Proteomics, drugs and nonlytic exocytosis: The pursuit of novel therapies against Cryptococcus neoformans

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen found worldwide that causes cryptococcosis, a potentially life-threatening disease in immunocompromised individuals. To gain insight into novel therapeutic targets, we did a proteomic analysis of the interaction between host macrophages and phagocytosed C. neoformans to characterize Cryptococcal protein regulation and secretion during infection. To differentiate actively secreted proteins from those passively shed, we compared the proteomic profiles between live and heat-killed C. neoformans. There were several proteins of note secreted by C. neoformans during infection such as urease, a well-characterized virulence factor, phospholipase D, a family member of another virulence factor phospholipase B, and transaldolase, which protects against oxidative stress. We also gain some perspective on the host macrophage response to Cryptococcal infection. Some examples of host macrophage proteins that were upregulated in live versus heat-killed yeast include cathepsins H, B and S, key proteases involved in the degradation within the phagolysosome. This same pattern was seen with Rab7a, a key regulator in endosomal maturation and endo-lysosomal trafficking. One interesting secreted Cryptococcal protein we identified in our proteomic screen was Yor1 which we discovered may play a role in a proposed virulence factor, nonlytic exocytosis. This phenomenon occurs when a phagocytosed pathogen can escape without harming the host phagocyte. It has been labeled as a possible method of systemic dissemination in cryptococcosis due to the "Trojan horse" nature of evading immune surveillance and freely traversing the host organism within a phagocyte prior to escape. We discovered that Yor1 mutants showed severely attenuated nonlytic exocytosis rates during murine macrophage infection leading us to pursue Yor1 as a regulatory candidate and a potential therapeutic target.;Keeping with the search for novel strategies against cryptococcosis, we evaluated a library of uncharacterized drugs as potential therapeutic agents. Growth inhibition assays yielded compounds with fungistatic and fungicidal activity. The most active compound, MMV665943 (DM262), was fungicidal against C. neoformans at concentrations 16-fold lower than fluconazole, a common antifungal used in the treatment of cryptococcosis. DM262 was also fungicidal at varying concentrations against other fungal pathogens: Cryptococcus gattii, Candida albicans and Lomentospora prolificans. Interestingly, C. gattii and L. prolificans are considered resistant to common drugs used in the treatment of fungal infections. In addition, there is also a rise in antifungal resistant cryptococcal isolates worldwide emphasizing the need for novel therapeutics.