Elucidating Mechanisms of Leishmania Signaling Pathways and Parasite Stress Response

In this dissertation, I address the significance of signal transduction cascades during Leishmania parasite differentiation for the purpose of identifying potential drug targets to be used for the treatment of leishmaniasis, a disease characterized by a spectrum of clinical manifestations ranging from ulcerative skin lesions to fatal visceral infections. To date, few anti-leishmanial drugs exist and most risk becoming ineffective due to emerging resistances; therefore, new drugs are urgently needed. During the infectious cycle, Leishmania differentiates from extracellular promastigotes in sandflies to pathogenic intracellular amastigotes in mammalian hosts. Differentiation is triggered by changes in environmental signals, namely pH and temperature, which are relayed through signal transduction pathways via reversible phosphorylation of kinases and phosphatases. Compared to higher eukaryotes, our knowledge of these signaling molecules in Leishmania is scarce despite their essential roles in the biology of the parasite. Mitogen-activated protein kinases (MAPKs) are conserved virtually across all eukaryotic organisms. Previous insight into the MAPK pathway in Leishmania revealed a downstream target of MAPK7, identified as LmaPA2G4. Upon investigating LmaPA2G4, we found it to be an essential gene potentially indicated in fundamental biological mechanisms, such as translation, therefore making it an attractive target for therapeutic intervention. Though protein kinases, and their downstream targets, have been more actively studied for the potential development of anti-parasitic therapeutics, our understanding of the biology of protein phosphatases in Leishmania is poor despite their implication in critical post-translational modifications and differentiation. Approximately 96-99% of proteins in eukaryotes are phosphorylated on Serine and Threonine (Ser/Thr) residues. Using bioinformatics and in silico analysis we identified protein phosphatase 5 (PP5), a Ser/Thr phosphatase belonging to the PPP family of phosphatases. PP5 is unique in that it differs from other phosphatases in its family due to its N-terminal tetratricopeptide repeat (TPR) domains, which are momentous in protein-protein interactions and auto-inhibition. Upon investigation, recombinant PP5 proved it to be a bona fide phosphatase that is enzymatically active. Further analysis showed PP5 to be developmentally regulated, with increased expression in metacyclic promastigotes, suggesting a possible role in metacyclogenesis. The generation of PP5 null mutant parasites revealed that despite its inessentiality, its removal significantly decreases parasite pathogenicity which, therefore, further implicates its potential future applicability as an anti-parasitic target. Altogether, this work demonstrates the importance of studying signal transduction events for the successful development of Leishmania-based therapeutics.