WorldWideScience

Sample records for k143r g464s g465s

  1. Dominant negative mutant of Plasmodium Rad51 causes reduced parasite burden in host by abrogating DNA double-strand break repair.

    Science.gov (United States)

    Roy, Nabamita; Bhattacharyya, Sunanda; Chakrabarty, Swati; Laskar, Shyamasree; Babu, Somepalli Mastan; Bhattacharyya, Mrinal Kanti

    2014-10-01

    Malaria parasites survive through repairing a plethora of DNA double-stranded breaks (DSBs) experienced during their asexual growth. In Plasmodium Rad51 mediated homologous recombination (HR) mechanism and homology-independent alternative end-joining mechanism have been identified. Here we address whether loss of HR activity can be compensated by other DSB repair mechanisms. Creating a transgenic Plasmodium line defective in HR function, we demonstrate that HR is the most important DSB repair pathway in malarial parasite. Using mouse malaria model we have characterized the dominant negative effect of PfRad51(K143R) mutant on Plasmodium DSB repair and host-parasite interaction. Our work illustrates that Plasmodium berghei harbouring the mutant protein (PfRad51(K143R)) failed to repair DSBs as evidenced by hypersensitivity to DNA-damaging agent. Mice infected with mutant parasites lived significantly longer with markedly reduced parasite burden. To better understand the effect of mutant PfRad51(K143R) on HR, we used yeast as a surrogate model and established that the presence of PfRad51(K143R) completely inhibited DNA repair, gene conversion and gene targeting. Biochemical experiment confirmed that very low level of mutant protein was sufficient for complete disruption of wild-type PfRad51 activity. Hence our work provides evidence that HR pathway of Plasmodium could be efficiently targeted to curb malaria.

  2. Detection of point mutations in genes related to fluconazole resistance in Candida albicans%白念珠菌耐氟康唑相关基因点突变的检测

    Institute of Scientific and Technical Information of China (English)

    景伟芳; 李春莉; 郭雅莉; 刘原君; 亓玉青; 刘全忠; 王惠平

    2013-01-01

    Objective To investigate the relationship of point mutations in ERG11,TAC1,MRR1 and UPC2 genes to fluconazole resistance in Candida albicans.Methods This study included 11 fluconazole-resistant Candida albicans strains.DNA was extracted from these Candida isolates by urea pyrolysis method.PCR was carried out to amplify the full-length EGR11 gene,as well as the corresponding fragments of TAC1,MRR1 and UPC2 genes followed by DNA sequencing.Results Eight mutations were detected in the ERG11 gene,including D116E,Y132H,Y132F,K143Q,T229A,E266D,G448E and G464S.Among them,K143Q was a novel mutation and had not been reported.A G648D mutation was detected in the UpC2 gene.No mutations were detected in the TAC1 or MRR1 gene.Conclusions There is a definite association between fluconazole resistance and ERG11 mutations,while further research is warranted to clarify the relationship between the mutations of TAC1,MRR1,UPC2 genes and resistance to fluconazole.%目的 探讨ERG11、TAC1、MRR1和UPC2点突变与白念珠菌氟康唑耐药之间的关系.方法 收集耐氟康唑的白念珠菌,尿素裂解法提取DNA,PCR扩增ERG11基因全长以及TAC1、MRR1和UPC2基因的相应片段,送交测序.结果 ERG11基因发现8种点突变,即D116E、Y132H、Y132F、K143Q、T229A、E266D、G448E和G464S,其中K143Q此前未见文献报道.UPC2基因发现1种点突变G648D.TAC1和MRR1基因中均未检测到相应点突变.结论 ERG11点突变与耐药的关系比较明确,TAC1、MRR1和UPC2点突变与耐药的关系有待进一步研究.

  3. ERG11 mutations associated with azole resistance in Candida albicans isolates from vulvovaginal candidosis patients

    Institute of Scientific and Technical Information of China (English)

    Bin; Wang; Li-Hua; Huang; Ji-Xue; Zhao; Man; Wei; Hua; Fang; Dan-Yang; Wang; Hong-Fa; Wang; Ji-Gang; Yin; Mei; Xiang

    2015-01-01

    Objective: To investigate the azole susceptibility of Candida albicans(C.albicans)from vulvovaginal candidosis patients and to analyze the relationship between ERG11 gene mutations in these isolates and azole resistance.Methods: Three hundred and two clinical isolates of Candida species were collected.Azole susceptibility was tested in vitro in microdilution studies. The ERG11 genes of 17 isolates of C. albicans(2 susceptibles, 5 dose-dependent resistants and 10 resistants) were amplified and sequenced.Results: Of the 302 isolates collected, 70.2% were C. albicans, of which 8.5%, 3.8% and4.2% were resistant to fluconazole, itraconazole and voriconazole, respectively. In total,27 missense mutations were detected in ERG11 genes from resistant/susceptible dosedependent isolates. Among them, Y132 H, A114 S, and Y257 H substitutions were most prevalent and were known to cause fluconazole resistance. G464 S and F72 S also have been proved to cause fluconazole resistance. Two novel substitutions(T285A, S457P) in hotspot regions were identified.Conclusions: Twenty seven mutations in the ERG11 gene were identified in azoleresistant C. albicans isolates, which indicated a possible relation with the increase in resistance to azole drugs and the recurrence of vulvovaginal candidosis. The relationship of two novel substitutions(T285A, S457P) with fluconazole resistance needs to be further verified by site-directed mutagenesis.

  4. ERG11 mutations associated with azole resistance in Candida albicans isolates from vulvovaginal candidosis patients

    Institute of Scientific and Technical Information of China (English)

    Bin Wang; Li-Hua Huang; Ji-Xue Zhao; Man Wei; Hua Fang; Dan-Yang Wang; Hong-Fa Wang; Ji-Gang Yin; Mei Xiang

    2015-01-01

    Objective:To investigate the azole susceptibility of Candida albicans (C. albicans) from vulvovaginal candidosis patients and to analyze the relationship between ERG11 gene mutations in these isolates and azole resistance. Methods:Three hundred and two clinical isolates of Candida species were collected. Azole susceptibility was tested in vitro in microdilution studies. The ERG11 genes of 17 isolates of C. albicans (2 susceptibles, 5 dose-dependent resistants and 10 resistants) were amplified and sequenced. Results:Of the 302 isolates collected, 70.2%were C. albicans, of which 8.5%, 3.8%and 4.2%were resistant to fluconazole, itraconazole and voriconazole, respectively. In total, 27 missense mutations were detected in ERG11 genes from resistant/susceptible dose-dependent isolates. Among them, Y132H, A114S, and Y257H substitutions were most prevalent and were known to cause fluconazole resistance. G464S and F72S also has been proved to cause fluconazole resistance. Two novel substitutions (T285A, S457P) in hotspot regions were identified. Conclusions:Twenty seven mutations in the ERG11 gene were identified in azole-resistant C. albicans isolates, which indicated a possible relation with the increase in resistance to azole drugs and the recurrence of vulvovaginal candidosis. The relationship of two novel substitutions (T285A, S457P) with fluconazole resistance needs to be further verified by site-directed mutagenesis.

  5. Signal transduction triggered by iron to induce the nuclear importation of a Myb3 transcription factor in the parasitic protozoan Trichomonas vaginalis.

    Science.gov (United States)

    Hsu, Hong-Ming; Lee, Yu; Hsu, Pang-Hung; Liu, Hsing-Wei; Chu, Chien-Hsin; Chou, Ya-Wen; Chen, Yet-Ran; Chen, Shu-Hui; Tai, Jung-Hsiang

    2014-10-17

    Iron was previously shown to induce rapid nuclear translocation of a Myb3 transcription factor in the protozoan parasite, Trichomonas vaginalis. In the present study, iron was found to induce a transient increase in cellular cAMP, followed by the nuclear influx of Myb3, whereas the latter was also induced by 8-bromo-cyclic AMP. Iron-inducible cAMP production and nuclear influx of Myb3 were inhibited by suramin and SQ22536, respective inhibitors of the Gα subunit of heterotrimeric G proteins and adenylyl cyclases. In contrast, the nuclear influx of Myb3 induced by iron or 8-bromo-cAMP was delayed or inhibited, respectively, by H89, the inhibitor of protein kinase A. Using liquid chromatography-coupled tandem mass spectrometry, Thr(156) and Lys(143) in Myb3 were found to be phosphorylated and ubiquitinated, respectively. These modifications were induced by iron and inhibited by H89, as shown by immunoprecipitation-coupled Western blotting. Iron-inducible ubiquitination and nuclear influx were aborted in T156A and K143R, but T156D was constitutively ubiquitinated and persistently localized to the nucleus. Myb3 was phosphorylated in vitro by the catalytic subunit of a T. vaginalis protein kinase A, TvPKAc. A transient interaction between TvPKAc and Myb3 and the phosphorylation of both proteins were induced in the parasite shortly after iron or 8-bromo-cAMP treatment. Together, these observations suggest that iron may induce production of cAMP and activation of TvPKAc, which then induces the phosphorylation of Myb3 and subsequent ubiquitination for accelerated nuclear influx. It is conceivable that iron probably exerts a much broader impact on the physiology of the parasite than previously thought to encounter environmental changes.