Global Effects of Inactivation of the Pyruvate Kinase Gene in the Mycobacterium tuberculosis Complex

To better understand the global effects of “natural” lesions in genes involved in the pyruvate metabolism in Mycobacterium bovis, null mutations were made in the Mycobacterium tuberculosis H37Rv ald and pykA genes to mimic the M. bovis situation. Like M. bovis, the M. tuberculosis ΔpykA mutant yielded dysgonic colonies on solid medium lacking pyruvate, whereas colony morphology was eugonic on pyruvate-containing medium. Global effects of the loss of the pykA gene, possibly underlying colony morphology, were investigated by using proteomics on cultures grown in the same conditions. The levels of Icd2 increased and those of Icl and PckA decreased in the ΔpykA knockout. Proteomics suggested that the synthesis of enzymes involved in fatty acid and lipid biosynthesis were decreased, whereas those involved in β-oxidation were increased in the M. tuberculosis ΔpykA mutant, as confirmed by direct assays for these activities. Thus, the loss of pykA from M. tuberculosis results in fatty acids being used principally for energy production, in contrast to the situation in the host when carbon from fatty acids is conserved through the glyoxylate cycle and gluconeogenesis; when an active pykA gene was introduced into M. bovis, the opposite effects occurred. Proteins involved in oxidative stress—AhpC, KatG, and SodA—showed increased synthesis in the ΔpykA mutant, and iron-regulated proteins were also affected. Ald levels were decreased in the ΔpykA knockout, explaining why an M. tuberculosis ΔpykA Δald double mutant showed little additional phenotypic effect. Overall, these data show that the loss of the pykA gene has powerful, global effects on proteins associated with central metabolism.Comparison of the genome sequences of Mycobacterium bovis and Mycobacterium tuberculosis revealed >99.95% identity at the nucleotide level; however, these pathogens differ in terms of host tropism, phenotype, and virulence (16). Eleven regions of difference (RD) were observed in the M. bovis genome (2 to 12.7 kb) compared to M. tuberculosis, while one region deleted from M. tuberculosis was present in M. bovis (5, 16). In addition to the RDs, there are over 2,400 single nucleotide polymorphisms (SNPs) between M. tuberculosis and M. bovis (16). Some SNPs cosegregate with regions of deletions or other genetic markers (5); one such SNP is in the pykA gene, which cosegregates with the RD9 deletion. This SNP results in an inactive pyruvate kinase (PykA) being produced due to a Glu220Asp mutation (20). Glu220 is in the active site of the enzyme (21, 24), and its substitution results in complete loss of the enzyme activity in M. bovis (20). Thus, the pykA SNP explains one of the classic distinctions between M. bovis and M. tuberculosis, namely, the requirement for pyruvate. Neither glycerol, the preferred carbon source for isolation of tubercle bacilli, nor glucose support the growth of M. bovis when they are not supplemented with pyruvate (38), due to the inactive pyruvate kinase.On the routinely used Middlebrook 7H11 agar, containing glycerol and oleate, M. bovis shows dysgonic colony morphology, whereas M. tuberculosis, in contrast, shows eugonic colony morphology with abundant growth. Complementation of M. bovis with the active pykA gene from M. tuberculosis restored the eugonic phenotype. Thus, loss of PykA activity commits M. bovis to using nonglycolytic substrates as carbon sources, such as lipids. This in itself is of biological significance since human M. tuberculosis switches to this kind of metabolism in experimentally infected animals or in macrophages (34, 35, 39). However, even with oleate (a lipid) as a sole carbon source which allows both species to grow, there was still a difference in colony morphology (20). This led us to consider that loss of the pykA gene had wider effects since PykA is not needed for energy production on oleate and has no role in gluconeogenesis (Fig. ​(Fig.1).1). Thus, we hypothesize that the loss of the pykA gene has global effects over and above the predicted effect of determining whether or not growth can take place on glycerol. To examine our hypothesis, we created a pykA mutant of M. tuberculosis to investigate the effect of pykA deletion by using isogenic strains. This builds upon our previous study in which we had complemented M. bovis with the (active) M. tuberculosis pykA gene (20). We also created a mutant in alanine dehydrogenase (H37Rv Δald) and a H37Rv Δald ΔpykA double mutant since M. bovis naturally lacks active ald and pykA genes (16). The global effects of these knockout mutations were then examined by their on growth on a range of carbon sources and on protein expression during growth on pyruvate, a gluconeogenic carbon source. A proteomic approach was chosen since it would reveal changes in all proteins, for example, regulatory proteins, enzymes, and stress proteins; key proteins, or effects of changes in their levels, could then be assayed for directly. These approaches revealed the major metabolic consequences resulting from pykA inactivation.Open in a separate windowFIG. 1.Pathways of carbon metabolism possible in strains with or without pyruvate kinase (PykA). Boxes denote substrates and/or products where arrows are used to denote pathways. Arrows to and from boxes are pathways; other arrows show reactions catalyzed by a single enzyme. Substrates are in text with serifs; pathways and enzymes in plain text. Colored arrows are used to denote glycolysis or gluconeogenesis in red, the tricarboxylic acid cycle in blue, and the glyoxylate cycle in magenta.