Rewiring of Signaling Networks Modulating Thermotolerance in the Human Pathogen Cryptococcus neoformans

Abstract

Thermotolerance is a crucial virulence attribute for human pathogens, including the fungus Cryptococcus neoformans that causes fatal meningitis in humans. Loss of the protein kinase Sch9 increases C. neoformans thermotolerance, but its regulatory mechanism has remained unknown. Here, we studied the Sch9-dependent and Sch9-independent signaling networks modulating C. neoformans thermotolerance by using genome-wide transcriptome analysis and reverse genetic approaches. During temperature upshift, genes encoding for molecular chaperones and heat shock proteins were upregulated, whereas those for translation, transcription, and sterol biosynthesis were highly suppressed. In this process, Sch9 regulated basal expression levels or induced/repressed expression levels of some temperature-responsive genes, including heat shock transcription factor (HSF1) and heat shock proteins (HSP104 and SSA1). Notably, we found that the HSF1 transcript abundance decreased but the Hsf1 protein became transiently phosphorylated during temperature upshift. Nevertheless, Hsf1 is essential for growth and its overexpression promoted C. neoformans thermotolerance. Transcriptome analysis using an HSF1 overexpressing strain revealed a dual role of Hsf1 in the oxidative stress response and thermotolerance. Chromatin immunoprecipitation demonstrated that Hsf1 binds to the step-type like heat shock element (HSE) of its target genes more efficiently than to the perfect-or gap-type HSE. This study provides insight into the thermotolerance of C. neoformans by elucidating the regulatory mechanisms of Sch9 and Hsf1 through the genome-scale identification of temperature-dependent genes. KEYWORDS Hsf1; Sch9; transcriptome analysis; high temperature; Cryptococcus neoformans R ESPONSE and adaptation to their host's physiological temperature, 37°, are key virulence attributes for most human fungal pathogens that infect from natural environments at ambient temperature. Cryptococcus neoformans is an example of such a pathogen. This basidiomycetous fungus exists in diverse environment niches and generates infectious yeast cells or spores through bisexual and unisexual differentiation, which are inhaled through the respiratory tract in the host and initially colonize the lungs (Idnurm et al. 2005;Lin and Heitman 2006). C. neoformans is subsequently disseminated into the bloodstream, breaches the central nervous system, and eventually elicits fatal meningoencephalitis, responsible for an estimated hundreds of thousands of deaths annually (Park et al. 2009). During this infectious process, the ability to sense and adapt to temperature upshift are crucial factors for the pathogen to establish the initial colonization of the lungs. Therefore, most mutants that are severely defective in thermotolerance tend to be highly attenuated in virulence. Due to such reasons, signaling cascades governing thermotolerance in fungi have been intensively investigated toward their exploitation as potential antifungal drug targets (Bahn et al. 2007;Bahn and Jung 2013). In...

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