Authors

M Abbasi¹; D S Pal¹; D K Mondal¹; R Datta*¹;  ¹ IISER Kolkata, India

Discussion

Intracellular parasite Leishmania efficiently proliferates within acidic phagolysosomes of human macrophage. The underlying mechanism by which Leishmania survive within phagolysosomal acidic environment is largely unknown.We have shown earlier, pharmacological inhibition of carbonic anhydrase (CA) activity resulted in faulty growth due to intracellular acidosis and cell death. Here we report presence of two CAs, LmCA1 and LmCA2, involved in acid acclimation of the parasite. Immunolocalization studies confirmed LmCA1 as cytosolic whereas LmCA2 as a plasma membrane-bound enzyme. For functional analysis targeted replacement of both genes with antibiotic selectable markers was attempted, but we failed to generate null mutants for any of these genes after repeated attempts, indicating functional importance of these genes. However we were able to generate LmCA1^+/-, LmCA2^+/- heterozygous and LmCA1^+/-: LmCA2^+/- double heterozygous strain. Comprehensive analysis of these mutant strains along with genetic complementation studies clearly shows that both the genes are crucial for parasite growth in acidic environment. The mutant strains also exhibited a marked decrease in virulence upon infection in J774A.1 macrophages which was reversed by lysosomal alkalization using chloroquine. Collectively, these data provide strong evidence that LmCAs play a determining role in parasite multiplication within acidic phagolysosomes of macrophages