| Background: Candida krusei is one of the five common pathogens of candidiasis,which can cause candidemia,endophthalmitis,arthritis,and endocarditis.Recently,it has attracted more and more attention,because of its high mortality and low response to standard antifungal therapy.As an emerging multidrug resistant pathogen,C.krusei mostly occurs in patients with severe immunodeficiency,hematological malignancies,and those receiving azole antifungal drugs,and is always associated with high lethality.Candida krusei is naturally resistant to fluconazole,an antifungal drug widely used in clinic,and has reduced sensitivity to amphotericin B.It is of great significance to explore the mechanism of drug resistance in C.krusei and actively search for potential therapeutic targets.The development of mass spectrometry-based proteomics and protein post-translational modification omics provides an effective approach to study the drug resistance of fungi.Our research aims to analyze the differentially expressed proteins between the induced drugresistant strain and its parental strain at the whole proteome level,explore the biological functions of these differential proteins,and find potential therapeutic targets.In addition,the differential expression of acylation between the resistant strain and its parental strain was also detected at the level of PTM,and the effect of acylation on acquired drug resistance of C.krusei was discussed.Methods:(1)The clinical isolates of C.krusei were collected from three tertiary hospitals in Shanghai.These strains were initially screened by CHROMagar Candida Media,and then further identified by internal transcribed spacer sequencing.According to the CLSI M27-A3 recommended by the Clinical and Laboratory Standards Institute,the microdilution method was performed to test the antifungal susceptibility of C.krusei against nine antifungal agents(echinocandins,polyenes,azoles,etc.).In addition,the susceptibility of all strains to AMB was confirmed by Etest.(2)The AMB sensitive strains were continuously subcultured in YPD liquid medium with AMB concentration cascade multiplication to induce AMB resistant strain in vitro.Each concentration was repeated at least five times.If the strains could still survive normally until the concentration of amphotericin B reached 32 μg/m L,it indicated that the induction of resistant strains was successful.And then,the resistant strain was passaged for 10 consecutive times on drug-free medium to detect the stability of the drug-resistant phenotype.Moreover,the changes of amphotericin B MIC values,growth rate,morphology and sensitivity to other antifungal drugs were compared before and after induction.(3)4D Label free quantitative proteomics were used to identify the differentially expressed proteins between the induced drug-resistant strain and its parental strain.Bioinformatics analysis,including GO analysis,subcellular localization,KEGG analysis,etc.was performed to analyze the functions of differential expressed proteins.Parallel reaction monitoring(PRM)was conducted to validate the results of the proteome.(4)The differential expression of the novel acylation between the induced resistant strain and its parental strain was evaluated by Western blot(WB).According to the results of WB,we initiated a first screening of lysine succinylation in these two strains to analyze the differential expressed succinylation sites.Bioinformatics analysis was performed to analyze the functions of differentially expressed succinylated proteins.Then,the checkerboard dilution method was applied to evaluated whether the histone acetyltransferase inhibitor C646 and AMB had synergistic effect based on the fractional inhibitory concentration index(FICI).Results:(1)A total of 21 isolates of C.krusei were obtained from clinical samples of patients in three hospitals.The patients were mainly distributed in general surgery(n = 6),infection department(n = 5)and ICU(n = 4).The strains were mainly isolated from blood specimens(n = 7),anastomotic drainage fluid(n = 6)and sputum(n = 5).The most common comorbidities were hematologic/solid neoplasms(n = 15),and other detailed clinical data were not available in our study.Among the nine antifungal agents tested in this study,all C.krusei strains showed high sensitivity to echinocinins,but all showed high MIC values for fluconazole with MIC no less than64 μg/m L.Five strains(5/21,23.81%)were considered as non-wild type(MIC = 0.5μg/m L)to caspofungin.Seven strains were considered as non-wild type(MIC = 1 μg/m L)to voriconazole.As for amphotericin B,all strains were regarded as wild-type(MIC ≤ 2 μg/ m L)which were confirmed by Etest,but four strains showed MIC values of 2 μg/ m L.(2)After the induction by amphotericin B,two C.krusei isolates acquired stable drug-resistant phenotypes.Under the long-term intervention of AMB,C.krusei underwent many changes to adapt to the selective pressure of amphotericin B.Compared with its parental strain,the induced drug-resistant strain showed reduced growth rates and prolonged lag period measured by the growth curve and colony morphology.Besides,the cell morphology of the resistant strain was irregular,and some cells appeared convex on their surface.In addition,Etest showed that the induced drug-resistant strain was more sensitive to azoles.(3)A total of 415 proteins were found differentially expressed between the resistant strain and its parental strain,among which 252 were significantly upregulated and 163 were significantly down-regulated.Cytochrome P450 lanosterol of14-alpha-demethylase(ERG11,encoding by ERG11),cytochrome P450 61(ERG5,encoding by ERG5),key enzymes of oxidative stress and other enzymes with metabolic activity,especially the key enzyme of acylation histone acetyltransferase type B catalytic subunit were significantly up-regulated in the resistant strain.Bioinformatics analysis showed that the differential expressed proteins were mainly involved in the amino acid biosynthesis and metabolism,sulfur metabolism and starch and sucrose metabolism.The results of PRM and proteomes are highly consistent,indicating that the results of proteomics are highly reliable.(4)WB showed that the overall levels of lysine succinylation,crotonylation and2-hydroxyisobutyrylation of the induced drug-resistant strains were significantly higher than those of its parental strain.383 differentially expressed succinylation sites were identified.Among them,344 succinylation sites in 134 proteins were upregulated,and 39 sites in 23 proteins were down-regulated,indicating that the overall level of succinylation in the resistant strain is indeed up-regulated.Bioinformatics analysis shows that the differential proteins are mainly involved in ribosome,glycolysis and gluconeogenesis,fructose and mannose metabolism and RNA degradation pathways.Moreover,we found that the histone acetyltransferase inhibitor C646 was able to synergize the antifungal effect of AMB against C.krusei with the FICI values under 0.5.Conclusion: In this study,we successfully induced the amphotericin B resistant C.krusei with parental strains using gradually increased concentration of AMB,which provided vital models to explore the mechanisms of drug resistance in the follow-up.Studies based on proteome and PRM showed that enzymes with metabolic activity such as ERG11 and ERG5 in ergosterol synthesis pathway were significantly overexpressed in drug-resistant strains,revealing the relationship between metabolism and amphotericin B resistance.In addition,we demonstrated that the level of acylation modification of induced drug-resistant strains was up-regulated,and many up-regulated succinylation sites with were identified.Our study confirmed for the first that the sensitivity of C.krusei to AMB could be promoted by inhibiting histone acyltransferase activity. |
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