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A proteomic landscape of Candida albicans in the stepwise evolution to fluconazole resistance

ABSTRACT: As an opportunistic fungal pathogen, Candida albicans is a major cause of superficial and systemic infections in immunocompromised patients. The increasing rate of azole resistance in C. albicans has brought further challenges to clinical therapy. In this study, we collect five isogenic C. albicans strains recovered over discrete intervals from an HIV-infected patient who suffered a two-year recurrent oropharyngeal candidiasis. Azole resistance was known from the clinical history to have developed gradually in this patient, and this was confirmed by MIC assay of each strain. Proteomic techniques can be used to investigate more comprehensively how resistance develops in pathogenic fungi over time. Our study is the first using tandem mass tag (TMT) labeling combined with liquid chromatography-mass spectrometry (LC-MS/MS) technology to investigate the acquired resistance mechanisms of serial C. albicans isolates at the proteomic level. A total of 4029 proteins have been identified, of which 3766 have been quantified. Compared with Ca1, bioinformatics analysis showed that differentially expressed proteins were mainly associated with the following aspects: down-regulation of glycolysis/gluconeogenesis, pyruvate metabolism, fatty acid degradation and oxidative stress response proteins in all four subsequent strains, but – remarkably – activation of amino acids metabolism in Ca8 and Ca14; increase in the protection against osmotic stress or excessive copper toxicity, up-regulation of respiratory chain activity, and suppression of iron transport in Ca17. By tracing proteomic alterations in this set of isogenic resistance isolates, we acquire mechanistic insight into the steps involved in the acquisition of azole resistance in C. albicans.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Candida Albicans (yeast)

TISSUE(S): Oral Mucosa

SUBMITTER: Nana Song

LAB HEAD: Xiaofang Li

PROVIDER: PXD031774 | Pride | 2022-02-23

REPOSITORIES: Pride

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Project description:In the pathogenic yeast Candida albicans, the zinc cluster transcription factor Upc2p has been shown to regulate expression of ERG11 and other genes involved in ergosterol biosynthesis upon exposure to azole antifungals. ERG11 encodes lanosterol demethylase, the target enzyme of this antifungal class. Over-expression of UPC2 reduces azole susceptibility, whereas its disruption results in hypersusceptibility to azoles and reduced accumulation of exogenous sterols. Constitutive up-regulation of ERG11 is a major cause of resistance to fluconazole in clinical isolates of C. albicans, yet the mechanism for this has yet to be determined. Using genome-wide gene expression profiling, we found UPC2 and other genes involved in ergosterol biosynthesis to be coordinately up-regulated with ERG11 in a fluconazole resistant clinical isolate as compared with a matched susceptible isolate from the same patient. Sequence analysis of the UPC2 alleles of these isolates revealed that the resistant isolate contained a single nucleotide substitution in one UPC2 allele that resulted in a G648D exchange in the encoded protein. Introduction of the mutated allele into a drug susceptible strain resulted in constitutive up-regulation of ERG11 and increased resistance to fluconazole. By comparing the gene expression profiles of the fluconazole resistant isolate and of strains carrying wild-type and mutated UPC2 alleles, we identified target genes that are controlled by Upc2p. Here we show for the first time that a gain-of-function mutation in UPC2 leads to increased expression of ERG11 and imparts resistance to fluconazole in clinical isolates of C. albicans. Keywords: genome-wide expression profiling Clinical isolates S1 (susceptible) and S2 (resistant), genome strain SC5314, UPC2 disruption strains (UPC2M4A and UPC2M4B), UPC2 re-integrant strains of non-mutated UPC2 allele (UPC2M2K21A and UPC2M2K21B), and UPC2 re-integrant strains of mutated UPC2 allele (UPC2M2K31A and UPC2M2K31B) were grown for 3 hours until log phase. RNA was extracted for each isolate/strain. Experiments were performed in duplicate.

Project description:In Candida albicans, the transcription factor Upc2 is central to the regulation of ergosterol biosynthesis. UPC2-activating mutations contribute to azole resistance, whereas disruption increases azole susceptibility. In the present study, we investigated the relationship of UPC2 to fluconazole susceptibility, particularly in azole-resistant strains. In addition to the reduced fluconazole MIC previously observed with UPC2 disruption, we observed a lower minimum fungicidal concentration (MFC) for a upc2M-NM-^T/M-NM-^T mutant than for its azole-susceptible parent, SC5314. Moreover, the upc2M-NM-^T/M-NM-^T mutant was unable to grow on a solid medium containing 10 M-BM-5g/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed higher fungistatic activity against the upc2M-NM-^T/M-NM-^T mutant than against SC5314. UPC2 disruption in strains carrying specific resistance mutations also resulted in reduced MICs and MFCs. UPC2 disruption in a highly azole resistant clinical isolate containing multiple resistance mechanisms likewise resulted in a reduced MIC and MFC. This mutant was unable to grow on a solid medium containing 10 M-BM-5g/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed increased fungistatic activity against the upc2M-NM-^T/M-NM-^T mutant in the resistant background. Microarray analysis showed attenuated induction by fluconazole of genes involved in sterol biosynthesis, iron transport, or iron homeostasis in the absence of UPC2. Taken together, these data demonstrate that the UPC2 transcriptional network is universally essential for azole resistance in C. albicans and represents an attractive target for enhancing azole antifungal activity. We examined the genome-wide gene expression profiles of the wild-type parent strain SC5314 and its upc2M-NM-^T/M-NM-^T derivative in response to fluconazole in order to identify genes whose expression in response to fluconazole is influenced by Upc2.

Project description:Azole resistance and varying degrees of cross-resistance to other members of the azole family in clinical isolates have been documented, which has necessitated additional and prolonged use of the antifungal agents available. 2-Amino-Nonyl-6-Methoxyl-Tetralin Muriate (10b), a novel chemical structural aminotetralin derivate, is synthesized as an antifungal agent and exibited strong antifungal activity. To further investigated the action mechanism, we used microarray analysis to investigate the genes expression profiles of C. albicans cells treated or untreated with 10b and found 957 genes were differentially expressed. Of them,457 showed a decrease in expression and 500 showed an increase in expression. 33 down-regulated genes were involved in glycolysis (e.g., PFK1, CDC19 and HXK2), fermentation (e.g., PDC11, ALD5 and ADH1) and respiratory electron transport chain (e.g., CBP3, COR1 and QCR8). 30 differentially expressed genes were found to relate to biofilm formation, filamentous or hyphal growth. It was noticed that striking up-regulation of SFL1 and marked down-regulation of YWP1 directly related to prevent C. albicans from changing its morphology from the yeast form to the hyphal. Two genes related to specifically hydrolyzing beta-1, 3 glucan (e.g., XOG1) and chitin (e.g., CHT1) were significantly increased. 40 overexpressed genes and 15 down-regulated genes were related to the lipid metabolic process. Of them, Eight were directly linked to ergosterol biosynthesis, including ERG2, ERG6 and ERG11. 99 genes related to translation were down-regulated following exposure to 10b, which account for 21.66% in down-regulated genes. This suggested that translation might be lower in SC5314 cells exposed to 10b than in control. Total RNA from the control SC5314 cells and 10b-treated SC5314 cells were used to generate target cDNA, and then hybridized to 8k Candida albicans Genome Array Genechips, representing about 7925 characterized Candida albicans genes. Two independent experiments were conducted. Reference strain was control SC5314 cells and test strain was SC5314 cells treated with 10b.

Project description:Azole antifungal agents such as fluconazole exhibit fungistatic activity against Candida albicans. Strategies to enhance azole antifungal activity would be therapeutically appealing. In an effort to identify transcriptional pathways that influence fluconazole susceptibility, we sought to identify transcription factors (TFs) involved in this process. From a collection of C. albicans strains disrupted for genes encoding TFs (Homann et al., PLoS Genet. 2009;5:e1000783), four exhibited a marked reduction in minimum fungicidal concentration (MFC) in both RPMI and YPD media. One of these, UPC2, has been previously characterized with regard to its role in azole susceptibility. Of mutants representing the three remaining TF genes of interest, one (CAS5) was unable to recover from fluconazole exposure at concentrations as low as 2 M-BM-5g/mL after 72 hours in YPD medium. This mutant also showed reduced susceptibility and a clear zone of inhibition by Etest, was unable to grow on solid media containing 10 M-BM-5g/mL fluconazole, and exhibited increased susceptibility by time-kill analysis. CAS5 disruption in highly azole-resistant clinical isolates exhibiting multiple resistance mechanisms did not alter susceptibility. However, CAS5 disruption in strains with specific resistance mutations in ergosterol biosynthesis or efflux pumps resulted in a moderate reduction in MIC and MFC. Genome-wide transcriptional analysis was performed in the presence of fluconazole and was consistent with the suggested role of CAS5 in cell wall organization while also suggesting a role in iron transport and homeostasis. These findings suggest that Cas5 regulates a transcriptional network that influences susceptibility of C. albicans to fluconazole. Further delineation of this transcriptional network may identify targets for potential co-therapeutic strategies to enhance the activity to the azole class of antifungals. We examined the genome-wide gene expression profiles of the wild-type parent strain SC5314 and its cas5M-NM-^T/M-NM-^T derivative in response to fluconazole in order to identify genes whose expression in response to fluconazole is influenced by Cas5.

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A proteomic landscape of Candida albicans in the stepwise evolution to fluconazole resistance

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