The Mechanistic Study Of NMA111 In Regulating Biofilm Formation In Candida Albicans

Background:Candida albicans is a conditional pathogenic fungus belonging to the phylum Ascomycota.It is widespread on the skin,oral cavity,gastrointestinal tract,urinary tract and mucosal surface of reproductive tract of humans or other mammals.For healthy hosts,Candida albicans is generally in a symbiotic state with the normal flora;but for people with weakened or defective immunity and normal flora disorders,Candida albicans may change from a symbiotic state to a disease-causing state,leading to superficial infections and even systemic infections with a high fatality rate in the host.Biofilm is one of the important pathogenic factors of Candida albicans and an important way for Candida albicans to infect the host.It plays a major role in Candida albicans drug resistance and host immune evasion.Therefore,it is also a big problem of the clinical treatments for Candida albicans infection.Nma111 is a protein with unknown function in Candida albicans.The Candida albicans Nma111 homologue has both serine protease activity and molecular chaperone activity in Saccharomyces cerevisiae,which plays a vital role in the survival of Saccharomyces cerevisiae in a stress environment.In bacteria and mammals,the homologue of Nma111 is named Htr A.For pathogenic bacteria,Htr A is an important pathogenic factor,which plays an important role in the process of pathogenic bacteria adapting to the host environment and invading host cells and tissues.At the same time,Htr A also plays an important role in the formation and spread of many pathogenic bacteria biofilms.In view of the important role of Nma111and its homolog Htr A in Saccharomyces cerevisiae and pathogenic bacteria,this research is devoted to exploring the pathogenicity relationship between Nma111 and Candida albicans and its mechanism.Objective:1.To explore the influence of NMA111 gene on the pathogenicity of Candida albicans and its pathway2.To explore the clear m0echanism of NMA111 gene affected biofilm formation of Candida albicans.Methods:1.The Candida albicans NMA111,BCR1 and TEC1 gene deletion strains were constructed by homologous recombination using Candida albicans SN152（leuΔ/Δ,hisΔ/Δ,argΔ/Δ）as the parent strain and named NC111（nma111Δ/Δ）,NC121（bcr1Δ/Δ）and NC131（tec1Δ/Δ）,respectively;The Candida albicans NMA111 gene complement strain and ALS1 gene overexpression strain were constructed using Candida albicans NC111（nma111Δ/Δ）as the parent strain,and named NC112（NMA111 AB）and NC113(nma111Δ/Δ+ALS1^OE),respectively;The Candida albicans NMA111 gene overexpression strain were constructed using Candida albicans NC121（bcr1Δ/Δ）or NC131（tec1Δ/Δ）as the parent strain and named NC123(bcr1Δ/Δ+NMA111^OE)and NC133(tec1Δ/Δ+NMA111^OE)respectively;2.The model of BALB/c mice systemic infection with Candida albicans was constructed by tail vein injection.The BALB/c mice were divided into 4 groups,namely WT（SN250）,NC111（nma111Δ/Δ）,NC112（NMA111 AB）,and PBS group respectively.The number of mice in the group is 8,of which 5 mice are used to determine the survival curve,and 3 mice are used to determine the kidney bacterial load.By comparing the survival curve and the kidney bacterial load of the mice in each group,verify the effect of NMA111 gene on the virulence of Candida albicans;3.Spider and RPMI1640 liquid medium and 10%FBS were used to induce the hyphae formation of Candida albicans SN250,NC111（nma111Δ/Δ）and NC112（NMA111 AB）at 37℃,exploring the effect of NMA111 gene on Candida albicans yeast phase-hypha phase transformation;The spider liquid medium was used to induce biofilm formation of Candida albicans in polystyrene twelve-well plate,verifying the effect of NMA111 gene on Candida albicans biofilm formation,overexpression of NMA111 gene on Candida albicans NC121（bcr1Δ/Δ）and NC131（tec1Δ/Δ）biofilm formation and ALS1 gene overexpression on Candida albicans NC111（nma111Δ/Δ）biofilm formation;The Candida albicans SN250,NC111（nma111Δ/Δ）and NC112（NMA111 AB）were inoculated into YPD liquid medium and cultured at 30℃200rpm.The OD₆₀₀absorbance of each group was measured every 1hour to determine the effect of NMA111 gene on the growth rate of Candida albicans;Using Spot assay to explore the effect of NMA111 gene on Candida albicans adaptability to stress environment;4.The Candida albicans SN152 and NC111（nma111Δ/Δ）were cultured for 48hours under biofilm induction conditions,and then samples were collected and subjected to TMT quantitative proteomics analysis to explore the differentially expressed proteins of SN152 v.s NC111（nma111Δ/Δ）.5.RT-qPCR was used to detect the NMA111 gene expression level of Candida albicans wild type strains SN250,NC121（bcr1Δ/Δ）and NC131（tec1Δ/Δ）under the conditions of biofilm formation,verifying the impact of transcription factor Tec1 and Bcr1 on the expression of NMA111 gene of Candida albicans;Results:1.Under the identification of nutritional type,PCR and RT-qPCR,we successfullyobtained the Candida albicans NC111（nma111Δ/Δ）,NC112（NMA111 AB）,NC113(nma111Δ/Δ+ALS1^OE),NC121（bcr1Δ/Δ）,NC123(bcr1Δ/Δ+NMA111^OE),NC131（tec1Δ/Δ）and NC131(tec1Δ/Δ+NMA111^OE)positive strains;2.The results of the BALB/c mouse systemic infection model showed that the survival rate of the NC111（nma111Δ/Δ）group was significantly higher than that of the WT（SN250）group（P<0.05）,while the survival rate of the NC112（NMA111 AB）group basically recovered to The level of wild-type,and the fungal burden in the kidneys of the NC111（nma111Δ/Δ）group decreased slightly compared with the WT（SN250）group and the NC112（NMA111 AB）group,but there was no statistical difference.The results showed the virulence of Candida albicans systemically infected BALB/c mice was significantly reduced after NMA111 gene knockout;3.The results of the hyphae induction experiment showed that compared with the WT（SN250）and NC112（NMA111 AB）group,the NC111（nma111Δ/Δ）group had no significant difference in the hyphae formation ability induced by Spider,RPMI1640and 10%fetal bovine serum liquid medium,indicating that NMA111 gene knockout does not affect the ability of Candida albicans to transform from yeast to hyphae;The results of the biofilm experiment showed that the biofilm formation ability of the Candida albicans NC111（nma111Δ/Δ）group was significantly defective compared with the WT（SN250）group（P<0.001）,while the biofilm formation of the NC112（NMA111 AB）group returned to the wild-type level,indicating that the NMA111 gene plays an important role in the biofilm formation of Candida albicans;The results of growth curve determination showed that the growth rate of NC111（nma111Δ/Δ）group was not significantly different from that of WT（SN250）group and NC112（NMA111 AB）group,which indicated that NMA111 gene knockout did not affect the growth rate of Candida albicans;Spot assay results showed that compared with the WT（SN250）and NC112（NMA111 AB）group,the the growth status of NC111（nma111Δ/Δ）group in acidic environment（p H=4.0）,alkaline environment（p H=9）,high calcium ion concentration environment（YPD+Ca Cl₂）,oxidative stress environment（The growth of YPD+H₂O₂）,cell wall stress environment（YPD+Congo red and YPD+CFW）and high temperature stress environment（42℃）were not significantly different,indicating that the NMA111 gene does not affect the adaptability of Candida albicans to the above stress environment;4.TMT quantitative proteomics results showed that there were 168 differentially expressed proteins in the SN152vs NC111（nma111Δ/Δ）comparison group,of which78 were up-regulated and 90 were down-regulated.We verified the transcription level of some differentially expressed protein-coding genes in this proteomics by RT-qPCR.RT-qPCR results showed that,except for THI13 and RHR2,the changes in m RNA levels of other genes were basically the same as the changes in protein levels.Then,we knocked out 15 genes encoding the most differentially expressed proteins in our proteomics results,and carried out nutritional type and PCR identification.After successful identification,the verification results of the biofilm formation ability of these gene knockout strains showed that there are 7 genes closely related to biofilm formation.The results of GO functional classification enrichment analysis show that,in the BP classification,the differentially expressed proteins are mainly distributed in cytoplasmic translation;in the CC classification,the differentially expressed proteins are mainly distributed in the ribosomal subunit and fungal cell wall（Fungal-type cell wall）and external encapsulating structure（External encapsulating structure）;in the MF classification,differentially expressed proteins are mainly distributed in the structure constituent of ribosome,unfolding protein binding and Isomerase activity.KEGG pathway enrichment analysis showed that differentially expressed proteins are mainly enriched in ribosomes,longevity regulating pathway-multiple species,and biosynthesis of valine,leucine and isoleucine pathway;5.The RT-qPCR results showed that under the conditions of biofilm formation,the expression of NMA111 gene of Candida albicans NC121（bcr1Δ/Δ）and NC131（tec1Δ/Δ）was significantly lower than that of the wild type strain SN250（P<0.01）;6.The test results of the biofilm formation ability of Candida albicans SN250,NC121（bcr1Δ/Δ）,NC123(bcr1Δ/Δ+NMA111^OE),NC131（tec1Δ/Δ）and NC133(tec1Δ/Δ+NMA111^OE)showed that Candida albicans NC121（bcr1Δ/Δ）and NC131（tec1Δ/Δ）biofilm formation ability compared with wild-type strains have significant defects（P<0.001）;after overexpression of NMA111 gene,Candida albicans NC123(bcr1Δ/Δ+NMA111^OE)biofilm formation Compared with the wild-type strain SN250,the ability has no significant difference,but the biofilm formation ability of Candida albicans NC133(tec1Δ/Δ+NMA111^OE)did not recover significantly;7.The test results of Candida albicans SN250,NC111（nma111Δ/Δ）and NC113(nma111Δ/Δ+ALS1^OE)biofilm formation ability showed that after ALS1 gene overexpression,NC113(nma111Δ/Δ+ALS1^OE)biofilm formation ability recovered significantly.Conclusions:1.NMA111 gene is involved in the formation of Candida albicans biofilm;2.NMA111 gene has significant effect on the virulence of Candida albicanssystemically infecting BALB/c mices;3.Under the regulation of the transcription factor Bcr1,Nma111 regulates the formation of Candida albicans biofilm by acting on the lectin-like adhesion factor Als1;...