Proteome signatures reveal homeostatic and adaptive oxidative responses by a putative co-chaperone, Wos2, to influence fungal virulence determinants in cryptococcosis.

The increasing prevalence of invasive fungal pathogens is dramatically changing the clinical landscape of infectious diseases, posing an imminent threat to public health. Specifically, Cryptococcus neoformans, the human opportunistic pathogen, expresses elaborate virulence mechanisms and is equipped with sophisticated adaptation strategies to survive in harsh host environments. This study extensively characterizes Wos2, an Hsp90 co-chaperone homolog, featuring bilateral functioning for both cryptococcal adaptation and the resulting virulence response. In this study, we evaluated the proteome and secretome signatures associated with wos2 deletion in enriched and infection-mimicking conditions to reveal Wos2-dependent regulation of the oxidative stress response through global translational reprogramming. The wos2Δ strain demonstrates 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 stress, and cell membrane stressors. We demonstrate the importance of Wos2 for intracellular lifestyle of C. neoformans during in vitro macrophage infection and provide evidence for reduced phagosomal replication levels associated with wos2Δ. Accordingly, wos2Δ featured significantly reduced virulence within impacting fungal burden in a murine model of cryptococcosis. Our study highlights a vulnerable point in the fungal chaperone network that offers a therapeutic opportunity to interfere with both fungal virulence and fitness.IMPORTANCEThe 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 increasing rates of resistance demand alternative therapeutic strategies, including an anti-virulence approach, which disarms the pathogen of critical virulence factors, empowering the host to remove the pathogens and clear the infection. To this end, we apply state-of-the-art mass spectrometry-based proteomics to evaluate the impact of a recently defined novel co-chaperone, Wos2, toward cryptococcal virulence using in vitro and in vivo models of infection. We explore global proteome and secretome remodeling driven by the protein and uncover the novel role in modulating the fungal oxidative stress response. Complementation of proteome findings with in vitro infectivity assays demonstrated the protective role of 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 infection.

The dichotomous effects of caffeine on homologous recombination in mammalian cells.

This study was initiated to examine the effects of caffeine on the DNA damage response (DDR) and homologous recombination (HR) in mammalian cells. A 5 mM caffeine treatment caused the cell cycle to stall at G2/M and cells eventually underwent apoptosis. Caffeine exposure also induced a strong DDR along with subsequent activation of wildtype p53 protein. An unexpected observation was the caffeine-induced depletion of Rad51 (and Brca2) proteins. Consequently, caffeine-treated cells were expected to be inefficient in HR. However, a dichotomy in the HR response of cells to caffeine treatment was revealed. Caffeine treatment rendered cells significantly better at performing the nascent DNA synthesis that accompanies the early strand invasion steps of HR. Additionally, caffeine treatment increased chromatin accessibility and elevated the efficiency of illegitimate recombination. Conversely, the increase in nascent DNA synthesis did not translate into a higher number of gene targeting events. Thus, prolonged caffeine exposure stalls the cell cycle, induces a p53-mediated apoptotic response and a down-regulation of critical HR proteins, and for reasons discussed, stimulates early steps of HR, but not the formation of complete recombination products.