Deciphering the Role of Beta Carbonic Anhydrase in Leishmania Major by Gene Knockout

Paul, Ritama (2014) Deciphering the Role of Beta Carbonic Anhydrase in Leishmania Major by Gene Knockout. Masters thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Carbonic anhydrases (CAs) are a group of metalloenzymes that catalyze reversible hydration of CO2. In organisms like humans they play a vital role in a variety of physiological processes including pH regulation, HCO3/CO2 transport as well as metabolic pathways such as gluconeogenesis, ureagenesis and lipogenesis. Moreover, recent studies have shown that bacterial pathogens such as Heliobacter pylori maintain their intracellular pH in acidic conditions due to interplay of their functional CAs. Our lab has recently identified and characterised a functional beta family CA (LmCA1) located in the cytosol of Leishmania promastigotes. Hence we hypothesize this enzyme might play an important role in amastigote survival within acidic lysosomes of host macrophages. To test the physiological role of LmCA1 in the parasite, we have tried to knock-out this gene from the diploid L.major by homologous recombination method. So far we have been successful in creating heterozygous LmCA1 cells (LmCA1+/-) and have characterized them. In vitro studies showed that LmCA1+/- cells show about 35% reduced activity (0.816 EU/mg±0.07) as compared to wild-type (WT) cells (1.203 EU/mg±0.01). Scanning electron microscopy (SEM) revealed that the heterozygous LmCA1 cells (9.48μm±2.3) show about 20% reduction in size as compared to WT cells (11.77μm±1.12) Although, no difference in growth kinetics was seen between LmCA1+/- and WT cells grown in media of neutral pH(pH 7.2), the LmCA1 heterozygous cells showed reduced growth rate with 50% reduction in average cell count after 3 days(3.87 x 106 cells/ml) as compared to WT cells(7.87 x 106 cells/ml) in acidic media (pH 5.5). This suggests that LmCA1 might have a role in intracellular pH maintenance of the parasite in acidic lysosomes. Therefore these phenotypes of LmCA1(+/-) cells along with the failure of our repeated attempts to obtain complete knockout cells (LmCA1-/-) suggest that LmCA1 is an important gene for Leishmania parasite.

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