Cryptococcus neoformans is an opportunistic yeast pathogen responsible for over 1 million infections and 600,000 deaths annually. In a published study, we identified a transcriptional regulator Gat201 that inhibits phagocytosis independently of the polysaccharide capsule. Previously, the major component of the polysaccharide capsule, glucoronoxylomannan (GXM), was believed to be the only mechanism by which the fungus could evade phagocytosis by macrophages. Our laboratory performed studies using custom microarrays to define which genes were controlled by Gat201 and screened each Gat201-regulated gene for its effect on phagocytosis. We determined that Blp1, a protein regulated by Gat201, inhibited C. neoformans phagocytosis by macrophages. I performed subsequent chromatin immunoprecipitation and sequencing (ChIP-Seq) experiments and discovered that two other proteins in the same family as Blp1 (Blp2 and Blp4) are targets of Gat201. Thus, my aims are as follows: (1) Determine whether Blp1, Blp2 and Blp4 contribute in a redundant manner to the GXM-independent phagocytosis inhibition controlled by Gat201 by constructing double and triple mutant strains and triply-misregulated Blp strains (Blp1 knockout, Blp2 and Blp4 overexpressed) in C. neoformans. Analysis of the phenotypes of these mutant strains will show whether Blp1, Blp2, and Blp4 function in the same virulence mechanism in vitro and in vivo. I anticipate this study will reveal some promising drug targets for treating cryptococcal infection. (2 & 3) Determine the molecular mechanism of Blp1 phagocytosis inhibition. Since Blp1 likely does not act through GXM, I will determine the novel mechanism by which this protein inhibits phagocytosis. I will test two competing hypotheses: Blp1 interacts with pathogen-associated molecular patterns (PAMPs) on C. neoformans so that macrophages do not recognize it as a pathogen or Blp1 engages inhibitory receptors on macrophages. I will test the first hypothesis by comparing phagocytosis levels of wildtype C. neoformans and the fungus lacking Blp proteins by wildtype macrophages and macrophages lacking the Syk activation factor in opsonized and unopsonized conditions. To test the second hypothesis, I will compare phagocytosis levels of wildtype and Blp-deficient C. neoformans by wildtype macrophages and macrophages lacking the SHP-1 inhibition factor in opsonized and unopsonized conditions. Together, these two aims will elucidate the mechanism by which C. neoformans protects itself from macrophages independently of its polysaccharide capsule.
The diagnosis and treatment of serious fungal infections by the human pathogen Cryptococcus neoformans is a major unmet clinical challenge. This study will determine the mechanism behind a recently discovered novel mechanism by which this pathogen causes disease. Additionally, this study will identify targets for drug development, expanding the limited drug choices treating fungal infections.
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