establishment-an-vitro-system-to-study-intracellular-behavior-candida

Establishment of an <em>In vitro</em> System to Study Intracellular Behavior of <em>Candida glabrata</em> in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Centre for DNA Fingerprinting and Diagnostics, Andhra Pradesh, India

Redox pathways play a key role in pathogenesis. Glutathione, a central molecule in redox homeostasis in yeasts, is an essential metabolite, but its requirements can be met either from endogenous biosynthesis or from the extracellular milieu. In this report we have examined the importance of glutathione biosynthesis in two major human opportunistic fungal pathogens, Candida albicans and Candida glabrata. As the genome sequence of C. glabrata had suggested the absence of glutathione transporters, we initially investigated exogenous glutathione utilization in C. glabrata by disruption of the MET15 gene, involved in methionine biosynthesis. We observed an organic sulphur auxotrophy in a C. glabrata met15Δ strain; however, unlike its Saccharomyces cerevisiae counterpart, the C. glabrata met15Δ strain was unable to grow on exogenous glutathione. This inability to grow on exogenous glutathione was demonstrated to be due to the lack of a functional glutathione transporter, despite the presence of a functional glutathione degradation machinery (the Dug pathway). In the absence of the ability to obtain glutathione from the extracellular medium, we examined and could demonstrate that γ-glutamyl cysteine synthase, the first enzyme of glutathione biosynthesis, was essential in C. glabrata. Further, although γ-glutamyl cysteine synthase has been reported to be non-essential in C. albicans, we report here for what is believed to be the first time that the enzyme is required for survival in human macrophages in vitro, as well as for virulence in a murine model of disseminated candidiasis. The essentiality of γ-glutamyl cysteine synthase in C. glabrata, and its essentiality for virulence in C. albicans, make the enzyme a strong candidate for antifungal development.

Glutathione biosynthesis in the yeast pathogens Candida glabrata and Candida albicans: essential in C. glabrata, and essential for virulence in C. albicans.

Fungal septicemia is an increasingly common complication of immunocompromised patients worldwide. Candida species are the leading cause of invasive mycoses with Candida glabrata being the second most frequently isolated Candida species from Intensive Care Unit patients. Despite its clinical importance, very little is known about the mechanisms that C. glabrata employs to survive the antimicrobial and immune response of the mammalian host. Here, to decipher the interaction of C. glabrata with the host immune cells, we have screened a library of 18,350 C. glabrata Tn7 insertion mutants for reduced survival in human THP-1 macrophages via signature-tagged mutagenesis approach. A total of 56 genes, belonging to diverse biological processes including chromatin organization and golgi vesicle transport, were identified which are required for survival and/or replication of C. glabrata in macrophages. We report for the first time that C. glabrata wild-type cells respond to the intracellular milieu of macrophage by modifying their chromatin structure and chromatin resistance to micrococcal nuclease digestion, altered epigenetic signature, decreased protein acetylation and increased cellular lysine deacetylase activity are the hall-marks of macrophage-internalized C. glabrata cells. Consistent with this, mutants defective in chromatin organization (Cgrsc3-aΔ, Cgrsc3-bΔ, Cgrsc3-aΔbΔ, Cgrtt109Δ) and DNA damage repair (Cgrtt107Δ, Cgsgs1Δ) showed attenuated virulence in the murine model of disseminated candidiasis. Further, genome-wide transcriptional profiling analysis on THP-1 macrophage-internalized yeasts revealed deregulation of energy metabolism in Cgrsc3-aΔ and Cgrtt109Δ mutants. Collectively, our findings establish chromatin remodeling as a central regulator of survival strategies which facilitates a reprogramming of cellular energy metabolism in macrophage-internalized C. glabrata cells and provide protection against DNA damage.

Article	Year
Glutathione biosynthesis in the yeast pathogens Candida glabrata and Candida albicans: essential in C. glabrata, and essential for virulence in C. albicans. Microbiology (Reading, England)\| PubMed ID: 20966090	2011
Functional genomic analysis of Candida glabrata-macrophage interaction: role of chromatin remodeling in virulence. PLoS pathogens\| PubMed ID: 22916016	2012

Maruti Nandan Rai

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