The microtubule quartet protein SNAP1 in Trypanosoma brucei facilitates flagellum and cell division plane positioning by promoting basal body segregation.

The unicellular protozoan Trypanosoma brucei has a single flagellum that is involved in cell motility, cell morphogenesis, and cell division. Inheritance of the newly assembled flagellum during the cell cycle requires its correct positioning, which depends on the faithful duplication or segregation of multiple flagellum-associated cytoskeletal structures, including the basal body, the flagellum attachment zone, and the hook complex. Along the flagellum attachment zone sites a set of four microtubules termed the microtubule quartet (MtQ), whose molecular function remains enigmatic. We recently reported that the MtQ-localized protein NHL1 interacts with the microtubule-binding protein TbSpef1 and regulates flagellum inheritance by promoting basal body rotation and segregation. Here, we identified a TbSpef1- and NHL1-associated protein named SNAP1, which co-localizes with NHL1 and TbSpef1 at the proximal portion of the MtQ, depends on TbSpef1 for localization and is required for NHL1 localization to the MtQ. Knockdown of SNAP1 impairs the rotation and segregation of the basal body, the elongation of the flagellum attachment zone filament, and the positioning of the newly assembled flagellum, thereby causing mis-placement of the cell division plane, a halt in cleavage furrow ingression, and an inhibition of cytokinesis completion. Together, these findings uncover a coordinating role of SNAP1 with TbSpef1 and NHL1 in facilitating flagellum positioning and cell division plane placement for the completion of cytokinesis.

Host cell protein LAMP-2 is the receptor for Trypanosoma cruzi surface molecule gp82 that mediates invasion.

Host cell invasion by Trypanosoma cruzi metacyclic trypomastigote (MT) is mediated by MT-specific surface molecule gp82, which binds to a still unidentified receptor, inducing lysosome spreading and exocytosis required for the parasitophorous vacuole formation. We examined the involvement of the major lysosome membrane-associated LAMP proteins in MT invasion. First, human epithelial HeLa cells were incubated with MT in the presence of antibody to LAMP-1 or LAMP-2. Antibody to LAMP-2, but not to LAMP-1, significantly reduced MT invasion. Next, HeLa cells depleted in LAMP-1 or LAMP-2 were generated. Cells deficient in LAMP-2, but not in LAMP-1, were significantly more resistant to MT invasion than wild-type controls. The possibility that LAMP-2 might be the receptor for gp82 was examined by co-immunoprecipitation assays. Protein A/G magnetic beads cross-linked with antibody directed to LAMP-1 or LAMP-2 were incubated with HeLa cell and MT detergent extracts. Gp82 bound to LAMP-2 but not to LAMP-1. Binding of the recombinant gp82 protein to wild-type and LAMP-1-deficient cells, which was dose dependent and saturable, had a similar profile and was much higher as compared with LAMP-2-depleted cells. These data indicate that MT invasion is accomplished through recognition of gp82 by its receptor LAMP-2.

Histone deacetylases inhibitors as new potential drugs against Leishmania braziliensis, the main causative agent of new world tegumentary leishmaniasis.

The protozoan parasite Leishmania braziliensis is a major causative agent of the neglected tropical diseases Cutaneous and Mucocutaneous Leishmaniases in the New World. There are no vaccines to prevent the infection and the treatment relies on few drugs that often display high toxicity and costs. Thus, chemotherapeutic alternatives are required. Histone Deacetylases (HDACs) are epigenetic enzymes involved in the control of chromatin structure. In this work, we tested an in-house library of 78 hydroxamic acid derivatives as putative inhibitors of L. braziliensis HDACs (HDACi). The compounds were evaluated in relation to the toxicity to the host cell macrophage and to the leishmanicidal effect against L. braziliensis during in vitro infection. Eight HDACi showed significant leishmanicidal effects and the top 5 compounds showed effective concentrations (EC50) in the range of 4.38 to 10.21 µM and selectivity indexes (SI) from of 6 to 21.7. Analyses by Transmission Electron Microscopy (TEM) indicated induction of apoptotic cell death of L. braziliensis amastigotes with a necrotic phenotype. An altered chromatin condensation pattern and cellular disorganization of intracellular amastigotes was also observed. A tight connection between the mitochondrion and nuclear protrusions, presumably of endoplasmic reticulum origin, was found in parasites but not in the host cell. In flow cytometry (FC) analyses, HDACi promoted parasite cell cycle arrest in the G2-M phase and no changes were found in macrophages. In addition, the direct effect of HDACi against the promastigotes showed apoptosis as the main mechanism of cell death. The FC results corroborate the TEM analyses indicating that the HDACi lead to changes in the cell cycle and induction of apoptosis of L. braziliensis. The production of nitric oxide by the infected macrophages was not altered after treatment with the top 5 compounds. Taken together, our results evidenced new HDACi as promising agents for the development of new treatments for American Tegumentary Leishmaniasis caused by L. braziliensis.