example, PEGylation reduces the immunogenicity of protein cage-based delivery systems and active targeting ligands can be attached via polymer chains to favour their accumulation in diseased tissue. Polymers within protein cages offer the possibility of increasing the loading density of drug molecules, nucleic acids, magnetic resonance imaging contrast agents or catalysts. Moreover, the interaction of protein cages and polymers can be used to modulate the size and shape of some viral capsids to generate structures that do not occur with native viruses. Another possibility is to use the interior of polymer cages as a confined reaction space for polymerization reactions such as atom transfer radical polymerization or rhodium-catalysed polymerization of phenylacetylene. The protein nanoreactors facilitate a higher degree of control over polymer synthesis. This review will summarize the hybrid structures that have been synthesized by polymerizing from protein cage-bound initiators, by conjugating polymers to protein cages, by embedding protein cages into bulk polymeric

A Novel Meloidogyne incognita Effector Misp12 Suppresses Plant Defense Response at Latter Stages of Nematode Parasitism

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Xie, Jialian; Li, Shaojun; Mo, Chenmi; Wang, Gaofeng; Xiao, Xueqiong; Xiao, Yannong

2016-01-01

Secreted effectors in plant root-knot nematodes (RKNs, or Meloidogyne spp.) play key roles in their parasite processes. Currently identified effectors mainly focus on the early stage of the nematode parasitism. There are only a few reports describing effectors that function in the latter stage. In this study, we identified a potential RKN effector gene, Misp12, that functioned during the latter stage of parasitism. Misp12 was unique in the Meloidogyne spp., and highly conserved in Meloidogyne incognita. It encoded a secretory protein that specifically expressed in the dorsal esophageal gland, and highly up-regulated during the female stages. Transient expression of Misp12-GUS-GFP in onion epidermal cell showed that Misp12 was localized in cytoplast. In addition, in planta RNA interference targeting Misp12 suppressed the expression of Misp12 in nematodes and attenuated parasitic ability of M. incognita. Furthermore, up-regulation of jasmonic acid (JA) and salicylic acid (SA) pathway defense-related genes in the virus-induced silencing of Misp12 plants, and down-regulation of SA pathway defense-related genes in Misp12-expressing plants indicated the gene might be associated with the suppression of the plant defense response. These results demonstrated that the novel nematode effector Misp12 played a critical role at latter parasitism of M. incognita. PMID:27446188

The Toolbox for Uncovering the Functions of Legionella Dot/Icm Type IVb Secretion System Effectors: Current State and Future Directions

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Gunnar N. Schroeder

2018-01-01

Full Text Available The defective in organelle trafficking/intracellular multiplication (Dot/Icm Type IVb secretion system (T4SS is the essential virulence factor for the intracellular life style and pathogenicity of Legionella species. Screens demonstrated that an individual L. pneumophila strain can use the Dot/Icm T4SS to translocate an unprecedented number of more than 300 proteins into host cells, where these, so called Icm/Dot-translocated substrates (IDTS or effectors, manipulate host cell functions to the benefit of the bacteria. Bioinformatic analysis of the pan-genus genome predicts at least 608 orthologous groups of putative effectors. Deciphering the function of these effectors is key to understanding Legionella pathogenesis; however, the analysis is challenging. Substantial functional redundancy renders classical, phenotypic screening of single gene deletion mutants mostly ineffective. Here, I review experimental approaches that were successfully used to identify, validate and functionally characterize T4SS effectors and highlight new methods, which promise to facilitate unlocking the secrets of Legionella's extraordinary weapons arsenal.

Peptide and low molecular weight proteins based kidney targeted drug delivery systems.

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Xu, Pengfei; Zhang, Hailiang; Dang, Ruili; Jiang, Pei

2018-05-30

Renal disease is a worldwide public health problem, and unfortunately, the therapeutic index of regular drugs is limited. Thus, it is a great need to develop effective treatment strategies. Among the reported strategies, kidney-targeted drug delivery system is a promising method to increase renal efficacy and reduce extra-renal toxicity. In recent years, working as vehicles for targeted drug delivery, low molecular weight proteins (LMWP) and peptide have received immense attention due to their many advantages, such as selective accumulation in kidney, high drug loading capability, control over routes of biodegradation, convenience in modification at the amino terminus, and good biocompatibility. In this review, we describe the current LMWP and peptide carriers for kidney targeted drug delivery systems. In addition, we discuss different linking strategies between carriers and drugs. Furthermore, we briefly outline the current status and attempt to give an outlook on the further study. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Expression, Delivery and Function of Insecticidal Proteins Expressed by Recombinant Baculoviruses

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Kroemer, Jeremy A.; Bonning, Bryony C.; Harrison, Robert L.

2015-01-01

Since the development of methods for inserting and expressing genes in baculoviruses, a line of research has focused on developing recombinant baculoviruses that express insecticidal peptides and proteins. These recombinant viruses have been engineered with the goal of improving their pesticidal potential by shortening the time required for infection to kill or incapacitate insect pests and reducing the quantity of crop damage as a consequence. A wide variety of neurotoxic peptides, proteins that regulate insect physiology, degradative enzymes, and other potentially insecticidal proteins have been evaluated for their capacity to reduce the survival time of baculovirus-infected lepidopteran host larvae. Researchers have investigated the factors involved in the efficient expression and delivery of baculovirus-encoded insecticidal peptides and proteins, with much effort dedicated to identifying ideal promoters for driving transcription and signal peptides that mediate secretion of the expressed target protein. Other factors, particularly translational efficiency of transcripts derived from recombinant insecticidal genes and post-translational folding and processing of insecticidal proteins, remain relatively unexplored. The discovery of RNA interference as a gene-specific regulation mechanism offers a new approach for improvement of baculovirus biopesticidal efficacy through genetic modification. PMID:25609310

Identification of proteins similar to AvrE type III effector proteins from ...

African Journals Online (AJOL)

Stephen Opiyo

GSE22274), and AraCyc databases, we highlighted 16 protein candidates from Arabidopsidis genome .... projection method similar to principal component analysis (PCA) .... RIN4 RIN4 (RPM1 INTERACTING PROTEIN 4); protein binding.

Genome-Wide Analysis of Type VI System Clusters and Effectors in Burkholderia Species

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Thao Thi Nguyen

2018-02-01

Full Text Available Type VI secretion system (T6SS has been discovered in a variety of gram-negative bacteria as a versatile weapon to stimulate the killing of eukaryotic cells or prokaryotic competitors. Type VI secretion effectors (T6SEs are well known as key virulence factors for important pathogenic bacteria. In many Burkholderia species, T6SS has evolved as the most complicated secretion pathway with distinguished types to translocate diverse T6SEs, suggesting their essential roles in this genus. Here we attempted to detect and characterize T6SSs and potential T6SEs in target genomes of plant-associated and environmental Burkholderia species based on computational analyses. In total, 66 potential functional T6SS clusters were found in 30 target Burkholderia bacterial genomes, of which 33% possess three or four clusters. The core proteins in each cluster were specified and phylogenetic trees of three components (i.e., TssC, TssD, TssL were constructed to elucidate the relationship among the identified T6SS clusters. Next, we identified 322 potential T6SEs in the target genomes based on homology searches and explored the important domains conserved in effector candidates. In addition, using the screening approach based on the profile hidden Markov model (pHMM of T6SEs that possess markers for type VI effectors (MIX motif (MIX T6SEs, 57 revealed proteins that were not included in training datasets were recognized as novel MIX T6SE candidates from the Burkholderia species. This approach could be useful to identify potential T6SEs from other bacterial genomes.

Three New Pierce's Disease Pathogenicity Effectors Identified Using Xylella fastidiosa Biocontrol Strain EB92-1.

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Zhang, Shujian; Chakrabarty, Pranjib K; Fleites, Laura A; Rayside, Patricia A; Hopkins, Donald L; Gabriel, Dean W

2015-01-01

Xylella fastidiosa (X. fastidiosa) infects a wide range of plant hosts and causes economically serious diseases, including Pierce's Disease (PD) of grapevines. X. fastidiosa biocontrol strain EB92-1 was isolated from elderberry and is infectious and persistent in grapevines but causes only very slight symptoms under ideal conditions. The draft genome of EB92-1 revealed that it appeared to be missing genes encoding 10 potential PD pathogenicity effectors found in Temecula1. Subsequent PCR and sequencing analyses confirmed that EB92-1 was missing the following predicted effectors found in Temecula1: two type II secreted enzymes, including a lipase (LipA; PD1703) and a serine protease (PD0956); two identical genes encoding proteins similar to Zonula occludens toxins (Zot; PD0915 and PD0928), and at least one relatively short, hemagglutinin-like protein (PD0986). Leaves of tobacco and citrus inoculated with cell-free, crude protein extracts of E. coli BL21(DE3) overexpressing PD1703 exhibited a hypersensitive response (HR) in less than 24 hours. When cloned into shuttle vector pBBR1MCS-5, PD1703 conferred strong secreted lipase activity to Xanthomonas citri, E. coli and X. fastidiosa EB92-1 in plate assays. EB92-1/PD1703 transformants also showed significantly increased disease symptoms on grapevines, characteristic of PD. Genes predicted to encode PD0928 (Zot) and a PD0986 (hemagglutinin) were also cloned into pBBR1MCS-5 and moved into EB92-1; both transformants also showed significantly increased symptoms on V. vinifera vines, characteristic of PD. Together, these results reveal that PD effectors include at least a lipase, two Zot-like toxins and a possibly redundant hemagglutinin, none of which are necessary for parasitic survival of X. fastidiosa populations in grapevines or elderberry.

Three New Pierce's Disease Pathogenicity Effectors Identified Using Xylella fastidiosa Biocontrol Strain EB92-1.

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Shujian Zhang

Full Text Available Xylella fastidiosa (X. fastidiosa infects a wide range of plant hosts and causes economically serious diseases, including Pierce's Disease (PD of grapevines. X. fastidiosa biocontrol strain EB92-1 was isolated from elderberry and is infectious and persistent in grapevines but causes only very slight symptoms under ideal conditions. The draft genome of EB92-1 revealed that it appeared to be missing genes encoding 10 potential PD pathogenicity effectors found in Temecula1. Subsequent PCR and sequencing analyses confirmed that EB92-1 was missing the following predicted effectors found in Temecula1: two type II secreted enzymes, including a lipase (LipA; PD1703 and a serine protease (PD0956; two identical genes encoding proteins similar to Zonula occludens toxins (Zot; PD0915 and PD0928, and at least one relatively short, hemagglutinin-like protein (PD0986. Leaves of tobacco and citrus inoculated with cell-free, crude protein extracts of E. coli BL21(DE3 overexpressing PD1703 exhibited a hypersensitive response (HR in less than 24 hours. When cloned into shuttle vector pBBR1MCS-5, PD1703 conferred strong secreted lipase activity to Xanthomonas citri, E. coli and X. fastidiosa EB92-1 in plate assays. EB92-1/PD1703 transformants also showed significantly increased disease symptoms on grapevines, characteristic of PD. Genes predicted to encode PD0928 (Zot and a PD0986 (hemagglutinin were also cloned into pBBR1MCS-5 and moved into EB92-1; both transformants also showed significantly increased symptoms on V. vinifera vines, characteristic of PD. Together, these results reveal that PD effectors include at least a lipase, two Zot-like toxins and a possibly redundant hemagglutinin, none of which are necessary for parasitic survival of X. fastidiosa populations in grapevines or elderberry.

USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level.

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Daniel Pouly

Full Text Available The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2, whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output effector. We report here that Usp2 is a clock output effector related to bodily Ca2+ homeostasis, a feature that is conserved across evolution. Drosophila with a whole-body knockdown of the orthologue of Usp2, CG14619 (dUsp2-kd, predominantly die during pupation but are rescued by dietary Ca2+ supplementation. Usp2-KO mice show hyperabsorption of dietary Ca2+ in small intestine, likely due to strong overexpression of the membrane scaffold protein NHERF4, a regulator of the Ca2+ channel TRPV6 mediating dietary Ca2+ uptake. In this tissue, USP2-45 is found in membrane fractions and negatively regulates NHERF4 protein abundance in a rhythmic manner at the protein level. In clock mutant animals (Cry1/Cry2-dKO, rhythmic USP2-45 expression is lost, as well as the one of NHERF4, confirming the inverse relationship between USP2-45 and NHERF4 protein levels. Finally, USP2-45 interacts in vitro with NHERF4 and endogenous Clathrin Heavy Chain. Taken together these data prompt us to define USP2-45 as the first clock output effector acting at the post-translational level at cell membranes and possibly regulating membrane permeability of Ca2+.

A salivary EF-hand calcium-binding protein of the brown planthopper Nilaparvata lugens functions as an effector for defense responses in rice

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Ye, Wenfeng; Yu, Haixin; Jian, Yukun; Zeng, Jiamei; Ji, Rui; Chen, Hongdan; Lou, Yonggen

2017-01-01

The brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), a major pest of rice in Asia, is able to successfully puncture sieve tubes in rice with its piercing stylet and then to ingest phloem sap. How BPH manages to continuously feed on rice remains unclear. Here, we cloned the gene NlSEF1, which is highly expressed in the salivary glands of BPH. The NlSEF1 protein has EF-hand Ca2+-binding activity and can be secreted into rice plants when BPH feed. Infestation of rice by BPH nymphs whose NlSEF1 was knocked down elicited higher levels of Ca2+ and H2O2 but not jasmonic acid, jasmonoyl-isoleucine (JA-Ile) and SA in rice than did infestation by control nymphs; Consistently, wounding plus the recombination protein NlSEF1 suppressed the production of H2O2 in rice. Bioassays revealed that NlSEF1-knockdown BPH nymphs had a higher mortality rate and lower feeding capacity on rice than control nymphs. These results indicate that the salivary protein in BPH, NlSEF1, functions as an effector and plays important roles in interactions between BPH and rice by mediating the plant’s defense responses. PMID:28098179

Transcriptome analysis of the fungal pathogen Fusarium oxysporum f. sp. medicaginis during colonisation of resistant and susceptible Medicago truncatula hosts identifies differential pathogenicity profiles and novel candidate effectors.

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Thatcher, Louise F; Williams, Angela H; Garg, Gagan; Buck, Sally-Anne G; Singh, Karam B

2016-11-03

Pathogenic members of the Fusarium oxysporum species complex are responsible for vascular wilt disease on many important crops including legumes, where they can be one of the most destructive disease causing necrotrophic fungi. We previously developed a model legume-infecting pathosystem based on the reference legume Medicago truncatula and a pathogenic F. oxysporum forma specialis (f. sp.) medicaginis (Fom). To dissect the molecular pathogenicity arsenal used by this root-infecting pathogen, we sequenced its transcriptome during infection of a susceptible and resistant host accession. High coverage RNA-Seq of Fom infected root samples harvested from susceptible (DZA315) or resistant (A17) M. truncatula seedlings at early or later stages of infection (2 or 7 days post infection (dpi)) and from vegetative (in vitro) samples facilitated the identification of unique and overlapping sets of in planta differentially expressed genes. This included enrichment, particularly in DZA315 in planta up-regulated datasets, for proteins associated with sugar, protein and plant cell wall metabolism, membrane transport, nutrient uptake and oxidative processes. Genes encoding effector-like proteins were identified, including homologues of the F. oxysporum f. sp. lycopersici Secreted In Xylem (SIX) proteins, and several novel candidate effectors based on predicted secretion, small protein size and high in-planta induced expression. The majority of the effector candidates contain no known protein domains but do share high similarity to predicted proteins predominantly from other F. oxysporum ff. spp. as well as other Fusaria (F. solani, F. fujikori, F. verticilloides, F. graminearum and F. pseudograminearum), and from another wilt pathogen of the same class, a Verticillium species. Overall, this suggests these novel effector candidates may play important roles in Fusaria and wilt pathogen virulence. Combining high coverage in planta RNA-Seq with knowledge of fungal pathogenicity

Denatured protein-coated docetaxel nanoparticles: Alterable drug state and cytosolic delivery.

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Zhang, Li; Xiao, Qingqing; Wang, Yiran; Zhang, Chenshuang; He, Wei; Yin, Lifang

2017-05-15

Many lead compounds have a low solubility in water, which substantially hinders their clinical application. Nanosuspensions have been considered a promising strategy for the delivery of water-insoluble drugs. Here, denatured soy protein isolate (SPI)-coated docetaxel nanosuspensions (DTX-NS) were developed using an anti-solvent precipitation-ultrasonication method to improve the water-solubility of DTX, thus improving its intracellular delivery. DTX-NS, with a diameter of 150-250nm and drug-loading up to 18.18%, were successfully prepared by coating drug particles with SPI. Interestingly, the drug state of DTX-NS was alterable. Amorphous drug nanoparticles were obtained at low drug-loading, whereas at a high drug-loading, the DTX-NS drug was mainly present in the crystalline state. Moreover, DTX-NS could be internalized at high levels by cancer cells and enter the cytosol by lysosomal escape, enhancing cell cytotoxicity and apoptosis compared with free DTX. Taken together, denatured SPI has a strong stabilization effect on nanosuspensions, and the drug state in SPI-coated nanosuspensions is alterable by changing the drug-loading. Moreover, DTX-NS could achieve cytosolic delivery, generating enhanced cell cytotoxicity against cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida.

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Chavarría, Max; Durante-Rodríguez, Gonzalo; Krell, Tino; Santiago, César; Brezovsky, Jan; Damborsky, Jiri; de Lorenzo, Víctor

2014-01-01

Fructose-1-phosphate (F1P) is the preferred effector of the catabolite repressor/activator (Cra) protein of the soil bacterium Pseudomonas putida but its ability to bind other metabolic intermediates in vivo is unclear. The Cra protein of this microorganism (Cra(PP)) was submitted to mobility shift assays with target DNA sequences (the PfruB promoter) and candidate effectors fructose-1,6-bisphosphate (FBP), glucose 6-phosphate (G6P), and fructose-6-phosphate (F6P). 1 mM F1P was sufficient to release most of the Cra protein from its operators but more than 10 mM of FBP or G6P was required to free the same complex. However, isothermal titration microcalorimetry failed to expose any specific interaction between Cra(PP) and FBP or G6P. To solve this paradox, transcriptional activity of a PfruB-lacZ fusion was measured in wild-type and ΔfruB cells growing on substrates that change the intracellular concentrations of F1P and FBP. The data indicated that PfruB activity was stimulated by fructose but not by glucose or succinate. This suggested that Cra(PP) represses expression in vivo of the cognate fruBKA operon in a fashion dependent just on F1P, ruling out any other physiological effector. Molecular docking and dynamic simulations of the Cra-agonist interaction indicated that both metabolites can bind the repressor, but the breach in the relative affinity of Cra(PP) for F1P vs FBP is three orders of magnitude larger than the equivalent distance in the Escherichia coli protein. This assigns the Cra protein of P. putida the sole role of transducing the presence of fructose in the medium into a variety of direct and indirect physiological responses.

The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1.

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Zhaohui Liu

2012-01-01

Full Text Available The wheat pathogen Stagonospora nodorum produces multiple necrotrophic effectors (also called host-selective toxins that promote disease by interacting with corresponding host sensitivity gene products. SnTox1 was the first necrotrophic effector identified in S. nodorum, and was shown to induce necrosis on wheat lines carrying Snn1. Here, we report the molecular cloning and validation of SnTox1 as well as the preliminary characterization of the mechanism underlying the SnTox1-Snn1 interaction which leads to susceptibility. SnTox1 was identified using bioinformatics tools and verified by heterologous expression in Pichia pastoris. SnTox1 encodes a 117 amino acid protein with the first 17 amino acids predicted as a signal peptide, and strikingly, the mature protein contains 16 cysteine residues, a common feature for some avirulence effectors. The transformation of SnTox1 into an avirulent S. nodorum isolate was sufficient to make the strain pathogenic. Additionally, the deletion of SnTox1 in virulent isolates rendered the SnTox1 mutated strains avirulent on the Snn1 differential wheat line. SnTox1 was present in 85% of a global collection of S. nodorum isolates. We identified a total of 11 protein isoforms and found evidence for strong diversifying selection operating on SnTox1. The SnTox1-Snn1 interaction results in an oxidative burst, DNA laddering, and pathogenesis related (PR gene expression, all hallmarks of a defense response. In the absence of light, the development of SnTox1-induced necrosis and disease symptoms were completely blocked. By comparing the infection processes of a GFP-tagged avirulent isolate and the same isolate transformed with SnTox1, we conclude that SnTox1 may play a critical role during fungal penetration. This research further demonstrates that necrotrophic fungal pathogens utilize small effector proteins to exploit plant resistance pathways for their colonization, which provides important insights into the molecular

The Genome Biology of Effector Gene Evolution in Filamentous Plant Pathogens.

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Sánchez-Vallet, Andrea; Fouché, Simone; Fudal, Isabelle; Hartmann, Fanny E; Soyer, Jessica L; Tellier, Aurélien; Croll, Daniel

2018-05-16

Filamentous pathogens, including fungi and oomycetes, pose major threats to global food security. Crop pathogens cause damage by secreting effectors that manipulate the host to the pathogen's advantage. Genes encoding such effectors are among the most rapidly evolving genes in pathogen genomes. Here, we review how the major characteristics of the emergence, function, and regulation of effector genes are tightly linked to the genomic compartments where these genes are located in pathogen genomes. The presence of repetitive elements in these compartments is associated with elevated rates of point mutations and sequence rearrangements with a major impact on effector diversification. The expression of many effectors converges on an epigenetic control mediated by the presence of repetitive elements. Population genomics analyses showed that rapidly evolving pathogens show high rates of turnover at effector loci and display a mosaic in effector presence-absence polymorphism among strains. We conclude that effective pathogen containment strategies require a thorough understanding of the effector genome biology and the pathogen's potential for rapid adaptation. Expected final online publication date for the Annual Review of Phytopathology Volume 56 is August 25, 2018. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen.

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Lu, Xunli; Kracher, Barbara; Saur, Isabel M L; Bauer, Saskia; Ellwood, Simon R; Wise, Roger; Yaeno, Takashi; Maekawa, Takaki; Schulze-Lefert, Paul

2016-10-18

Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVR a gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVR a genes and identified AVR a1 and AVR a13 , encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVR a1 and AVR a13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVR A1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVR A1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVR A1 Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation.

SseK3 Is a Salmonella Effector That Binds TRIM32 and Modulates the Host's NF-κB Signalling Activity.

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Zhe Yang

Full Text Available Salmonella Typhimurium employs an array of type III secretion system effectors that facilitate intracellular survival and replication during infection. The Salmonella effector SseK3 was originally identified due to amino acid sequence similarity with NleB; an effector secreted by EPEC/EHEC that possesses N-acetylglucoasmine (GlcNAc transferase activity and modifies death domain containing proteins to block extrinsic apoptosis. In this study, immunoprecipitation of SseK3 defined a novel molecular interaction between SseK3 and the host protein, TRIM32, an E3 ubiquitin ligase. The conserved DxD motif within SseK3, which is essential for the GlcNAc transferase activity of NleB, was required for TRIM32 binding and for the capacity of SseK3 to suppress TNF-stimulated activation of NF-κB pathway. However, we did not detect GlcNAc modification of TRIM32 by SseK3, nor did the SseK3-TRIM32 interaction impact on TRIM32 ubiquitination that is associated with its activation. In addition, lack of sseK3 in Salmonella had no effect on production of the NF-κB dependent cytokine, IL-8, in HeLa cells even though TRIM32 knockdown suppressed TNF-induced NF-κB activity. Ectopically expressed SseK3 partially co-localises with TRIM32 at the trans-Golgi network, but SseK3 is not recruited to Salmonella induced vacuoles or Salmonella induced filaments during Salmonella infection. Our study has identified a novel effector-host protein interaction and suggests that SseK3 may influence NF-κB activity. However, the lack of GlcNAc modification of TRIM32 suggests that SseK3 has further, as yet unidentified, host targets.

The role of type III effectors from Xanthomonas axonopodis pv. manihotis in virulence and suppression of plant immunity.

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Medina, Cesar Augusto; Reyes, Paola Andrea; Trujillo, Cesar Augusto; Gonzalez, Juan Luis; Bejarano, David Alejandro; Montenegro, Nathaly Andrea; Jacobs, Jonathan M; Joe, Anna; Restrepo, Silvia; Alfano, James R; Bernal, Adriana

2018-03-01

Xanthomonas axonopodis pv. manihotis (Xam) causes cassava bacterial blight, the most important bacterial disease of cassava. Xam, like other Xanthomonas species, requires type III effectors (T3Es) for maximal virulence. Xam strain CIO151 possesses 17 predicted T3Es belonging to the Xanthomonas outer protein (Xop) class. This work aimed to characterize nine Xop effectors present in Xam CIO151 for their role in virulence and modulation of plant immunity. Our findings demonstrate the importance of XopZ, XopX, XopAO1 and AvrBs2 for full virulence, as well as a redundant function in virulence between XopN and XopQ in susceptible cassava plants. We tested their role in pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) using heterologous systems. AvrBs2, XopR and XopAO1 are capable of suppressing PTI. ETI suppression activity was only detected for XopE4 and XopAO1. These results demonstrate the overall importance and diversity in functions of major virulence effectors AvrBs2 and XopAO1 in Xam during cassava infection. © 2017 BSPP AND JOHN WILEY & SONS LTD.

Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

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Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

2011-09-27

The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

Effective delivery of recombinant proteins to rod photoreceptors via lipid nanovesicles

Energy Technology Data Exchange (ETDEWEB)

Asteriti, Sabrina [Dept. of Translational Research, University of Pisa, Pisa (Italy); Dal Cortivo, Giuditta [Dept. of Life Sciences and Reproduction, University of Verona, Strada Le Grazie 8, Verona (Italy); Pontelli, Valeria [Dept. of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, Verona (Italy); Cangiano, Lorenzo [Dept. of Translational Research, University of Pisa, Pisa (Italy); Buffelli, Mario, E-mail: mario.buffelli@univr.it [Dept. of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, Verona (Italy); Center for Biomedical Computing, University of Verona, Strada le Grazie 8, 37134 Verona (Italy); Dell’Orco, Daniele, E-mail: daniele.dellorco@univr.it [Dept. of Life Sciences and Reproduction, University of Verona, Strada Le Grazie 8, Verona (Italy); Center for Biomedical Computing, University of Verona, Strada le Grazie 8, 37134 Verona (Italy)

2015-06-12

The potential of liposomes to deliver functional proteins in retinal photoreceptors and modulate their physiological response was investigated by two experimental approaches. First, we treated isolated mouse retinas with liposomes encapsulating either recoverin, an important endogenous protein operating in visual phototransduction, or antibodies against recoverin. We then intravitrally injected in vivo liposomes encapsulating either rhodamin B or recoverin and we investigated the distribution in retina sections by confocal microscopy. The content of liposomes was found to be released in higher amount in the photoreceptor layer than in the other regions of the retina and the functional effects of the release were in line with the current model of phototransduction. Our study sets the basis for quantitative investigations aimed at assessing the potential of intraocular protein delivery via biocompatible nanovesicles, with promising implications for the treatment of retinal diseases affecting the photoreceptor layer. - Highlights: • Recombinant proteins encapsulated in nano-sized liposomes injected intravitreally reach retinal photoreceptors. • The phototransduction cascade in rods is modulated by the liposome content. • Mathematical modeling predicts the alteration of the photoresponses following liposome fusion.

Principles and applications of TAL effectors for plant physiology and metabolism.

Science.gov (United States)

Bogdanove, Adam J

2014-06-01

Recent advances in DNA targeting allow unprecedented control over gene function and expression. Targeting based on TAL effectors is arguably the most promising for systems biology and metabolic engineering. Multiple, orthogonal TAL-effector reagents of different types can be used in the same cell. Furthermore, variation in base preferences of the individual structural repeats that make up the TAL effector DNA recognition domain makes targeting stringency tunable. Realized applications range from genome editing to epigenome modification to targeted gene regulation to chromatin labeling and capture. The principles that govern TAL effector DNA recognition make TAL effectors well suited for applications relevant to plant physiology and metabolism. TAL effector targeting has merits that are distinct from those of the RNA-based DNA targeting CRISPR/Cas9 system. Copyright © 2014 Elsevier Ltd. All rights reserved.

Water-in-Oil Microemulsions for Protein Delivery: Loading Optimization and Stability.

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Perinelli, Diego R; Cespi, Marco; Pucciarelli, Stefania; Vincenzetti, Silvia; Casettari, Luca; Lam, Jenny K W; Logrippo, Serena; Canala, Elisa; Soliman, Mahmoud E; Bonacucina, Giulia; Palmieri, Giovanni F

2017-01-01

Microemulsions are attractive delivery systems for therapeutic proteins and peptides due to their ability to enhance bioavailability. Although different proteins and peptides have been successfully delivered through such ternary systems, no information can be found about protein loading and the formulation stability when such microemulsions are prepared with pharmaceuticallyapproved oils and surfactants. The aim of this work was to optimise a ternary system consisting of water/ ethyl oleate/Span® 80-Tween® 80 and to determine its protein loading capacity and stability, using bovine serum albumin (BSA) as a model of biomolecule. The optimization was carried out using a Central Composite Design and all the prepared formulations were characterised through dynamic light scattering, rheology, optical and polarized microscopy. Subsequently, the maximum loading capacity was determined and the stability of the final microemulsion with the highest content of protein was followed over six months. To investigate the structural features of the protein, BSA was recovered from the microemulsion and analysed through fluorescence spectroscopy. After incorporation of the protein in the microemulsion, a decrease of its aqueous solubility was observed. However, the formulation remained stable over six months and the native-like state of the recovered protein was demonstrated by fluorescence spectroscopy Conclusion: This study demonstrated the feasibility of preparing microemulsions with the highest content of protein and their long-term stability. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Oxysterols and Their Cellular Effectors

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Eija Nissilä

2012-02-01

Full Text Available Oxysterols are oxidized 27-carbon cholesterol derivatives or by-products of cholesterol biosynthesis, with a spectrum of biologic activities. Several oxysterols have cytotoxic and pro-apoptotic activities, the ability to interfere with the lateral domain organization, and packing of membrane lipids. These properties may account for their suggested roles in the pathology of diseases such as atherosclerosis, age-onset macular degeneration and Alzheimer’s disease. Oxysterols also have the capacity to induce inflammatory responses and play roles in cell differentiation processes. The functions of oxysterols as intermediates in the synthesis of bile acids and steroid hormones, and as readily transportable forms of sterol, are well established. Furthermore, their actions as endogenous regulators of gene expression in lipid metabolism via liver X receptors and the Insig (insulin-induced gene proteins have been investigated in detail. The cytoplasmic oxysterol-binding protein (OSBP homologues form a group of oxysterol/cholesterol sensors that has recently attracted a lot of attention. However, their mode of action is, as yet, poorly understood. Retinoic acid receptor-related orphan receptors (ROR α and γ, and Epstein-Barr virus induced gene 2 (EBI2 have been identified as novel oxysterol receptors, revealing new physiologic oxysterol effector mechanisms in development, metabolism, and immunity, and evoking enhanced interest in these compounds in the field of biomedicine.

Nodulation outer proteins: double-edged swords of symbiotic rhizobia.

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Staehelin, Christian; Krishnan, Hari B

2015-09-15

Rhizobia are nitrogen-fixing bacteria that establish a nodule symbiosis with legumes. Nodule formation depends on signals and surface determinants produced by both symbiotic partners. Among them, rhizobial Nops (nodulation outer proteins) play a crucial symbiotic role in many strain-host combinations. Nops are defined as proteins secreted via a rhizobial T3SS (type III secretion system). Functional T3SSs have been characterized in many rhizobial strains. Nops have been identified using various genetic, biochemical, proteomic, genomic and experimental approaches. Certain Nops represent extracellular components of the T3SS, which are visible in electron micrographs as bacterial surface appendages called T3 (type III) pili. Other Nops are T3 effector proteins that can be translocated into plant cells. Rhizobial T3 effectors manipulate cellular processes in host cells to suppress plant defence responses against rhizobia and to promote symbiosis-related processes. Accordingly, mutant strains deficient in synthesis or secretion of T3 effectors show reduced symbiotic properties on certain host plants. On the other hand, direct or indirect recognition of T3 effectors by plant cells expressing specific R (resistance) proteins can result in effector triggered defence responses that negatively affect rhizobial infection. Hence Nops are double-edged swords that may promote establishment of symbiosis with one legume (symbiotic factors) and impair symbiotic processes when bacteria are inoculated on another legume species (asymbiotic factors). In the present review, we provide an overview of our current understanding of Nops. We summarize their symbiotic effects, their biochemical properties and their possible modes of action. Finally, we discuss future perspectives in the field of T3 effector research. © 2015 Authors; published by Portland Press Limited.

Rapid and long-term induction of effector immediate early genes (BDNF, Neuritin and Arc) in peri-infarct cortex and dentate gyrus after ischemic injury in rat brain

DEFF Research Database (Denmark)

Rickhag, Karl Mattias; Teilum, Maria; Wieloch, Tadeusz

2007-01-01

including cerebral cortex and hippocampus. Brain-derived neurotrophic factor (BDNF), Neuritin and Activity-regulated cytoskeleton-associated protein (Arc) belong to a subgroup of immediate early genes implicated in synaptic plasticity known as effector immediate early genes. Here, we investigated...... at 0-6 h of reperfusion for Neuritin and 0-12 h of reperfusion for Arc while BDNF was induced 0-9 h of reperfusion. Our study demonstrates a rapid and long-term activation of effector immediate early genes in distinct brain areas following ischemic injury in rat. Effector gene activation may be part...

A comprehensive screening platform for aerosolizable protein formulations for intranasal and pulmonary drug delivery.

Science.gov (United States)

Röhm, Martina; Carle, Stefan; Maigler, Frank; Flamm, Johannes; Kramer, Viktoria; Mavoungou, Chrystelle; Schmid, Otmar; Schindowski, Katharina

2017-10-30

Aerosolized administration of biopharmaceuticals to the airways is a promising route for nasal and pulmonary drug delivery, but - in contrast to small molecules - little is known about the effects of aerosolization on safety and efficacy of biopharmaceuticals. Proteins are sensitive against aerosolization-associated shear stress. Tailored formulations can shield proteins and enhance permeation, but formulation development requires extensive screening approaches. Thus, the aim of this study was to develop a cell-based in vitro technology platform that includes screening of protein quality after aerosolization and transepithelial permeation. For efficient screening, a previously published aerosolization-surrogate assay was used in a design of experiments approach to screen suitable formulations for an IgG and its antigen-binding fragment (Fab) as exemplary biopharmaceuticals. Efficient, dose-controlled aerosol-cell delivery was performed with the ALICE-CLOUD system containing RPMI 2650 epithelial cells at the air-liquid interface. We could demonstrate that our technology platform allows for rapid and efficient screening of formulations consisting of different excipients (here: arginine, cyclodextrin, polysorbate, sorbitol, and trehalose) to minimize aerosolization-induced protein aggregation and maximize permeation through an in vitro epithelial cell barrier. Formulations reduced aggregation of native Fab and IgG relative to vehicle up to 50% and enhanced transepithelial permeation rate up to 2.8-fold. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida.

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Thorpe, Peter; Mantelin, Sophie; Cock, Peter Ja; Blok, Vivian C; Coke, Mirela C; Eves-van den Akker, Sebastian; Guzeeva, Elena; Lilley, Catherine J; Smant, Geert; Reid, Adam J; Wright, Kathryn M; Urwin, Peter E; Jones, John T

2014-10-23

The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure - the syncytium - which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium. The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure. This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Multiple candidate effectors from the oomycete pathogen Hyaloperonospora arabidopsidis suppress host plant immunity.

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Georgina Fabro

2011-11-01

Full Text Available Oomycete pathogens cause diverse plant diseases. To successfully colonize their hosts, they deliver a suite of effector proteins that can attenuate plant defenses. In the oomycete downy mildews, effectors carry a signal peptide and an RxLR motif. Hyaloperonospora arabidopsidis (Hpa causes downy mildew on the model plant Arabidopsis thaliana (Arabidopsis. We investigated if candidate effectors predicted in the genome sequence of Hpa isolate Emoy2 (HaRxLs were able to manipulate host defenses in different Arabidopsis accessions. We developed a rapid and sensitive screening method to test HaRxLs by delivering them via the bacterial type-three secretion system (TTSS of Pseudomonas syringae pv tomato DC3000-LUX (Pst-LUX and assessing changes in Pst-LUX growth in planta on 12 Arabidopsis accessions. The majority (~70% of the 64 candidates tested positively contributed to Pst-LUX growth on more than one accession indicating that Hpa virulence likely involves multiple effectors with weak accession-specific effects. Further screening with a Pst mutant (ΔCEL showed that HaRxLs that allow enhanced Pst-LUX growth usually suppress callose deposition, a hallmark of pathogen-associated molecular pattern (PAMP-triggered immunity (PTI. We found that HaRxLs are rarely strong avirulence determinants. Although some decreased Pst-LUX growth in particular accessions, none activated macroscopic cell death. Fewer HaRxLs conferred enhanced Pst growth on turnip, a non-host for Hpa, while several reduced it, consistent with the idea that turnip's non-host resistance against Hpa could involve a combination of recognized HaRxLs and ineffective HaRxLs. We verified our results by constitutively expressing in Arabidopsis a sub-set of HaRxLs. Several transgenic lines showed increased susceptibility to Hpa and attenuation of Arabidopsis PTI responses, confirming the HaRxLs' role in Hpa virulence. This study shows TTSS screening system provides a useful tool to test whether

Fibre optic sensor on robot end effector for flexible assembly

International Nuclear Information System (INIS)

Yung, K.L.; Lau, W.S.; Choi, C.K.; Shan, Y.Y.

1995-01-01

A fibre optic sensor system was constructed for use on robot end effectors for flexible assembly. The sensor detected the deviations between robot end effector and the workpiece. The signal was fed back to robot controller to shift the end effector until the centre of end effector and the centre of workpiece were aligned at the correct orientation. Then workpiece can be grasped symmetrically. Sensor fusion concept was used to guard against sensor system failure. Fuzzy linguistic variable and control rule concept were introduced in the sensor integration. The experimental setup for the sensor integrated system was shown. The accuracy was also discussed

Vaccine delivery system for tuberculosis based on nano-sized hepatitis B virus core protein particles

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Dhanasooraj D

2013-02-01

Full Text Available Dhananjayan Dhanasooraj, R Ajay Kumar, Sathish MundayoorMycobacterium Research Group, Rajiv Gandhi Centre for Biotechnology, Kerala, IndiaAbstract: Nano-sized hepatitis B virus core virus-like particles (HBc-VLP are suitable for uptake by antigen-presenting cells. Mycobacterium tuberculosis antigen culture filtrate protein 10 (CFP-10 is an important vaccine candidate against tuberculosis. The purified antigen shows low immune response without adjuvant and tends to have low protective efficacy. The present study is based on the assumption that expression of these proteins on HBc nanoparticles would provide higher protection when compared to the native antigen alone. The cfp-10 gene was expressed as a fusion on the major immunodominant region of HBc-VLP, and the immune response in Balb/c mice was studied and compared to pure proteins, a mixture of antigens, and fusion protein-VLP, all without using any adjuvant. The humoral, cytokine, and splenocyte cell proliferation responses suggested that the HBc-VLP bearing CFP-10 generated an antigen-specific immune response in a Th1-dependent manner. By virtue of its self-adjuvant nature and ability to form nano-sized particles, HBc-VLPs are an excellent vaccine delivery system for use with subunit protein antigens identified in the course of recent vaccine research.Keywords: Mycobacterium tuberculosis, VLP, hepatitis B virus core particle, CFP-10, self-adjuvant, vaccine delivery

Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease.

Science.gov (United States)

Dinca, Ana; Chien, Wei-Ming; Chin, Michael T

2016-02-22

Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.

Positive selection and intragenic recombination contribute to high allelic diversity in effector genes of Mycosphaerella fijiensis, causal agent of the black leaf streak disease of banana

NARCIS (Netherlands)

Stergiopoulos, I.; Cordovez da Cunha, V.; Okmen, B.; Beenen, H.G.; Kema, G.H.J.; Wit, de P.J.G.M.

2014-01-01

Previously, we have determined the nonhost-mediated recognition of the MfAvr4 and MfEcp2 effector proteins from the banana pathogen Mycosphaerella fijiensis in tomato, by the cognate Cf-4 and Cf-Ecp2 resistance proteins, respectively. These two resistance proteins could thus mediate resistance

Applications and Challenges for Use of Cell-Penetrating Peptides as Delivery Vectors for Peptide and Protein Cargos

Directory of Open Access Journals (Sweden)

Mie Kristensen

2016-01-01

Full Text Available The hydrophilic nature of peptides and proteins renders them impermeable to cell membranes. Thus, in order to successfully deliver peptide and protein-based therapeutics across the plasma membrane or epithelial and endothelial barriers, a permeation enhancing strategy must be employed. Cell-penetrating peptides (CPPs constitute a promising tool and have shown applications for peptide and protein delivery into cells as well as across various epithelia and the blood-brain barrier (BBB. CPP-mediated delivery of peptides and proteins may be pursued via covalent conjugation of the CPP to the cargo peptide or protein or via physical complexation obtained by simple bulk-mixing of the CPP with its cargo. Both approaches have their pros and cons, and which is the better choice likely relates to the physicochemical properties of the CPP and its cargo as well as the route of administration, the specific barrier and the target cell. Besides the physical barrier, a metabolic barrier must be taken into consideration when applying peptide-based delivery vectors, such as the CPPs, and stability-enhancing strategies are commonly employed to prolong the CPP half-life. The mechanisms by which CPPs translocate cell membranes are believed to involve both endocytosis and direct translocation, but are still widely investigated and discussed. The fact that multiple factors influence the mechanisms responsible for cellular CPP internalization and the lack of sensitive methods for detection of the CPP, and in some cases the cargo, further complicates the design and conduction of conclusive mechanistic studies.

A multi-layered mechanistic modelling approach to understand how effector genes extend beyond phytoplasma to modulate plant hosts, insect vectors and the environment.

Science.gov (United States)

Tomkins, Melissa; Kliot, Adi; Marée, Athanasius Fm; Hogenhout, Saskia A

2018-03-13

Members of the Candidatus genus Phytoplasma are small bacterial pathogens that hijack their plant hosts via the secretion of virulence proteins (effectors) leading to a fascinating array of plant phenotypes, such as witch's brooms (stem proliferations) and phyllody (retrograde development of flowers into vegetative tissues). Phytoplasma depend on insect vectors for transmission, and interestingly, these insect vectors were found to be (in)directly attracted to plants with these phenotypes. Therefore, phytoplasma effectors appear to reprogram plant development and defence to lure insect vectors, similarly to social engineering malware, which employs tricks to lure people to infected computers and webpages. A multi-layered mechanistic modelling approach will enable a better understanding of how phytoplasma effector-mediated modulations of plant host development and insect vector behaviour contribute to phytoplasma spread, and ultimately to predict the long reach of phytoplasma effector genes. Copyright © 2018. Published by Elsevier Ltd.

Nanorobotic end-effectors: Design, fabrication, and in situ characterization

Science.gov (United States)

Fan, Zheng

Nano-robotic end-effectors have promising applications for nano-fabrication, nano-manufacturing, nano-optics, nano-medical, and nano-sensing; however, low performances of the conventional end-effectors have prevented the widespread utilization of them in various fields. There are two major difficulties in developing the end-effectors: their nano-fabrication and their advanced characterization in the nanoscale. Here we introduce six types of end-effectors: the nanotube fountain pen (NFP), the super-fine nanoprobe, the metal-filled carbon nanotube (m CNT)-based sphere-on-pillar (SOP) nanoantennas, the tunneling nanosensor, and the nanowire-based memristor. The investigations on the NFP are focused on nano-fluidics and nano-fabrications. The NFP could direct write metallic "inks" and fabricating complex metal nanostructures from 0D to 3D with a position servo control, which is critically important to future large-scale, high-throughput nanodevice production. With the help of NFP, we could fabricate the end-effectors such as super-fine nanoprobe and m CNT-based SOP nanoantennas. Those end-effectors are able to detect local flaws or characterize the electrical/mechanical properties of the nanostructure. Moreover, using electron-energy-loss-spectroscopy (EELS) technique during the operation of the SOP optical antenna opens a new basis for the application of nano-robotic end-effectors. The technique allows advanced characterization of the physical changes, such as carrier diffusion, that are directly responsible for the device's properties. As the device was coupled with characterization techniques of scanning-trasmission-electron-microscopy (STEM), the development of tunneling nanosensor advances this field of science into quantum world. Furthermore, the combined STEM-EELS technique plays an important role in our understanding of the memristive switching performance in the nanowire-based memristor. The developments of those nano-robotic end-effectors expend the study

The Fusarium oxysporum effector Six6 contributes to virulence and suppresses I-2-mediated cell death.

Science.gov (United States)

Gawehns, F; Houterman, P M; Ichou, F Ait; Michielse, C B; Hijdra, M; Cornelissen, B J C; Rep, M; Takken, F L W

2014-04-01

Plant pathogens secrete effectors to manipulate their host and facilitate colonization. Fusarium oxysporum f. sp. lycopersici is the causal agent of Fusarium wilt disease in tomato. Upon infection, F. oxysporum f. sp. lycopersici secretes numerous small proteins into the xylem sap (Six proteins). Most Six proteins are unique to F. oxysporum, but Six6 is an exception; a homolog is also present in two Colletotrichum spp. SIX6 expression was found to require living host cells and a knockout of SIX6 in F. oxysporum f. sp. lycopersici compromised virulence, classifying it as a genuine effector. Heterologous expression of SIX6 did not affect growth of Agrobacterium tumefaciens in Nicotiana benthamiana leaves or susceptibility of Arabidopsis thaliana toward Verticillium dahliae, Pseudomonas syringae, or F. oxysporum, suggesting a specific function for F. oxysporum f. sp. lycopersici Six6 in the F. oxysporum f. sp. lycopersici- tomato pathosystem. Remarkably, Six6 was found to specifically suppress I-2-mediated cell death (I2CD) upon transient expression in N. benthamiana, whereas it did not compromise the activity of other cell-death-inducing genes. Still, this I2CD suppressing activity of Six6 does not allow the fungus to overcome I-2 resistance in tomato, suggesting that I-2-mediated resistance is independent from cell death.

Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst.

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Zárský, Viktor; Potocký, Martin

2010-04-01

The Rho/Rop small GTPase regulatory module is central for initiating exocytotically ACDs (active cortical domains) in plant cell cortex, and a growing array of Rop regulators and effectors are being discovered in plants. Structural membrane phospholipids are important constituents of cells as well as signals, and phospholipid-modifying enzymes are well known effectors of small GTPases. We have shown that PLDs (phospholipases D) and their product, PA (phosphatidic acid), belong to the regulators of the secretory pathway in plants. We have also shown that specific NOXs (NADPH oxidases) producing ROS (reactive oxygen species) are involved in cell growth as exemplified by pollen tubes and root hairs. Most plant cells exhibit several distinct plasma membrane domains (ACDs), established and maintained by endocytosis/exocytosis-driven membrane protein recycling. We proposed recently the concept of a 'recycling domain' (RD), uniting the ACD and the connected endosomal recycling compartment (endosome), as a dynamic spatiotemporal entity. We have described a putative GTPase-effector complex exocyst involved in exocytic vesicle tethering in plants. Owing to the multiplicity of its Exo70 subunits, this complex, along with many RabA GTPases (putative recycling endosome organizers), may belong to core regulators of RD organization in plants.

Investigation of a bio-inspired lift-enhancing effector on a 2D airfoil.

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Johnston, Joe; Gopalarathnam, Ashok

2012-09-01

A flap mounted on the upper surface of an airfoil, called a 'lift-enhancing effector', has been shown in wind tunnel tests to have a similar function to a bird's covert feathers, which rise off the wing's surface in response to separated flows. The effector, fabricated from a thin Mylar sheet, is allowed to rotate freely about its leading edge. The tests were performed in the NCSU subsonic wind tunnel at a chord Reynolds number of 4 × 10(5). The maximum lift coefficient with the effector was the same as that for the clean airfoil, but was maintained over an angle-of-attack range from 12° to almost 20°, resulting in a very gentle stall behavior. To better understand the aerodynamics and to estimate the deployment angle of the free-moving effector, fixed-angle effectors fabricated out of stiff wood were also tested. A progressive increase in the stall angle of attack with increasing effector angle was observed, with diminishing returns beyond the effector angle of 60°. Drag tests on both the free-moving and fixed effectors showed a marked improvement in drag at high angles of attack. Oil flow visualization on the airfoil with and without the fixed-angle effectors proved that the effector causes the separation point to move aft on the airfoil, as compared to the clean airfoil. This is thought to be the main mechanism by which an effector improves both lift and drag. A comparison of the fixed-effector results with those from the free-effector tests shows that the free effector's deployment angle is between 30° and 45°. When operating at and beyond the clean airfoil's stall angle, the free effector automatically deploys to progressively higher angles with increasing angles of attack. This slows down the rapid upstream movement of the separation point and avoids the severe reduction in the lift coefficient and an increase in the drag coefficient that are seen on the clean airfoil at the onset of stall. Thus, the effector postpones the stall by 4-8° and makes the

The Pseudomonas syringae type III effector HopG1 targets mitochondria, alters plant development, and suppresses plant innate immunity

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Block, Anna; Guo, Ming; Li, Guangyong; Elowsky, Christian; Clemente, Thomas E.; Alfano, James R.

2009-01-01

Summary The bacterial plant pathogen Pseudomonas syringae uses a type III protein secretion system to inject type III effectors into plant cells. Primary targets of these effectors appear to be effector-triggered immunity (ETI) and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). The type III effector HopG1 is a suppressor of ETI that is broadly conserved in bacterial plant pathogens. Here we show that HopG1 from P. syringae pv. tomato DC3000 also suppresses PTI. Interestingly, HopG1 localizes to plant mitochondria, suggesting that its suppression of innate immunity may be linked to a perturbation of mitochondrial function. While HopG1 possesses no obvious mitochondrial signal peptide, its N-terminal two-thirds was sufficient for mitochondrial localization. A HopG1-GFP fusion lacking HopG1’s N-terminal 13 amino acids was not localized to the mitochondria reflecting the importance of the N-terminus for targeting. Constitutive expression of HopG1 in Arabidopsis thaliana, Nicotiana tabacum (tobacco) and Lycopersicon esculentum (tomato) dramatically alters plant development resulting in dwarfism, increased branching and infertility. Constitutive expression of HopG1 in planta leads to reduced respiration rates and an increased basal level of reactive oxygen species. These findings suggest that HopG1’s target is mitochondrial and that effector/target interaction promotes disease by disrupting mitochondrial functions. PMID:19863557

Design and force analysis of end-effector for plug seedling transplanter.

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Jiang, Zhuohua; Hu, Yang; Jiang, Huanyu; Tong, Junhua

2017-01-01

Automatic transplanters have been very important in greenhouses since the popularization of seedling nurseries. End-effector development is a key technology for transplanting plug seedlings. Most existing end-effectors have problems with holding root plugs or releasing plugs. An efficient end-effector driven by a linear pneumatic cylinder was designed in this study, which could hold root plugs firmly and release plugs easily. This end-effector with four needles could clamp the plug simultaneously while the needles penetrate into the substrate. The depth and verticality of the needles could be adjusted conveniently for different seedling trays. The effectiveness of this end-effector was tested by a combinational trial examining three seedling nursery factors (the moisture content of the substrate, substrate bulk density and the volume proportion of substrate ingredients). Results showed that the total transplanting success rate for the end-effector was 100%, and the root plug harm rate was below 17%. A force measure system with tension and pressure transducers was installed on the designed end-effector. The adhesive force FL between the root plug and the cell of seedling trays and the extrusion force FK on the root plug were measured and analyzed. The results showed that all three variable factors and their interactions had significant effects on the extrusion force. Each factor had a significant effect on adhesive force. Additionally, it was found that the end-effector did not perform very well when the value of FK/FL was beyond the range of 5.99~8.67. This could provide a scientific basis for end-effector application in transplanting.

Polysaccharides-based polyelectrolyte nanoparticles as protein drugs delivery system

Energy Technology Data Exchange (ETDEWEB)

Shu Shujun; Sun Lei; Zhang Xinge, E-mail: zhangxinge@nankai.edu.cn [Nankai University, Key Laboratory of Functional Polymer Materials Ministry of Education, Institute of Polymer Chemistry (China); Wu Zhongming [Tianjin Medical University, Metabolic Diseases Hospital (China); Wang Zhen; Li Chaoxing, E-mail: lcx@nankai.edu.cn [Nankai University, Key Laboratory of Functional Polymer Materials Ministry of Education, Institute of Polymer Chemistry (China)

2011-09-15

Polysaccharides-based nanoparticles were prepared by synthesized quaternized chitosan and dextran sulfate through simple ionic-gelation self-assembled method. Introduction of quaternized groups was intended to increase water solubility of chitosan and make the nanoparticles have broader pH sensitive range which can remain more stable in physiological pH and decrease the loss of protein drugs caused by the gastric cavity. The load of BSA was affected by molecular parameter, i.e., degree of substitution, and average molecular weight of quaternized chitosan, as well as concentration of BSA. Fast release occurred in phosphate buffer solution (pH 7.4) while the release was slow in hydrochloric acid (pH 1.4). The drug release mechanism is Fickian diffusion through release kinetics analysis. Cell uptake demonstrated nanoparicles can internalize into Caco-2 cells, which suggested that nanoparticles had good biocompatibility. No significant conformation change was noted for the released BSA in comparison with native BSA using circular dichroism spectroscopy. This kind of novel composite nanoparticles may be a promising delivery system for oral protein and peptide drugs.

Noninvasive delivery systems for peptides and proteins in osteoporosis therapy: a retroperspective.

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Hoyer, Herbert; Perera, Glen; Bernkop-Schnürch, Andreas

2010-01-01

The aim of this review is to provide the reader general and inspiring prospects in various attempts to make noninvasive delivery systems of calcitonin and teriparatide feasible and as convenient as possible. Calcitonin and teriparatide play an important role in both calcium homeostasis and bone remodelling. Currently calcitonin is available as a subcutaneous injection and as a nasal spray whereas teriparatide is administered subcutaneously. In the past few years, an increasing number of articles about drug delivery systems for calcitonin and teriparatide have been published. These delivery systems have been developed to overcome the inherent barriers for the uptake across the diverse membranes on the various routes for protein and peptide delivery. Co-administration of permeation enhancers, mucoadhesive agents, viscosity modifying agents, multifunctional polymers, protease inhibitors as well as encapsulation and chemical modification are utilized in order to improve calcitonin and teriparatide absorption after oral, nasal, pulmonal, or buccal administration. The majority of research groups have been working on the development of formulations based on the encapsulation of molecules in biodegradable and biocompatible polymeric nanoparticles. However these observations are based on data obtained under different experimental conditions. Hence, it is difficult to compare the obtained results in order to draw general conclusions about the most promising characteristics required for oral and nasal formulations for these peptides.

The Ustilago maydis effector Pep1 suppresses plant immunity by inhibition of host peroxidase activity.

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Christoph Hemetsberger

Full Text Available The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1 as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δpep1 mutant penetrations, H₂O₂ strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δpep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction.

Food-grade protein-based nanoparticles and microparticles for bioactive delivery: fabrication, characterization, and utilization.

Science.gov (United States)

Davidov-Pardo, Gabriel; Joye, Iris J; McClements, David Julian

2015-01-01

Proteins can be used to fabricate nanoparticles and microparticles suitable for use as delivery systems for bioactive compounds in pharmaceutical, food, cosmetic, and other products. Food proteins originate from various animal or vegetal sources and exhibit a wide diversity of molecular and physicochemical characteristics, e.g., molecular weight, conformation, flexibility, polarity, charge, isoelectric point, solubility, and interactions. As a result, protein particles can be assembled using numerous different preparation methods, from one or more types of protein or from a combination of a protein and another type of biopolymer (usually a polysaccharide). The final characteristics of the particles produced are determined by the proteins and/or polysaccharides used, as well as the fabrication techniques employed. This chapter provides an overview of the functional properties of food proteins that can be used to assemble nanoparticles and microparticles, the fabrication techniques available to create those particles, the factors that influence their stability, and their potential applications within the food industry. © 2015 Elsevier Inc. All rights reserved.

Design and force analysis of end-effector for plug seedling transplanter.