Loss of direct binding of Leishmania profilin with actin adversely affected its functions and interactions with other cellular proteins

Abstract

Profilins are actin binding proteins that play a central role in regulation of actin remodeling in all eukaryotic organisms. Leishmania, a family of protozoan parasites that cause leishmaniasis, express a single homolog of profilin, which differs from other eukaryotic profilins in that it contains an extra stretch of 20 amino acids (actin binding domain) through which it directly binds actin. We therefore considered it of interest to analyze the role of direct binding of profilin with actin in interactions and functions of profilin in L. donovani promastigotes. For this, we deleted the actin binding domain in L. donovani profilin (LdPfn), and then carried out comparative analysis of LdPfn and truncated LdPfn ({delta}LdPfn) interactomes by affinity pull-down and mass spectrometry. To assess the effect of deletion of the actin binding domain on the LdPfn functions, we expressed GFP conjugates of LdPfn and {delta}LdPfn in the wild type Leishmania cells and then carried out comparative analysis of their growth, cell division cycle and intracellular vesicle trafficking activity. Results revealed that expression of GFP-{delta}LdPfn in the wild type cells adversely affected their cell growth, intracellular vesicle trafficking and G1-to-S and S-to-G2/M phase transitions during their cell division cycle. Also, there was a complete loss of LdPfn binding to several cellular proteins including actin and mitochondrial outer membrane protein, porin (LdPorin) after deleting its actin binding domain. Further, to assess the effect of lack of LdPorin binding to {delta}LdPfn on the mitochondrial functions, we measured the cellular ATP levels in wild-type and transgenic promastigotes. The ATP levels were significantly increased by expressing GFP-{delta}LdPfn in wild-type cells. These results taken together strongly indicate that LdPfn-driven actin remodeling besides playing a pivotal role in regulation of intracellular vesicle trafficking and cell division cycle, it also plays an important role in regulation of Leishmania mitochondrial activity.