Research Article: Glucose Phosphorylation Is Required for Mycobacterium tuberculosis Persistence in Mice

Date Published: January 10, 2013

Publisher: Public Library of Science

Author(s): Joeli Marrero, Carolina Trujillo, Kyu Y. Rhee, Sabine Ehrt, Marcel A. Behr.

http://doi.org/10.1371/journal.ppat.1003116

Abstract

Mycobacterium tuberculosis (Mtb) is thought to preferentially rely on fatty acid metabolism to both establish and maintain chronic infections. Its metabolic network, however, allows efficient co-catabolism of multiple carbon substrates. To gain insight into the importance of carbohydrate substrates for Mtb pathogenesis we evaluated the role of glucose phosphorylation, the first reaction in glycolysis. We discovered that Mtb expresses two functional glucokinases. Mtb required the polyphosphate glucokinase PPGK for normal growth on glucose, while its second glucokinase GLKA was dispensable. 13C-based metabolomic profiling revealed that both enzymes are capable of incorporating glucose into Mtb’s central carbon metabolism, with PPGK serving as dominant glucokinase in wild type (wt) Mtb. When both glucokinase genes, ppgK and glkA, were deleted from its genome, Mtb was unable to use external glucose as substrate for growth or metabolism. Characterization of the glucokinase mutants in mouse infections demonstrated that glucose phosphorylation is dispensable for establishing infection in mice. Surprisingly, however, the glucokinase double mutant failed to persist normally in lungs, which suggests that Mtb has access to glucose in vivo and relies on glucose phosphorylation to survive during chronic mouse infections.

Partial Text

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a disease that killed 1.5 million people in 2010 worldwide [1]. The development of new and effective antimycobacterial drugs hinges on our ability to understand how Mtb establishes and maintains chronic infections. Mtb’s metabolic adaptations are central to its pathogenicity and prevailing evidence has strongly implicated fatty acids and lipids for biomass and energy production during infection [2]–[6]. In contrast, the importance of other carbon sources including carbohydrates during in vivo growth and persistence of Mtb remains uncertain. Mtb possesses carbohydrate transporters and the enzymes required to metabolize sugars [7], [8], but it is unclear whether it utilizes these sugar metabolism pathways to support growth in vivo. Mtb is not thought to encounter a sugar-rich environment inside the host, given that expression of sugar catabolism genes was not induced during infection of macrophages and mice [9], [10]. However, sugar transport appears important in Mtb pathogenesis as mutants with transposon insertions in carbohydrate transport systems were attenuated in mouse spleens [11]. The LpqY-SugA-SugB-SugC carbohydrate transporter was confirmed to be indispensable for normal growth in mouse lungs and spleens [12]. This transporter was shown to be highly specific for uptake of the disaccharide trehalose. Trehalose is not present in mammals, but can be released by Mtb from trehalose-containing cell wall glycolipids and recycled [12]. The intracellular fate of recycled trehalose remains to be identified, and it is unknown which other sugars are metabolized by Mtb to maintain fitness during infection.

That Mtb can metabolize glucose to support its growth in liquid culture has been established over 80 years ago [25]. Whether glucose metabolism is important for the generation of biomass during in vivo growth or required during chronic infections was unknown. Here, we investigated the role of Mtb’s glucokinases catalyzing the generation of glucose-6-P, the first step in glycolysis and essential for glucose metabolism. We found that Mtb expresses two functional glucokinase ortholgues PPGK and Rv0650, which we named GLKA consistent with the M. smegmatis homolog [15]. Protein sequence analysis revealed that PPGK and GLKA belong to the ROK (repressor, ORF, kinase) family, which includes glucokinases and transcriptional repressors [26]. Bioinformatic analysis did not identify a canonical helix-turn-helix motif in PPGK or GLKA commonly found at the amino terminus of transcriptional repressors of the ROK family, suggesting that they do not act as transcriptional regulators. PPGK and GLKA have, however, conserved glucokinase features including the catalytic active aspartate residue, ATP binding domain, and glucose binding sites (Figure S2) [27]. PPGK contains five glucose binding sites and two ATP binding domains spanning amino acid residues 22–31 and 114–156 [13]. Similar to GLKA from M. smegmatis, Mtb GLKA contains only three of the five amino acid residues involved in glucose binding and only a single ATP binding domain spanning amino acids 4–17 [15]. This might explain why this enzyme is less efficient than PPGK in incorporating glucose into Mtb’s central carbon metabolism and not sufficient to sustain robust growth on glucose in the absence of PPGK. Mtb lacking both PPGK and GLKA glucokinases could not metabolize glucose, which established that Mtb does not have any alternative pathway for glucose assimilation under the conditions tested.

 

Source:

http://doi.org/10.1371/journal.ppat.1003116

 

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