Functional partnership between carbonic anhydrase and malic enzyme in promoting gluconeogenesis in Leishmania major

Abstract

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. One perplexing aspect of gluconeogenesis in Leishmania is the lack of the crucial pyruvate carboxylase (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 raises question about the mechanism of pyruvate entry into the gluconeogenic route. We report 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 bypass pathway. Additionally, our study establishes LmCA1 and LmME as promising therapeutic targets.