Flagellar membrane targeting, immune evasion, and protein palmitoylation in the protozoan parasite Trypanosoma brucei

Trypanosomes are unicellular eukaryotic parasites responsible for tremendous morbidity and mortality worldwide. The research described herein focuses on Trypanosoma brucei, the etiologic agent of African sleeping sickness and a model organism for the related Trypanosoma cruzi and Leishmania spp., which cause Chagas disease and the leishmaniases, respectively. Trypanosomes express a family of myristoylated and palmitoylated calcium-binding proteins, the calflagins in T. brucei, which specifically localize to the flagellar membrane. To define the molecular determinants of calflagin localization, point mutations were introduced into the calflagin amino terminus. Assessment of point mutant acylation state and localization demonstrated the sufficiency of myristoylation for plasma membrane localization, and the requirement of secondary palmitoylation for specific sorting to the flagellar membrane. Contemporaneous with calflagin flagellar targeting upon palmitoylation was an association with lipid raft microdomains, which are enriched in the flagellar membrane. To identify and localize the enzyme responsible for calflagin palmitoylation and trafficking, a genome-wide screen for likely palmitoyl acyltransferases (PATs) was performed, followed by generation of knock-down mutants for each of twelve candidates. Testing each of these mutants led to the identification of the single enzyme mediating calflagin palmitoylation, TbPAT7. Calflagin palmitoylation by TbPAT7 is the first PAT-substrate pair identified in a protozoan organism and the first palmitoyl acyltransferase in any system shown to confer flagellar targeting. Surprisingly, TbPAT7, like its substrates, was found to localize to the flagellar membrane. These findings support a model, likely applicable to all ciliated eukaryotic cells, wherein enzyme-specific palmitoylation at the flagellar membrane favors the retention of protein substrates through interactions with the unique lipid composition of this organelle.;To determine the biologic function of the calflagins, calflagin deficient mutants were engineered and subjected to extensive phenotypic analysis. Despite being unaffected in terms of their in vitro viability, growth, motility, and morphology, the virulence of these mutants was greatly attenuated in a murine model of infection. Compared to parental wild type cells, which proliferated unchecked to cause 100% host mortality within 10 days post-infection, calflagin mutants demonstrated a normal initial rise in parasitemia, followed by clearance to undetectable levels within 8 days post-infection. Outgrowth of parasite populations was associated with antigenic variation of the VSG surface coat, indicating that the initial population had been cleared in an antigen-specific manner. A mechanism for the increased susceptibility of calflagin mutants to the early host response was suggested by a relevant in vitro experiment. When surface-labeled with anti-VSG antibodies, calflagin mutants showed delayed endocytosis and degradation of these antibodies, a deficit that is expected to render parasites susceptible to host humoral immunity. Moreover, the localization of the calflagins was shown to be critical for their function, since inhibition of TbPAT7, which causes a redistribution of calflagins to the pellicular rather than flagellar membrane, likewise prolonged host survival and suppressed parasitemias during infection. These finding implicate the calflagins as important virulence factors through their contribution to evasion of host immunity.;With a specific role established for palmitoylation in the flagellar trafficking of calflagins, examination of protein palmitoylation was then extended to the global level, first by characterization of mutants for each of the twelve T. brucei PATs, and then by identification of the total cellular palmitoyl proteome. Despite the lethality of chemical pan-PAT inhibition, no single enzyme appears essential to parasite growth, indicating either enzymatic compensation or non-canonical PAT activity for those substrates essential to parasite proliferation. Acyl-biotin exchange and streptavidin chromatography enabled the purification and identification of cellular T. brucei palmitoyl proteins, including 3 of 4 previously described palmitoyl proteins and another 49 novel palmitoyl proteins. The identities of these proteins implicate palmitoylation in broad aspects of trypanosome biology, including parasite metabolism, subcellular trafficking, and cell signaling. Unexpectedly, the identification of certain novel palmitoyl proteins furthermore offers an explanation for previously enigmatic processes, including the chain length specificity of fatty acyl CoA synthetases and the dynamic regulation of folate/pterin transporters.;Taken together, the research described in this thesis represents a substantial and novel contribution to the field of trypanosome biology, identifying a novel pathway for flagellar/ciliary trafficking, elucidating an important role for calcium-signaling proteins in immune evasion and pathogenesis, and providing the first global analysis of the enzyme mediators and protein substrates of palmitoylation in these pathogenic parasites.