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The involvement of the Candida glabrata trehalase enzymes in stress resistance and gut colonization.

ABSTRACT: Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo to determine the role of these enzymes in virulence. Ath1 was found to be located in the periplasm and was essential for growth on trehalose as sole carbon source, while Nth1 on the other hand was important for oxidative stress resistance, an observation which was consistent by the lower survival rate of the NTH1 deletion strain in human macrophages. No significant phenotype was observed for Nth2. The triple deletion strain was unable to establish a stable colonization of the gastrointestinal (GI) tract in mice indicating the importance of having trehalase activity for colonization in the gut.

SUBMITTER: Van Ende M

PROVIDER: S-EPMC7808424 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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The involvement of the Candida glabrata trehalase enzymes in stress resistance and gut colonization.

Van Ende Mieke M Timmermans Bea B Vanreppelen Giel G Siscar-Lewin Sofía S Fischer Daniel D Wijnants Stefanie S Romero Celia Lobo CL Yazdani Saleh S Rogiers Ona O Demuyser Liesbeth L Van Zeebroeck Griet G Cen Yuke Y Kuchler Karl K Brunke Sascha S Van Dijck Patrick P

Virulence 20211201 1

Candida glabrata is an opportunistic human fungal pathogen and is frequently present in the human microbiome. It has a high relative resistance to environmental stresses and several antifungal drugs. An important component involved in microbial stress tolerance is trehalose. In this work, we characterized the three C. glabrata trehalase enzymes Ath1, Nth1 and Nth2. Single, double and triple deletion strains were constructed and characterized both in vitro and in vivo ...[more]

PMID: 33356857

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Project description:Candida glabrata is a high-performance microbial cell factory for the production of organic acids. To elucidate the role of the C. glabrata Mediator tail subunit Med2 (CgMed2) at pH 2.0, we deleted or overexpressed CgMed2 and used transcriptome analysis to identify genes that are regulated by CgMed2. At pH 2.0, the deletion of CgMed2 resulted in a cell growth decrease of 26.1% and a survival decrease of 32.3%. Overexpression of CgMed2 increased cell growth by 12.4% and cell survival by 5.9% compared to the wild-type strain. Transcriptome and phenotypic analyses identified CgYap6 as a transcription factor involved in acid pH stress tolerance. Deletion of CgYap6 caused growth defects, whereas its overexpression enhanced cell growth at pH 2.0. Furthermore, total glycerophospholipid content and membrane integrity decreased by 33.4% and 21.8%, respectively, in the CgMed2Δ strain; however, overexpression of CgMed2 increased the total glycerophospholipid content and membrane integrity by 24.7% and 12.1%, respectively, compared with those of the wild-type strain at pH 2.0. These results demonstrated that under acid pH stress, CgMed2 physically interacts with CgYap6, which translocates from the cytoplasm to the nucleus after being phosphorylated by the protein kinase CgYak1. Once in the nucleus, CgYap6 recruits CgMed2 to express glycerophospholipid-related genes. Our study elucidated the function of CgMed2 under acid pH stress and provides a potential strategy to equip Candida glabrata with low-pH resistance during organic acid fermentation.IMPORTANCE This study investigated the function of the Mediator tail subunit CgMed2 in C. glabrata under low-pH stress. The protein kinase CgYak1 activates CgYap6 for the recruitment of CgMed2, which in turn increases glycerophospholipid content and membrane integrity to confer low-pH stress tolerance. This study establishes a new link between the Mediator tail subunit and transcription factors. Overall, these findings indicate that CgMed2 is a novel target to induce the low-pH stress response in C. glabrata.

Project description:Microarray was used to analyze azole resistance of Candida glabrata oropharyngeal isolates from 7 hematopoietic stem cell transplant recipients receiving fluconazole prophylaxis. Transcriptional profiling of the sequential-paired clinical isolates by microarray revealed 19 genes upregulated in the majority of resistant isolates compared to their paired-susceptible isolates. All seven resistant isolates had greater than two fold upregulation of CgPDR1, a master transcriptional regulator of PDR network, and all 7 resistant isolates showed upregulation of known CgPDR1-target genes. The altered transcriptome can be explained in part by the observation that all 7 resistant isolates had acquired a single nonsynonymous mutation in their CgPDR1 ORF. Four mutations occurred in the regulatory domain (L280P, L344S, G348A, S391L) and one in the activation domain (G943S) while two mutations (N764I, R772I) occurred in an undefined region. Association of azole resistance and the CgPDR1 mutations was investigated in the same genetic background by introducing the CgPDR1 sequences from one sensitive and five resistant isolates into a laboratory azole-sensitive strain (cgpdr1) via integrative transformation. The cgpdr1 strain was restored to wild-type fluconazole susceptibility when transformed with CgPDR1 from the susceptible isolate but became resistant when transformed with CgPDR1 from the resistant isolates. However, despite the identical genetic background, upregulation of CgPDR1 and CgPDR1-target genes varied between the 5 transformants, independent of the domain locations in which the mutations occurred. In sum, gain-of-function mutations in CgPDR1 not only contributed to the clinical azole resistance but different mutations had varying degrees of impact on the CgPDR1-target genes. Two groups (C1S/84 and C16R/84) consisted of 4 biological replicates in which a complemented strain sample was paired with the laboratory wild type strain 84 sample. Each group included 1 reciprocally labeled sample.

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The involvement of the Candida glabrata trehalase enzymes in stress resistance and gut colonization.

Publications

The involvement of the <i>Candida glabrata</i> trehalase enzymes in stress resistance and gut colonization.

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