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Fungal diseases impact the lives of a millions of people across the globe, and with our current repertoire of therapeutic options dwindling, effective treatment strategies are urgently needed. Critically, the emergence of azole-resistant isolates in the clinic following prolonged treatment regimes, environmental fungicide exposure, and fungal evolution, threatens the outcome of current therapeutics, further endangering the survival of infected individuals. Here, we investigate the underpinnings of antifungal resistance using quantitative proteomics to discover protein-level signatures of fluconazole (FLC) resistance in the opportunistic human fungal pathogen, Cryptococcus neoformans . We explore ClpX, an ATP-dependent unfoldase, as a target to overcome FLC resistance and explore target efficacy through macrophage and murine models of cryptococcal infection. Here we show that disruption of ClpX, following gene deletion or targeted inhibition, re-introduces FLC susceptibility into resistant strains, rendering FLC treatment effective once again. Further, we identify and experimentally confirm mechanisms by which ClpX influences susceptibility to FLC, through association with both heme biosynthesis and ergosterol production. Overall, our results contribute to the understanding of mechanisms driving FLC resistance in a globally important fungal pathogen, and we provide avenues for targeting proteins as a therapeutic strategy to reverse antifungal resistance. Here, the authors reveal that the ATP-dependent unfoldase ClpX is a key driver of fluconazole resistance in Cryptococcus neoformans , and show that targeting ClpX restores drug susceptibility, offering a potential therapeutic strategy to reverse antifungal resistance and to render current drugs effective.

Background Fungal infections impact over 25% of the global population. For the opportunistic fungal pathogen, Cryptococcus neoformans , infection leads to cryptococcosis. In the presence of the host, disease is enabled by elaboration of sophisticated virulence determinants, including polysaccharide capsule, melanin, thermotolerance, and extracellular enzymes. Conversely, the host protects itself from fungal invasion by regulating and sequestering transition metals (e.g., iron, zinc, copper) important for microbial growth and survival. Results Here, we explore the intricate relationship between zinc availability and fungal virulence via mass spectrometry-based quantitative proteomics. We observe a core proteome along with a distinct zinc-regulated protein-level signature demonstrating a shift away from transport and ion binding under zinc-replete conditions towards transcription and metal acquisition under zinc-limited conditions. In addition, we revealed a novel connection among zinc availability, thermotolerance, as well as capsule and melanin production through the detection of a Wos2 ortholog in the secretome under replete conditions. Conclusions Overall, we provide new biological insight into cellular remodeling at the protein level of C. neoformans under regulated zinc conditions and uncover a novel connection between zinc homeostasis and fungal virulence determinants.

Intracellular bacterial pathogens have evolved sophisticated infection strategies, including the release and secretion of virulence factors to interfere with host cell functions and to perturb immune responses. For Salmonella enterica serovar Typhimurium (S. Typhimurium), the type III secretion systems encoded on Salmonella pathogenicity islands (SPI) 1 and 2 mediates invasion of the bacterium into innate immune cells and regulates bacterial replication and survival within the hostile environment of the host, respectively. Here, we explore the temporal and strain-specific dual perspective response of both the host and pathogen during cellular infection via quantitative proteomics. We report time- and pathogenicity island-specific expression and secretion of infection-associated proteins (i.e., SL1344_1263, SL1344_3112, SL1344_1563, and YnhG) and regulated immune response proteins in macrophage, including Cd86, Cd40, Casp4, C3, IL-1?, and Cd69). Through intracellular macrophage and in vivo murine models of infection, we reveal a role in virulence for three of the bacterial proteins (SL1344_1263, SL1344_1563, and YnhG), defining their importance as novel T3SS effectors. We characterize the temporal intra- and extracellular production of the effectors and identify their interaction networks in host cells, representing inhibitory and stimulatory pathways mounted by invading bacterial pathogens. Competing Interest Statement The authors have declared no competing interest.