Study of small GTPase Rab11 in Leishmania mexicana

NICOLÁS AGUSTÍN MÉNDEZ; GIANNINA CARLEVARO; JUAN MUCCI

Ciudad Autonoma de Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedades de Biociencias de Argentina

Leishmaniasis is a human disease produced by species of the genus Leishmania, that infect 1 million individuals per year. As Trypanosoma cruzi and Trypanosoma brucei, Leishmania parasites express surface GPI-anchored virulence factors with key roles in infection.In T. cruzi, the small GTPase Rab11 and the contractile vacuole complex (CVC) are key elements in the transport pathway of these proteins to the surface, whereas in T. brucei Rab11 mediates endosomal recycling, as in higher eukaryotes. The function of Rab11 in Leishmania has not been studied yet. To investigate this, we overexpressed (OE) a GFP-LmRab11 fusion protein gene in Leishmania mexicana, cloned in the pNUS-nGFP vector. Expression was verified by western blot or flow cytometry and the overexpressed populations showed no growth differences with wild type and GFPOE control parasites.We found that the GFP-Rab11 signal is localized within a small intracellular region, as in T. cruzi, compatible with the location of the CVC. We observed morphological changes in Rab11OE parasites with respect to controls. Scanning electron microscopy revealed that Rab11OE parasites have less extracellular vesicles than the wild type.To explore the effects of interfering with the Rab11 pathway, we transfected L. mexicana with mCherry-Rab11 ?dominant-negative? (DN): an enzymatically inactive version of the GTPase by the S21N substitution. Despite numerous attempts, it was not possible to obtain Rab11DNOE populations. However, simultaneous transfection with GFP-Rab11 was successful. As expected, we observed that the active GTPase localizes in the CVC region, while the Rab11DN signal is distributed throughout the cytoplasm. These results show that expression of Rab11DN is deleterious, as it was only possible by co-expression in Rab11OE background.This effect is not observed in T. cruzi and suggests that Rab11DN interferes with vital processes in Leishmania biology.