Investigating the Role of Leishmania major Carbonic Anhydrase and Malic Enzyme in Gluconeogenesis

Mondal, Dipon Kumar (2020) Investigating the Role of Leishmania major Carbonic Anhydrase and Malic Enzyme in Gluconeogenesis. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Dipon Kumar Mondal (14RS030)) 14RS030.pdf - Submitted Version Restricted to Repository staff only Download (5MB)

Abstract

Leishmania are protozoan parasites from the group of trypanosomatid protozoa which are also the causative agent of wide spectrum of diseases collectively known as leishmaniasis. It has been and still, one of the major health problem around the globe that mainly affects the impoverished regions of the world. According to the recent report of WHO, over 1 billion people are residing in the area of active Leishmania transmission with limited healthcare facilities. Unavailability of an effective vaccine, toxic side effects and increasing resistance to the existing drugs warrants consolidated efforts towards the identification of unique molecular target and discovery of novel antileishmanial agents. Leishmania has a remarkable ability to proliferate under widely fluctuating levels of essential nutrients, such as glucose. For this the parasite is heavily dependent on its gluconeogenic machinery. In mammalian cells, mitochondrial CA-V has been established to supply the crucial bicarbonate molecule to the pyruvate carboxylase (PC) reaction to initiate gluconeogenesis. One perplexing aspect of gluconeogenesis in Leishmania is the lack of the PC gene. PC-catalyzed conversion of pyruvate to oxaloacetate is a key entry point through which gluconeogenic amino acids are funnelled into this pathway. Absence of PC in Leishmania thus raised an intriguing question about the mechanism of pyruvate entry and involvement of LmCAs into the gluconeogenic route. We established here that this task is accomplished in Leishmania major through a novel functional partnership between its mitochondrial malic enzyme (LmME) and cytosolic carbonic anhydrase (LmCA1). Using a combination of pharmacological inhibition studies with genetic manipulation, we showed that both these enzymes are necessary in promoting gluconeogenesis and supporting parasite growth under glucose limiting condition. Functional crosstalk between LmME and LmCA1 was evident when it was observed that the growth retardation caused by inhibition of any one of these enzymes could be protected to a significant extent by overexpressing the other enzyme. We also found that while LmCA1 exhibited constitutive expression, LmME protein level was strongly upregulated in low glucose condition. Notably, both LmME and LmCA1 were found to be important for survival of Leishmania amastigotes within host macrophages. Taken together, our results indicate that LmCA1 by virtue of its CO2 concentrating ability stimulates LmME-catalyzed pyruvate carboxylation, thereby driving gluconeogenesis through pyruvate-malate-oxaloacetate, a bypass pathway. Additionally, our study also established that both the LmCA1 and LmME can be considered as promising therapeutic targets against leishmaniasis.

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