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 thatMtbrelies mainly on fatty acid catabolism in the host. However,Mtbalso maintains a functional glycolytic pathway and its role in the cellular metabolism ofMtbhas yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and theMtbgenome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show thatpykAencodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion ofpykApreventsMtbgrowth 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 α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism inMtb.

Keywords: Mycobacterium tuberculosis; allosteric regulation; bacterial metabolism; fatty acid metabolism; microbial pathogenesis; pyruvate kinase; tricarboxylic acid cycle (TCA cycle) (Krebs cycle).

FIGURE 1.

Activation of PK by allosteric effectors. A, activity of PK in the presence of 5 mm P-enolpyruvate (PEP) and 0.02–5.2 mm ADP. B, activity of Mtb PK alone (circles) with 0.5 mm Glc-6-P (squares) or 2 mm AMP (triangles) in the presence of 5 mm ADP and 2.5 nm PK. C, activation of PK by Glc-6-P in the presence of 5 mm ADP, 0.4 mm P-enolpyruvate, 2.5 nm PK, and 0–4 mm Glc-6-P. D, activation of PK by AMP in the presence of 5 mm ADP, 0.4 mm P-enolpyruvate, 2.5 nm PK, and 0–4 mm AMP. E, metabolic profile of Mtb grown in HdB with glucose (black bars) or acetate (green bars) as a sole source of carbon. n = 6, statistical significance was determined by t test. * represents p value < 0.005.

FIGURE 2.

Growth curves of WT (black), ΔpykA (red), and ΔpykAcomp (blue), in the presence of (A) glucose, (B) glycerol, (C) acetate, (D) pyruvate, (E) glucose + pyruvate, (F) glucose + acetate and (G) acetate + pyruvate. Error bars represent standard deviations from 3 biological replicates. OD measurement were stopped when cultures started to clump. Ace, acetate; pyr, pyruvate; gly, glycerol; glu, glucose.

FIGURE 3.

A, cfu count of ΔpykA grown on acetate (light green), glucose (black), or no carbon (dark green). B, methylglyoxal quantification of WT, ΔpykA, and ΔpykAcomp grown HdB supplemented with glucose and acetate. n = 3, significance was determined by t test, * represents p value < 0.005.

FIGURE 4.

Growth curves of WT (black), ΔpykA (red), and ΔpykAcomp (blue) in the presence of (A) glucose + propionate, (B) glucose + butyrate, (C) propionate, and (D) butyrate. Error bars represent standard deviations from 3 biological replicates. C3, propionate; C4, butyrate.

FIGURE 5.

Metabolic profiling of WT (black), ΔpykA (red), and ΔpykAcomp (blue) growing in HdB solid media supplemented with acetate or a combination of acetate and glucose. n = 3, significance was tested by using t test. * represents p value < 0.005.

FIGURE 6.

Upper panel: A, ICDH activity in whole cell lysate in the presence (triangles) or absence (circles) of 10 mm P-enolpyruvate (PEP). Data were fitted to Equation 1 to obtain K_m values of 0.027 and 0.190 mm in the absence and presence of P-enolpyruvate, respectively. k_cat values were 0.008 and 0.0125 mm min⁻¹ mg of protein⁻¹ in the absence or presence of P-enolpyruvate, respectively. Lower panel: growth curves of WT (black), ΔpykA (red), and ΔpykAcomp (blue) in the presence of propionate + glutamate or butyrate + glutamate (C). Error bars represent standard deviations from 3 biological replicates. C3, propionate; C4, butyrate; glut, glutamate.

FIGURE 7.

A, survival plot of SCID mice infected with low dose aerosol of CDC 1551 (black), ΔpykA (red), and ΔpykAcomp (blue). SCID mouse survival was significantly different between ΔpykA and CDC 1551 or complemented strain (p value < 0.01, Gehan-Breslow-Wilcoxon test). B, lung burden of CDC 1551, ΔpykA, and ΔpykAcomp in C57BL/6 mice infected with low dose aerosol.