Mechanisms of Protein Localization in Cryptococcus neoformans Mediate Virulence and Immune Recognition

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease and death in immunocompromised populations, in particular among those with advanced HIV infection. This fungus is found ubiquitously in the environment and acquired through inhalation into the respiratory tract followed by dissemination to the central nervous system in immunocompromised individuals. The ability for C. neoformans to sense and adapt to the host environment is crucial to its success as a pathogen. Many C. neoformans proteins require proper subcellular localization for their function, and as such this fungus carefully regulates the localization of proteins involved in important cellular processes related to host adaptation. Fungal growth and morphogenesis, as well as thermotolerance and virulence are controlled by conserved Ras-like GTPases. These proteins require proper localization for full function and are directed to cellular membranes through the posttranslational modification process known as prenylation. Using the tools of fungal genetics and molecular biology, we establish that the C. neoformans RAM1 gene encoding the farnesyltransferase -subunit is required for thermotolerance and pathogenesis. We also identified and characterized post-prenylation protease and carboxyl methyltransferase enzymes in C. neoformans, demonstrating that these later steps have only subtle effects on stress response and fungal virulence. By fluorescent microscopy and molecular biology, we show that Ram1 is required for proper subcellular localization of Ras1, but not Cdc42, and that the post-prenylation processing steps are dispensable for the localization of these substrate proteins. C. neoformans dramatically alters its cell wall upon entering the host in order to facilitate immune avoidance. Using the tools of forward genetics, we identified a novel cell wall regulatory protein, Mar1. We have demonstrated that this protein is required for capsule attachment and full virulence in mouse models of infection. Using staining and biochemical techniques, we have characterized the cell wall of mar1∆ mutant cells, and by fluorescent microscopy we have demonstrated that the -(1,3)-glucan synthase catalytic subunit, Fks1, is mislocalized in mar1∆ cells. Using in vitro co-culture models, we have determined that the mar1∆ cell wall induces increased macrophage activation that is dependent on the Card9 and MyD88 adaptor proteins, as well as the Dectin-1 and TLR-2 pattern recognition receptors. To further understand the impact of the Mar1 protein on the host-pathogen interaction, we used in vivo mouse models to characterize the pathogenesis and immune response to this strain. Using histopathology and light microscopy, we have shown that mar1∆ cells induce granulomas in the lungs of infected mice, an in in vitro co-culture models we have demonstrated that the mar1∆ strain induces increased markers angiogenesis. Finally using immunization strategies, we show that the mar1∆ strain does not induce a protective response against a secondary lethal challenge. Lastly, using bioinformatics tools and batch sampling, we developed a novel computational tool to more efficiently analyze mutants of interest generated by forward genetic screens. We demonstrate the efficacy of this tool through proof of principle experiments that led us to the discovery of the Mar1 protein described above. Additional projects in our lab and others have already utilized this mutant analysis tool in C. neoformans and we propose that it can be ultimately applied to a wide range of experimental systems and methods of mutagenesis, facilitating future microbial genetic screens.