Polyphosphatases have a polyphosphate-independent influence on the virulence of <i>Cryptococcus neoformans</i>

<i>Cryptococcus neoformans,</i> an invasive basidiomycete fungal pathogen, causes one of the most prevalent, life-threatening diseases in immunocompromised individuals and accounts for ~19% of AIDS-associated deaths. Therefore, understanding the pathogenesis of <i>C. neoformans</i> and its interactions with the host immune system is critical for developing therapeutics against cryptococcosis. Previous studies demonstrated that <i>C. neoformans</i> cells lacking polyphosphate (polyP), an immunomodulatory polyanionic storage molecule, display altered cell surface architecture but unimpaired virulence in a murine model of cryptococcosis. However, the relevance of cell surface changes and the role of hyperaccumulation of polyP in the virulence of <i>C. neoformans</i> remain unclear. Here we show that mutants with abundant polyP due to loss of the polyphosphatases Xpp1 and Epp1 are attenuated for virulence. The double mutant differed from the wild type during disease by demonstrating a higher fungal burden in disseminated organs at the experimental endpoint and by provoking an altered immune response. An analysis of triple mutants lacking the polyphosphatases and the Vtc4 protein for polyP synthesis also caused attenuated virulence in mice, thus suggesting an influence of Xpp1 and/or Epp1 independent of polyP levels. A more detailed characterization revealed that Xpp1 and Epp1 play multiple roles by contributing to the organization of the cell surface, virulence factor production, the response to stress, and mitochondrial function. Overall, we conclude that polyphosphatases have additional functions in the pathobiology of <i>C. neoformans</i> beyond an influence on polyP levels.IMPORTANCECryptococcus neoformans causes one of the most prevalent fungal diseases in people with compromised immune systems and accounts for ~19% of AIDS-associated deaths worldwide. The continual increase in the incidence of fungal infections and limited treatment options necessitate the development of new antifungal drugs and improved diagnostics. Polyphosphate (polyP), an under-explored biopolymer, functions as a storage molecule, modulates the host immune response, and contributes to the ability of some fungal and bacterial pathogens to cause disease. However, the role of polyP in cryptococcal disease remains unclear. In this study, we report that the polyphosphatase enzymes that regulate polyP synthesis and turnover contribute to the virulence of <i>C. neoformans</i> in a mouse model of cryptococcosis. The polyphosphatases influenced the survival of <i>C. neoformans</i> in macrophages and altered the host immune response. In addition, the mutants lacking the enzymes have changes in cell surface architecture and size, as well as defects in both mitochondrial function and the stress response. By using mutants defective in the polyphosphatases and polyP synthesis, we demonstrate that many of the phenotypic contributions of the polyphosphatases are independent of polyP.