Research On The Function Of Genes Wsc3 And MpkA In Talaromyces Marneffei

Talaromyces mameffei is a kind of fungus and can be induced by temperature.It show the mold shape at 25? and show the yeast shape at 37?or in the human body,shape of yeast for its pathogenesis.It can cause severe and disseminated Penicilliosis Mameffei.It is easy to invade patients with low immunity,especially AIDS patients,but the infection rate of people with normal immunity increased significantly in recent years,if not treated effectively,the case fatality rate is very high.In China,the epidemic areas of the disease are mainly concentrated in guangxi,guangzhou,yunnan,Hong Kong and other regions.The most common route of transmission is infection by inhalation of T.marneffei spores through the respiratory tract.Many studies have shown that the morphological transformation of dimorphic fungi from molds phase to yeast phase is necessary for its toxicity and this property is closely related to its pathogenicity.They can express many virulence factors in' yeast phase.The transformation of T.mameffei from mold phase to pathogenic yeast phase not only changes cell morphology,but also changes in cell wall composition,antigen molecules and expression of toxic characteristics.However,the molecular of its dimorphic switching mechanism and pathogenicity is not well understood.Cell wall integrity and stress response component(Wsc)protein mediated CWI-MAPK signaling pathway plays an important role in fungal cells' response to environmental stress.Studies have shown that WSC protein-mediated CWI-MAPK signaling pathway is involved in response to environmental pressures such as heat,H2O2,ethanol and hypoosmosis in yeast,and eventually produces cell wall synthase to maintain the integrity of cell wall to adapt to environmental changes.Wsc3 protein is one of the sensing proteins on yeast cell membrane to detect the disturbance of cell wall by environmental pressure.When conducting Pathway significance enrichment analysis of differentially expressed genes in different growth phases,it was found that the stress response sensor protein gene Wsc3,which is homologous to the MAPK Pathway and the yeast,was significantly down-regulated in the yeast phase.Based on this result,we speculated that WSC protein-mediated CWI-MAPK signaling pathway may play an important role in the dimorphic switching mechanism and pathogenicity regulation of T.mameffei,and these two pathways may be inhibited by adaptive feedback after playing a role.In recent years,the dimorphic switching mechanism of T.mameffei has attracted more and more attention from researchers,most of whom believe that this mechanism is closely related to its pathogenicity.Therefore,our research group sequenced the chain specific transcriptome of the standard strain of T.marneffei.The analysis found that the expression of Wsc3 and MpkA in the CWI-MAPK signaling pathway was significantly different during the dimorphic switching process of four different phases.The results showed that the expression of Wsc3 and MpkA gene was low in yeast phase and the transformation of mycelium to yeast,but increased in mycelial phase and the transformation of yeast to mycelium,especially the gene of Wsc3 in the CWI signaling pathway.The differential expression of Wsc3 and MpkA genes suggests that they may play a role in the dimorphic switching mechanism of T.marneffei.Therefore,by studying the functions of these two genes,we want to explore the role and characteristics of CWI-MAPK signaling pathway in the dimorphic switching process and pathogenesis of T.mameffei,laying an important foundation for further understanding the pathogenesis of this fungus.Objective:By studying the functions of Wsc3 and MpkA genes of CWI-MAPK signaling pathway in different phases,it is elucidated further that the functional characteristics of CWI-MAPK signaling pathway in T.marneffei,its dimorphic switching mechanism and its pathogenic mechanism.Methods:Using the homologous recombination to construct the gene deletion mutant,and analyze the function of genes in various aspects after compared with the Spm4 strain.Observing their morphology and transformation process on different culture medium at 25? and 37? and the changes of the strain's sensitivity to various stress agents,such as cell wall pressure,oxidative stress,osmotic pressure,drugs,temperature,etc.The purpose is to preliminarily explore and understand the function of the target gene.Results:(1)The target gene Wsc3,MpkA deletion mutant strain was successfully constructed;(2)Compared with the wild strains Spm4 and MpkA gene deletion strains,the sensitivity of cell wall pressure SDS,Congo red,salt pressure NaNO2 and anti-fungal agents micafengein increased,and the sensitivity to cell wall pressure CFW,oxidative pressure,osmotic pressure D-Sorbitol,salt pressure KCl and NaCl and anti-fungal agents fluconazole increased slightly;(3)Wsc3 gene deletion strain only cell wall pressure Congo red increased significantly,slightly increased sensitivity to cell wall pressure SDS,CFW,other stress changes were not obvious.Conclusions:(1)Based on the gene homologous recombination,the mutant strains of Wsc3 and MpkA gene deletion were successfully constructed by using the method of fracture marker gene;(2)The Wsc3 and MpkA genes of mameffei were involved in the regulation of cell wall pressure,oxidative pressure,salt pressure,high osmotic pressure and temperature pressure sensitivity,and the MpkA gene had a more obvious effect.