Function And Mechanism Of Small Heat Shock Protein Fmp28 On Pathogenicity In Candida Albicans

Background:Small heat shock proteins（s Hsp）are a class of highly conserved proteins with a molecular weight of less than 30 k D,DNL type zinc finger,and can be used as molecular chaperones to assist protein transportation and prevent the aggregation of harmful proteins.Under certain stress conditions such as heat,permeation,and blocked cell wall synthesis,the expression of this type of protein in Candida albicans is significantly upregulated,thereby maintaining its survival and infectivity.Therefore,these small molecules of heat shock proteins can be used as virulence factor to promote the morphological transformation,biofilm formation and adhesion ability of Candida albicans,suggesting that small molecules of heat shock proteins may become potential targets of antifungal drugs.In this study,we identified a new protein Fmp28of Candida albicans with a molecular weight of 21 k D,which belongs to the s Hsp subfamily.It is predicted that Fmp28 is a heat shock protein with molecular chaperones activity in Candida albicans.At present,there is little research on this protein in Candida albicans,and it is still unknown whether it affects the virulence of Candida albicans and the related regulatory mechanisms.Objectives:To clarify the biological function of Fmp28 and explore its role as a virulence factor in the pathogenesis of Candida albicans.Methods:1.Using homologous recombination genetic methods to construct a series of strains of Candida albicans,such as knockout strains,complement strains,overexpression strains,and labeled strains:using homologous recombination genetic method,construct knockout strain fmp28Δ/Δand labeled strain Fmp28-3HA using SN152 as the parent strain;with fmp28Δ/Δas the parent strain,construct a complementary strain fmp28AB and an overexpression strains fmp28Δ/Δ+als3^OE,fmp28Δ/Δ+pra1^OE and fmp28Δ/Δ+csa1^OE;using SN250 as the parent strain,overexpression strains fmp28^OE,als3^OE,pra1^OE,and csa1^OE were constructed;2.The biological function of Fmp28 as a heat shock protein molecule was confirmed using the following methods:compare the difference in growth rate between WT（SN250）,fmp28Δ/Δ,fmp28AB and fmp28^OE by measuring the growth curve and point plate experiments;the m RNA level of fmp28 and the protein expression of Fmp28 under heat stress,osmotic pressure and cell wall pressure were detected by RT-q PCR and Western Blot experiments,respectively,to verify the biological function of Fmp28 as a heat shock protein;3.Explore the relationship between Candida albicans Fmp28 and pathogenic function using the following methods:（1）Compare the sensitivity of WT（SN250）,fmp28Δ/Δand fmp28AB to cell wall interference agents（CGR,CFW,Caspofungin）under 30℃and 37℃conditions through point plate experiments,and verify the effect of Fmp28 on Candida albicans’resistance to cell wall pressure disturbance under heat shock conditions;（2）Inoculate WT（SN250）,fmp28Δ/Δand fmp28AB on RPMI 1640 and Spider liquid and solid media for mycelial induction,and compare the effects of Fmp28 on the occurrence and extension of Candida albicans hyphae at 37℃;（3）Inoculate strains WT（SN250）,fmp28Δ/Δ,fmp28AB and fmp28^OE in Spider liquid culture based on polystyrene twelve well plate induction,compare its adhesion ability and biofilm formation,and verify the promoting effect of Fmp28 on Candida albicans adhesion ability and biofilm formation at 37℃;（4）Incubate strains WT（SN250）and fmp28Δ/Δwith HUVEC cells for 1 hour,2hours,and 3 hours,respectively,to verify the effect of Fmp28 on the adhesion ability of Candida albicans to endothelial cells at 37℃;（5）By constructing murine systemic infection model and Galleria mellonella model,compare the survival curves,kidney and liver fungal burden of mice infected with strains WT（SN250）,fmp28Δ/Δand fmp28AB,the promotion effect of Fmp28 on the virulence of Candida albicans was verified;4.Explore the pathogenic mechanism of Candida Fmp28 using the following methods:（1）Inoculate strains WT（SN250）and fmp28Δ/Δinto RPMI 1640 liquid medium and induce it for 2h,extract RNA samples and send them to transcriptome for sequencing analysis,compare the expression level of differentially expressed genes,and screen out genes related to biofilm,adhesion ability and virulence;（2）The results of transcriptome analysis were verified by RT q PCR,and the differences of biofilm formation and adhesion between fmp28Δ/Δbetween strains fmp28Δ/Δ+als3^OE,fmp28Δ/Δ+pra1^OE and fmp28Δ/Δ+csa1^OE were compared,confirming the pathway of fmp28 regulating the adhesion process and biofilm formation of Candida albicans;（3）By constructing murine systemic infection model and Galleria mellonella model,the survival curves,kidney and liver fungal burden,serum biochemical indicators（UREA）,and renal injury pathological scores of mice infected with WT（SN250）,fmp28Δ/Δ,fmp28AB and fmp28Δ/Δ+als3^OE were compared to verify that Fmp28 regulates the expression of als3 and affects the virulence of Candida albicans;Results:1.All genetic recombination strains involved in the project like fmp28Δ/Δ,fmp28AB,fmp28^OE,fmp28Δ/Δ+als3^OE,fmp28Δ/Δ+pra1^OE,fmp28Δ/Δ+csa1^OE,als3^OE,pra1^OE,csa1^OE and Fmp28-3HA are successfully constructed;2.The growth curve results shows that fmp28Δ/Δhas no growth difference at30℃compared with SN250,fmp28AB and fmp28^OE,but shows obvious growth defects at 37℃（P<0.05）;when Candida albicans is subjected to heat stress,osmotic pressure and cell wall pressure,the expression of Fmp28 protein and the m RNA level of its coding gene fmp28 increased;3.The results show:（1）Compared with SN250 and fmp28AB,the hyphal length of fmp28Δ/Δis significantly shortened in liquid medium,while on solid culture medium,the colony morphology is irregular,with no obvious folds in the center of the colony,rough edges,less mycelium formation,and significantly reduced yeast hyphal state conversion ability;（2）Under thermal pressure（37℃）,add exogenous cell wall interference reagents such as CGR and fmp28Δ/ΔThe growth defect is more significant,and the sensitivity to cell wall pressure is significantly increased;（3）Compared with SN250 and fmp28AB,using polystyrene as the adhesive medium,fmp28Δ/Δshowed significant defects in adhesion ability（P<0.001）;if HUVEC cells are used as adhesion mediators,the adhesion rates of fmp28Δ/Δwere significantly lower than those of SN250（P<0.001）at 1,2,and 3 hours;（4）Under Spider induction conditions,the biofilm formation detected by crystal violet staining and dry weight method of fmp28Δ/Δwas significantly reduced compared with SN250 and fmp28AB（P<0.001）;（5）In the murine systemic infection model,under both high-dose（5×10⁶ cfu）and low-dose（5×10⁵ cfu）,the mortality rate of fmp28Δ/Δsignificantly decreased,the time of death of fmp28Δ/Δsignificantly lagged behind,and the renal fugal burden of fmp28Δ/Δsignificantly decreased（P<0.05）,fmp28Δ/Δshowed significant toxicity defects compared with SN250 and fmp28AB（P<0.001）;4.The experimental results of Fmp28 regulating the virulence of Candida albicans show that:（1）The transcriptome data combined with CGD database analysis shows that Fmp28 may affect the biofilm formation,adhesion process and toxicity of Candida albicans by regulating als3,pra1 and csa1;the results of RT-q PCR show that the m RNA levels of als3,pra1 and csa1 of strain fmp28Δ/Δare significantly down-regulated;（2）By comparing the adhesion ability and biofilm formation of fmp28Δ/Δand fmp28Δ/Δ+als3^OE,fmp28Δ/Δ+pra1^OE,fmp28Δ/Δ+csa1^OE（P<0.05）,it is determined that als3,pra1,and csa1 are located downstream of fmp28.Fmp28 promotes the adhesion process of Candida albicans by regulating the expression of als3,pra1,and csa1,then promoting biofilm formation;（3）The results of survival curves of galleria mellonella model and murine systemic infection model show that compared with group SN250 and group fmp28AB,the mortality of group fmp28Δ/Δdecreases and the time of death lags,while the mortality of group fmp28Δ/Δ+als3^OE increases and the time of death advances;in addition,the fungal burden of fmp28Δ/Δ+als3^OE in kidney and liver shows no difference from that of group fmp28Δ/Δ;the results of UREA level of cytokines related to kidney and pathological evaluation of kidney injury show that the degree of kidney injury in group fmp28Δ/Δ+als3^OE is increased compared with that in group fmp28Δ/Δ（P<0.05）;these results show that the increased expression level of als3could restore the toxicity defect caused by fmp28 deletion to a certain extent.In the virulence regulation pathway,als3 is located downstream of fmp28.Conclusion:1.Fmp28 is an important member of small heat shock protein of Candida albicans;2.The deletion of fmp28 leads to the defects of Candida albicans hypha,biofilm,adhesion,increased sensitivity to cell wall pressure under heat shock,and virulence defects in the systemic infection model;3.Fmp28 regulates the expression of Als3 and promotes virulence of Candida albicans.