Investigating the role of the SNARE protein, Tlg2, in Leishmania mexicana

Soluble N-ethylmaleimide-sensitive factor adaptor proteins receptors (SNARE) proteins are essential components of intracellular membrane trafficking yet are critically understudied in trypanosomatid parasites. Although 27 SNARE-domain containing proteins have been identified in Leishmania major, no functional characterisation has yet been undertaken. In yeast, Tlg2 is a SNARE protein that forms part of the trans-Golgi network and mediates fusion events between endosomes and the late Golgi. It also plays a role in autophagy, although the mechanisms behind this are not fully understood. Bioinformatic analysis of the Leishmania mexicana genome revealed homologues of Tlg2, its SNARE complex partners and two Sec1/Munc18 (SM) Vps45 regulatory proteins. Overexpression of ectopic Tlg2 in L. mexicana promastigotes did not result in any observed phenotypes but demonstrated that the protein is exclusively expressed during logarithmic growth. In contrast, tlg2 null mutants displayed growth and morphological phenotypes, as well as delayed expression of SHERP mRNA (a marker for metacyclogenesis). Addback of an ectopic wild-type Tlg2 (Tlg2WT) rescued the null mutants. Addback of a F10A/L11A mutant (Tlg2FL) that is predicted to disrupt a high-affinity pocket-mode of binding to Vps45, was unable to rescue the null mutant phenotypes, suggesting a role for the N-terminal motif in protein function. L. mexicana tlg2∆ displayed decreased virulence in macrophage infection assays and a change in Atg8 expression (an autophagy marker) at early promastigote lifecycle stages. Finally, use of immunoprecipitation assays revealed that Tlg2WT formed protein complexes with several Golgi and endosomal-associating proteins, whereas Tlg2FL associated more with proteins involved in translation, protein degradation and stress response. Taken together, this data demonstrates a role for Tlg2 in parasite growth and differentiation, with the N-terminal motif playing an important part in this SNARE protein’s function.