Studying the Function of Leishmania major Carbonic Anhydrases by Targeted Gene Manipulation

Varghese, Binitha Anu (2015) Studying the Function of Leishmania major Carbonic Anhydrases by Targeted Gene Manipulation. Masters thesis, Indian Institute of Science Education and Research Kolkata.

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Abstract

Objectives: Carbonic anhydrases (CAs) are a family of metalloenzymes that catalyze reversible hydration of CO₂. They play a crucial role in various physiological processes including pH regulation, HCO₃⁻/CO₂ transport and in metabolic pathways of gluconeogenesis, ureagenesis, and lipogenesis. Recently, our lab identified two CA isoforms in Leishmania major (L. major). One of them is a cytosolic CA (LmCA1) and the other is a plasma membrane-bound CA (LmCA2).Treatment with CA inhibitors (dithiocarbamates) caused parasite death at submicromolar concentrations, which indicates LmCAs are crucial for parasite survival. So the objective of this work was to investigate the actual function of LmCAs in parasite physiology by targeted gene manipulation. Methods: Two mutant strains were generated for the study by targeted gene replacement technique; a heterozygous strain where one of the alleles of LmCA2 was deleted (LmCA2⁺/⁻) and a double heterozygous strain where one allele of both LmCA1 and LmCA2 was deleted (LmCA1⁺/⁻:LmCA2⁺/⁻).The LmCA2+/- was generated by electroporating LmCA2 deletion cassette into wild-type (WT) L. major from pXG-HYG vector and selection under hygromycin B antibiotic. LmCA1⁺/⁻:LmCA2⁺/⁻ was established by electroporating LmCA2 deletion cassette into LmCA1 heterozygous strain (LmCA1⁺/⁻) and selection under both hygromycin B and G418-sulfate antibiotics. In addition, a genetically modified L. major was also generated, which can function as a tetracycline inducible system for gene knockdown. It is established by incorporating T7 RNA polymerase and a tetracycline repressor protein encoding gene into WT L. major genomic DNA using targeted gene replacement technique. Growth and morphology of LmCA-mutant was determined by haemocytometer-based cell counting and field emission scanning electron microscopy (FESEM). Susceptibility to acidic pH was investigated by growing cells in acidic culture media (pH 5.5), followed by haemocytometer-based cell counting and intracellular pH measurement by pH-sensitive fluorescent dye BCECF-AM. Susceptibility to CA inhibitor was studied by growing parasite in increasing concentration of drug (zineb), followed by haemocytometer-based cell counting. Leishmania infected macrophages (RAW264.7) were employed to evaluate the infectivity of LmCA-mutant strains. Results: We were successfully able to generate a LmCA2 heterozygous strain (LmCA2⁺/⁻) and a double heterozygous strain (LmCA1⁺/⁻:LmCA2⁺/⁻) and compared both the strains with WT L. major and already generated LmCA1 heterozygous strain (LmCA1⁺/⁻). These LmCA- mutant strains showed significant reduction in total CA activity (25-59%) as compared to WT cells. However, growth rates of LmCA1⁺/⁻, LmCA2⁺/⁻ and LmCA1⁺/⁻:LmCA2⁺/⁻ were comparable to WT cells at normal pH (7.4). Amongst these strains, LmCA1⁺/⁻:LmCA2⁺/⁻ cells showed markedly increased susceptibility to CA inhibitor (zineb) compared to the WT cells (LD50 reduced from 0.599 μM to 0.3 μM). Additionally, FESEM revealed a significant reduction (17-46%) in their cell sizes indicating that the cells were under some kind of metabolic stress. In acidic pH (5.5), all the mutant strains showed a profound reduction in growth rate (22-57%) as compared to WT cells. Staining these cells with BCECF-AM revealed their intracellular pH (pHi) was significantly lowered with respect to WT cells and LmCA1⁺/⁻:LmCA2⁺/⁻ was having the highest difference (0.25 units). However, addition of exogenous HCO3- reverted their pHi back to physiological pH and also resulted in almost complete revival of cell growth. Interestingly, when macrophages (RAW264.7) were infected with WT and LmCA1⁺/⁻:LmCA2⁺/⁻ parasite, LmCA1⁺/⁻:LmCA2⁺/⁻ showed a 37% reduction in intracellular parasite load compared to WT cells, suggesting that the infectivity of the double heterozygous strain is significantly reduced. The tetracycline inducible knockdown L.major strain was also successfully established and its verification is underway. It will be used for studying the function of LmCAs upon overexpression and inducible knockdown of the respective genes. Conclusions: The interplay of LmCA1 and LmCA2 helps in intracellular pH maintenance via bicarbonate buffering in L. major, thereby helping them to thrive in the acidic phagolysosomes of host macrophages.

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