SakA Gene Regulates Stress Response,Drug Sensitivity And Virulence Of Talaromyces Marneffei

ObjectiveDespite being the most important dimorphic fungus causing disseminated mycosis in South East Asia,the pathogenic mechanisms of Talaromyces marneffei are still unclear.This organism must be able to adapt to stress changes in the intracellular host environment,and then evade or tolerate the host immune defence system.The high-osmolarity glycerol（HOG）mitogen-activated protein kinase（HOG-MAPK）signaling pathway plays a crucial role in regulating morphology,growth,and adaptation to stress and virulence in a number of fungal pathogens.The stress-activated kinase（SakA）gene is the core gene in HOG-MAPK pathway.Here,we builded the SakA mutant strains（ΔsakA）.By comparing the differences between wild-type stains（FRR）andΔsakA in phenotype characteristics,the osmotic,oxidative and temperature stress resistance,drug sensitivity,and establishing murine model of disseminated penicilliosis marneffei to explore the role of sakA gene in regulates stress response,drug sensitivity and virulence of T.marneffei.And it is benefit to promote our understanding of the pathogenesis of T.marneffei one step further.MethodsWe observed the phenotype features and surveyed of germination rates FRR andΔsakA at 25℃、37℃、39℃,and compared the growth in the culture medium containing different concentration of KCl,sorbitol,H₂O₂,Congo red as well as SDS.Antifungal drug sensitivity test in vitro were evaluated by the microdilution method referring to the M38-A2protocol recommended by the National committee for clinical laboratory standards（NCCLS）with appropriate modifications.Then,we established murine model of disseminated penicilliosis marneffei,and infected with FRR andΔsakA strains after immunosuppressing mice to investigate the differences of survival and survival time between each group.The significance of the differences in the data about relative expression was determined by Student’s t test.Statistical analysis was done with the SPSS packatge（version 17.0）,P value of<0.05 was considered significant.ResultsAt 37℃and 39℃,both the growth and germination rates ofΔsakA strains were reduced,however there was no difference in growth between the FRR andΔsakA strains at 25℃.The results showedΔsak A strains with significant growth inhibition comparing with FRR in the culture medium containing different concentration of KCl,sorbitol,H₂O₂,Congo red as well as SDS.In vitro drug susceptibility test,ΔsakA strains appeared to be more sensitive to micafungin and caspofungin at yeast.Furthermore,deletion of SakA resulted in a drastic increase in the mean survival time of systemically infected mice.The mortality rate of mice infected with FRR was 100%in comparision with an apparent decline in mortality rate of mice infected withΔsakA,and FRR strains exhibited more aggressive thanΔsakA strainsConclusionThe core gene Sak A in HOG-MAPK signaling pathways involved in the osmotic,oxidative and temperature stress response,it suggested SakA gene may play an important role in adapting to different environmental stress,and it was essential in growth,morphology,asexual development and the integrity of the cell wall.The results also demonstrated that SakA gene contributed to drug resistance in T.marneffei,and it may be involved in the mechanism of that T.marneffei yeast phase is insensitive to echinocandin antifungal agent.In addition,the lack of SakA gene could lead the dramatic attenuation in virulence and may contribute to an altered immune response in mice.We concluded that SakA gene is a virulence factor in murine model of disseminated penicilliosis marneffe.