STUDY OF SMALL GTPASE RAB11 IN Leishmania mexicana

MENDEZ N; CARLEVARO G; MUCCI J

Encuentro; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017

Leishmaniasis is a human disease produced by species of thegenus Leishmania, that infect 1 million individuals per year. AsTrypanosoma cruzi and Trypanosoma brucei, Leishmania parasitesexpress surface GPI-anchored virulence factors with key roles ininfection.In T. cruzi, the small GTPase Rab11 and the contractile vacuolecomplex (CVC) are key elements in the transport pathway of theseproteins to the surface, whereas in T. brucei Rab11 mediates endosomalrecycling, as in higher eukaryotes. The function of Rab11 inLeishmania has not been studied yet. To investigate this, we overexpressed(OE) a GFP-LmRab11 fusion protein gene in Leishmaniamexicana, cloned in the pNUS-nGFP vector. Expression wasverified by western blot or flow cytometry and the overexpressedpopulations showed no growth differences with wild type and GFPOEcontrol parasites.We found that the GFP-Rab11 signal is localized within a smallintracellular region, as in T. cruzi, compatible with the location ofthe CVC. We observed morphological changes in Rab11OE parasiteswith respect to controls. Scanning electron microscopy revealed thatRab11OE parasites have less extracellular vesicles than the wild type.To explore the effects of interfering with the Rab11 pathway, wetransfected L. mexicana with mCherry-Rab11 ?dominant-negative?(DN): an enzymatically inactive version of the GTPase by the S21Nsubstitution. Despite numerous attempts, it was not possible to obtainRab11DNOE populations. However, simultaneous transfectionwith GFP-Rab11 was successful. As expected, we observed thatthe active GTPase localizes in the CVC region, while the Rab11DNsignal is distributed throughout the cytoplasm. These results showthat expression of Rab11DN is deleterious, as it was only possibleby co-expression in Rab11OE background.