Integration of homeostatic and adaptive oxidative responses by a putative co-chaperone, Wos2, drives fungal virulence in cryptococcosis

The increasing prevalence of invasive fungal pathogens are dramatically changing the clinical landscape of infectious diseases and are an imminent burden to public health that lack the resources (i.e., robust antifungals) to tackle this threat. Specifically, the human opportunistic pathogen, Cryptococcus neoformans, expresses elaborate virulence mechanisms and is equipped with sophisticated adaptation strategies to survive in harsh host environments. In this study, we extensively characterize Wos2, an Hsp90 co-chaperone homologue, featuring bilateral functioning for both cryptococcal adaptation and virulence strategies. Here, we evaluated the proteome and secretome signatures of Wos2 in enriched and infection-mimicking conditions to reveal a Wos2-dependent regulation of oxidative stress response. The wos2{Delta} strain reports defective intracellular and extracellular antioxidant protection systems measurable through a decreased abundance of critical antioxidant enzymes and reduced growth in the presence of peroxide stress. Additional Wos2-associated stress phenotypes were observed upon fungal challenge with heat shock, osmotic, and cell wall stressors. We demonstrate the importance of Wos2 for C. neoformans intracellular lifestyle during in vitro macrophage infection and provide evidence for wos2{Delta} reduced phagosomal replication levels. Accordingly, wos2{Delta} featured significantly reduced virulence in a murine model of cryptococcosis. Our study highlights a vulnerable point in the fungal chaperone network that offers a powerful druggable opportunity to interfere with both virulence and fitness. Author SummaryThe global impact of fungal pathogens, both emerging and emerged, is undeniable and the alarming increase in antifungal resistance rates hampers our ability to protect the global population from deadly infections. For cryptococcal infections, a limited arsenal of antifungals and resistance demands alternative therapeutic strategies, including an anti-virulence approach, which disarms the pathogen of critical virulence factors, empowering the host to remove the pathogen and clear the infection. To this end, we apply state-of-the-art mass spectrometry-based proteomics to interrogate the impact of a recently defined novel co-chaperone, Wos2, towards cryptococcal virulence using in vitro and in vivo models of infection. We defined global proteome and secretome remodeling driven by the protein and uncovered a novel role in modulating the fungal oxidative stress response. Complementation of the proteome findings with in vitro infectivity assays demonstrated a protective role for Wos2 within the macrophage phagosome, influencing fungal replication and survival. These results underscore differential cryptococcal survivability and weakened patterns of dissemination in the absence of wos2. Overall, our study establishes Wos2 as an important contributor to fungal pathogenesis and warrants further research into critical proteins within global stress response networks as potential druggable targets to reduce fungal virulence and clear the infection.