Srinivasan Ramakrishnan

The bloodstream stage of Trypanosoma brucei , the causative agent of African trypanosomiasis, is characterized by its high rate of endocytosis, which is involved in remodeling of its surface coat. Here we present evidence that RNAi‐mediated expression down‐regulation of vacuolar protein sorting 41 (Vps41), a component of the homotypic fusion and vacuole protein sorting (HOPS) complex, leads to a strong inhibition of endocytosis, vesicle accumulation, enlargement of the flagellar pocket (“big eye” phenotype), and dramatic effect on cell growth. Unexpectedly, other functions described for Vps41 in mammalian cells and yeasts, such as delivery of proteins to lysosomes, and lysosome‐related organelles (acidocalcisomes) were unaffected, indicating that in trypanosomes post‐Golgi trafficking is distinct from that of mammalian cells and yeasts. The essentiality of TbVps41 suggests that it is a potential drug target.

Trypanosoma cruziis a protist parasite and the causative agent of American trypanosomiasis or Chagas disease. The parasite life cycle in its mammalian host includes an intracellular stage, and glycosylated proteins play a key role in host-parasite interaction facilitating adhesion, invasion and immune evasion. Here, we report that a Golgi-localized Mn²⁺-Ca²⁺/H⁺exchanger ofT.cruzi(TcGDT1) is required for efficient protein glycosylation, host cell invasion, and intracellular replication. The Golgi localization was determined by immunofluorescence and electron microscopy assays. TcGDT1 was able to complement the growth defect ofSaccharomyces cerevisiae nullmutants of its orthologScGDT1but ablation ofTcGDT1by CRISPR/Cas9 did not affect the growth of the insect stage of the parasite. The defect in protein glycosylation was rescued by Mn²⁺supplementation to the growth medium, underscoring the importance of this transition metal for Golgi glycosylation of proteins.

Significance: Cysteine fatty acylation (S-fatty acylation) regulates the stability, trafficking, and activity of proteins in eukaryotes. Current methods to study S-fatty acylation rely on the metabolic incorporation of fatty acid analogs or selective chemical labeling and affinity purification, which limits the detection of endogenous S-fatty acylated protein isoforms and levels. Here we demonstrate that the biochemical exchange of acyl groups on cysteines with defined mass-tags enables the direct visualization of endogenous S-fatty acylated protein levels. The application of this mass-tag labeling method revealed that site-specific S-fatty acylation of interferon-induced transmembrane protein 3 controls the antiviral activity of this IFN-induced effector.

Summary: Apicomplexa are parasitic protozoa that cause important human diseases including malaria, cryptosporidiosis and toxoplasmosis. The replication of these parasites within their target host cell is dependent on both salvage as well as de novo synthesis of fatty acids. In Toxoplasma gondii, fatty acid synthesis via the apicoplast‐localized FASII is essential for pathogenesis, while the role of two other fatty acid biosynthetic complexes remains unclear. Here, we demonstrate that the ER‐localized fatty acid elongation (ELO) complexes are essential for parasite growth. Conditional knockdown of the nonredundant hydroxyacyl‐CoA dehydratase and enoyl‐CoA reductase enzymes in the ELO pathway severely repressed intracellular parasite growth. ¹³C‐glucose and ¹³C‐acetate labeling and comprehensive lipidomic analyses of these mutants showed a selective defect in synthesis of unsaturated long and very long‐chain fatty acids (LCFAs and VLCFAs) and depletion of phosphatidylinositol and phosphatidylethanolamine species containing unsaturated LCFAs and VLCFAs. This requirement for ELO pathway was bypassed by supplementing the media with specific fatty acids, indicating active but inefficient import of host fatty acids. Our experiments highlight a gap between the fatty acid needs of the parasite and availability of specific fatty acids in the host cell that the parasite has to close using a dedicated synthesis and modification pathway.

Intracellular pathogens have complex metabolic interactions with their host cells to ensure a steady supply of energy and anabolic building blocks for rapid growth. Here we use the obligate intracellular parasite Toxoplasma gondii to probe this interaction for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Synthesis of precursors for isoprenoids in Apicomplexa occurs in the apicoplast and is essential. To synthesize longer isoprenoids from these precursors, T. gondii expresses a bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase (TgFPPS). In this work we construct and characterize T. gondii null mutants for this enzyme. Surprisingly, these mutants have only a mild growth phenotype and an isoprenoid composition similar to wild type parasites. However, when extracellular, the loss of the enzyme becomes phenotypically apparent. This strongly suggests that intracellular parasite salvage FPP and/or geranylgeranyl diphosphate (GGPP) from the host. We test this hypothesis using inhibitors of host cell isoprenoid synthesis. Mammals use the mevalonate pathway, which is susceptible to statins. We document strong synergy between statin treatment and pharmacological or genetic interference with the parasite isoprenoid pathway. Mice can be cured with atorvastatin (Lipitor) from a lethal infection with the TgFPPs mutant. We propose a double-hit strategy combining inhibitors of host and parasite pathways as a novel therapeutic approach against Apicomplexan parasites. Toxoplasma gondii is an obligate intracellular parasite and is not able to replicate outside the host cell. The parasite lives in a specialized parasitophorous vacuole in contact with the host cytoplasm through the parasitophorous vacuole membrane. It is highly likely that a very active exchange of metabolites occurs between parasite and host cell. We present evidence for this exchange for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Our work shows that intracellular T. gondii tachyzoites are able to salvage farnesyl diphosphate (FPP) and/or geranylgeranyl diphosphate (GGPP) from the host, and the parasite is able to grow even when its endogenous production is shut down. However, when extracellular, the parasite depends entirely on its own production of isoprenoids. We propose to use a combination of inhibitors that would hit both the host and the parasite pathways as a novel therapeutic approach against Toxoplasma gondii that could also work against other Apicomplexan parasites.