The Leish niche: the secretome of Leishmania and its role in parasite virulence

Leishmaniasis is a neglected tropical parasitic disease that causes several debilitating manifestations. No commercially available vaccine exists against this disease, and treatment strategies are far from ideal with the emergence of resistance, coupled with toxic side effects of many of the drugs available. Rational drug design relies on knowledge of the cell biology of the parasite and the interplay between the parasite and its hosts. Production of secreted proteins, the secretome, has become a known strategy for parasite invasion and persistence in host cells, however, host-parasite interaction is still not well defined. Virulence factors secreted by the parasite mediate the host-parasite interaction and create a niche permissive for parasite proliferation. They therefore represent potential therapeutic targets and vaccine candidates. Here, the use of secretomics was implemented to investigate these virulence factors. Parasite conditioned culture supernatant, containing the secretome, was characterised by morphological, immunochemical and proteomic analyses. Here, we optimised and extended current methods and applied them to the medically relevant amastigote stage. Method development and validation was implemented to extract a reproducible secretome in vitro. Induction of cell stress was managed and cell viability maintained to minimise interference of intracellular proteins. A total of 256 proteins were reproducibly identified in the secretome of promastigotes and 36 proteins were reproducibly identified in the secretome of amastigotes. Analysis of their protein abundance index (emPAI) allowed comparison of the relative abundance of proteins and functions of the secretome throughout the parasite life cycle. Differences in the putative functions of nutrient salvage, protease production and antioxidant activity were observed. Analyses reveal that many proteins lack a signal peptide and as such are thought to be released by nonclassical secretion mechanisms. Several exosome-associated proteins and membrane proteins were also detected in the secretome, suggesting the occurrence of secretion by exosomes or microvesicles. Extended comparative analyses between the secretome of parasites with differing phenotypes allowed us to infer functionality of the secretome in the parasite’s survival but also variations within the same species which result in differing disease outcomes. Dysregulation in the secretion of various proteins in attenuated parasites implicates these proteins in the virulence of the parasite. An increase in the secretion of pro-inflammatory mediators and destructive proteases by parasites isolated from patients with chronic cutaneous leishmaniasis compared to those from patients with self-healing lesions, indicates the role of the parasite in the chronicity of cutaneous leishmaniasis. Here, we demonstrate an applicable method for the study of the Leishmania mexicana promastigote and amastigote secretome. Results suggest that the secretome plays a role in disease progression and virulence. Proteomic analyses of the secretome, like this study presented here, provide crucial information on the host:parasite interaction for the identification of therapeutic targets and potential vaccine candidates for the provision of safer treatments and new vaccines for eradication of this disease.