Identification and Characterization of a Novel α-Carbonic Anhydrase from Leishmania major to Evaluate its Role in Leishmania Physiology

Abbasi, Mazharul (2019) Identification and Characterization of a Novel α-Carbonic Anhydrase from Leishmania major to Evaluate its Role in Leishmania Physiology. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Mazharul Abbasi (12RS038)) 12RS038.pdf - Submitted Version Restricted to Repository staff only Download (19MB)

Abstract

Leishmaniasis is a vector borne parasitic disease caused by intracellular pathogen Leishmania. It has been, and still, one of the major health problem around the globe, mainly affecting the underdeveloped regions of the world. Currently, over 1 billion people are residing in the area of active Leishmania transmission. Unavailability of a vaccine and increasing signs of resistance to few existing drugs warrants consolidated effort towards discovery of novel anti-leishmanial drug targets. Leishmania parasites proliferate efficiently within acidic phagolysosomes of macrophages. The underlying mechanism by which Leishmania can survive within phagolysosomal acidic environment is largely unknown. Recently, carbonic anhydrases (CAs) have been studied in a number of pathogenic microorganisms and have been proposed as suitable drug target due to their crucial role in growth/survival and virulence of these pathogens. We discovered that L. major expresses two CAs (LmCAs); a β-family LmCA1, present in chromosome 6 and an α-family LmCA2 in chromosome 28. Using pharmacological inhibition studies with potent CA inhibitors, maneb and zineb, we have demonstrated that CA activity is essential for parasite growth and viability. Coupling pharmacological inhibition data with in silico binding mode analysis of CA inhibitors, we have established that LmCA2 is structurally distinct from human α-CA and can be selectively targeted by CA inhibitors. These encouraging results prompted us to investigate the role of LmCA2 in Leishmania physiology. Immunofluorescence imaging and subcellular fractionation studies showed that LmCA2 is localized in the plasma membrane of the parasites. For functional analysis targeted replacement of both alleles of LmCA2 with antibiotic selectable markers was attempted. But we failed to generate null mutant for this gene after repeated attempts indicating functional indispensability of LmCA2 in Leishmania parasites. However, we were successful in generating LmCA2⁺/⁻ and LmCA1⁺/⁻:LmCA2⁺/⁻ heterozygous mutant and their respective complementation strains. The heterozygous strains grew normally in neutral medium but suffered from intracellular acidosis-induced cell cycle arrest, growth retardation and morphological abnormalities in acidic pH. They also showed reduced parasite load and infectivity in macrophage cell line, which was completely rescued in macrophages with alkaline lysosomes further confirming the role of LmCA2 in acidic environment. Our data suggests that LmCA2 is crucial for cytosolic buffering of L. major and plays a determining role in proliferation of Leishmania in acidic milieu in vitro and within phagolysosomes of host macrophages. Therefore, LmCA2 can be a novel drug target against Leishmania parasites.

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