Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests that Mtb relies mainly on fatty acid catabolism in the host. However, Mtb also maintains a functional glycolytic pathway and its role in the cellular metabolism of Mtb has yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and the Mtb genome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show that pykA encodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion of pykA prevents Mtb growth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the ␣-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism in Mtb.Mycobacterium tuberculosis (Mtb) 3 pathogenesis has been studied for decades, however, our knowledge concerning the metabolism and physiology of the bacterium during host infection is still limited (1-4). Specifically, we lack understanding of nutrient availability in the host microenvironments during the different phases of infection and consequently, which nutrients (e.g. carbon and nitrogen sources) are used by the bacterium for growth and maintenance of replicative and non-replicative states (5). In vitro evidence suggests that Mtb does not utilize carbon catabolite repression, a regulatory mechanism that allows bacteria and single-cell eukaryotes to gain growth advantage through prioritized metabolism of one carbon source over the other (6), but rather co-catabolizes multiple carbon sources at once (4). Several lines of evidence suggest that Mtb relies mainly on fatty acid metabolism in the non-replicative state within the host (1, 7-12). During growth on fatty acids Mtb bypasses the carbon dioxide releasing steps of the TCA cycle by running the glyoxylate shunt to conserve carbon (4). Nevertheless, Mtb maintains a functional and intact glycolytic pathway, suggesting that glycolysis plays a role under certain in vivo conditions. Recent studies with the adenosine triphosphate (ATP) synthase inhibitor, Bedaquiline, showed a delayed cidal effect when Mtb was grown on fermentable carbon sources (13), suggesting that Mtb can produce ATP through substrate level phosphorylation when oxidative-phosph...

Mycobacterium tuberculosis in the Face of Host-Imposed Nutrient Limitation

“…Taken together and based on the current literature, we know that several amino acids can be taken up by the pathogen from the host, but none of them is essential for replication and survival in the host. This suggests that M. tuberculosis uses nitrogen cocatabolism for nitrogen assimilation, much like its strategy of carbon cocatabolism (33, 34). …”

“…M. bovis BCG Δ gdh was also unable to grow with cholesterol as the sole carbon source (89), indicating that glutamate anaplerosis (glutamate feeds into the TCA cycle via alpha-ketoglutarate) is important during growth on lipids and fatty acids. In fact, an M. tuberculosis strain (Δ pykA ) deficient for growth on fermentable carbon sources was recently shown to be unable to grow on fatty acids unless the medium was supplemented with glutamate (34). However, the pykA mutant was not attenuated in a mouse model, suggesting that metabolites such as glutamate or acetate can be scavenged from the host (34).…”

“…In fact, an M. tuberculosis strain (Δ pykA ) deficient for growth on fermentable carbon sources was recently shown to be unable to grow on fatty acids unless the medium was supplemented with glutamate (34). However, the pykA mutant was not attenuated in a mouse model, suggesting that metabolites such as glutamate or acetate can be scavenged from the host (34). An isotopologue labeling experiment in human THP-1 macrophages suggests M. tuberculosis’ s ability to scavenge glutamate (30), but proof of glutamate utilization during animal infection is still missing.…”

Model-based integration of genomics and metabolomics reveals SNP functionality in Mycobacterium tuberculosis

“…This SNP was shown to abolish the activity of PykA in animaladapted strains, rendering them unable to grow on glucose as the sole carbon source 42,43 . Deleting pykA in the laboratory strain H37Rv also leads to glucose toxicity, presumably due to accumulation of methylglyoxal 45 . While our analysis classified the pykA E220D SNP as functional, it predicted only a relatively small flux effect under the tested conditions in E200Dcarrying MTBC strains (Fig.…”

Mycobacterium tuberculosisin the Face of Host-Imposed Nutrient Limitation

“…M. bovis BCG Δgdh was also unable to grow with cholesterol as the sole carbon source (89), indicating that glutamate anaplerosis (glutamate feeds into the TCA cycle via alpha-ketoglutarate) is important during growth on lipids and fatty acids. In fact, an M. tuberculosis strain (ΔpykA) deficient for growth on fermentable carbon sources was recently shown to be unable to grow on fatty acids unless the medium was supplemented with glutamate (34). However, the pykA mutant was not attenuated in a mouse model, suggesting that metabolites such as glutamate or acetate can be scavenged from the host (34).…”