The goal of this proposal is to decipher the mechanism by which fungi are recognized by the innate immune system and the mechanisms used by fungi to evade recognition. Pathogenic fungi such as Candida albicans, and the non-pathogen, S. cerevisiae camouflage their surface immune determinants from macrophages and neutrophils by genetic and structural variation. Recent analysis of the genome sequence of Candida albicans suggests that it encodes the crucial proteins of the RNAi system (missing from Saccharomyces) that could be a key contributor to genetic variation. Sequence analysis reveals that C. albicans has homologues of Argonaute and Dicer, two key components of an inhibitory RNA pathway, known in other systems to suppress the transposition of mobile genetic elements. Moreover, it also produces small RNAs, likely interfering RNAs, which have homology to the LINE-1 retrotransposon, Zorro-3. Knockouts of the putative Argonaute and Dicer genes will determine whether these proteins constitute elements of the pathway to create these RNAs. These mutants together with a tagged Zorro-3 element will permit determination of whether loss of this system enhances transposition and whether this RNAi system affects morphogenesis and virulence of Candida. Both in vitro and in vivo experiments are designed to identify the components of the innate immune system that recognize Candida. An in vitro screen utilizes a murine RNAi library to identify macrophage genes responsible for phagocytosis and filamentation of Candida. In concert with this screen Candida mutants will be constructed that have unmasked key signature molecules on their surface. These mutants will be used together with these knockdowns to reconstruct both the pathways from receptor to cytokine production and the specific receptors for these cell surface signatures. The in vivo experiments are designed to identify the function of the 2-glucan receptor, Dectin-1 in recognizing fungi in transgenic mice tagged with a short sequence, the """"""""Sortag"""""""". These mice expressing a sortagged Dectin-1 from its own promoter will permit identification of proteins that interact with Dectin-1, the ensemble of different immune cells that express Dectin-1, and the function(s) recognized in vivo for fungal recognition. The availability of sortagged Dectin-1 mice will provide a unique opportunity to identify new targets for anti-fungal agents.
Our defense against fungal disease involves recognition of the invading pathogen by our immune system. The research proposed will reveal the mechanism of this detection system and the strategies used by the fungi to avoid recognition. To avoid recognition the pathogen changes or masks the identifying signature. Understanding this encounter will guide the design of new antibiotics.
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