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Individual Mycobacterium tuberculosis universal stress protein homologues are dispensable in vitro.

ABSTRACT: Mycobacterium tuberculosis has 10 universal stress proteins, whose function is unknown. However, proteomic and transcriptomic analyses have shown that a number of usp genes are significantly upregulated under hypoxic conditions and in response to nitric oxide and carbon monoxide, as well as during M. tuberculosis infection of macrophage cell lines. Six of these USPs are part of the DosR regulon and this, along with their expression pattern and the phenotypes of usp mutants in other bacterial species, suggests a potential role in the persistence and/or intracellular survival of Mtb. Knock-out mutants of individual usp genes encoding the USPs Rv1996, Rv2005c, Rv2026c and Rv2028c were generated and their growth and survival under hypoxic and other stress conditions examined. Although the majority of usp genes are highly induced in hypoxic conditions, mutation did not affect the long term survival of Mtb under these conditions, or in response to a range of stress conditions chosen to represent the environmental onslaughts experienced by the bacillus during an infection, nor during infection of mouse and human - derived macrophage cell lines. The possibility remains that these USPs are functionally redundant in Mtb.

SUBMITTER: Hingley-Wilson SM

PROVIDER: S-EPMC2914252 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Individual Mycobacterium tuberculosis universal stress protein homologues are dispensable in vitro.

Hingley-Wilson S M SM Lougheed K E A KE Ferguson K K Leiva S S Williams H D HD

Tuberculosis (Edinburgh, Scotland) 20100611 4

Mycobacterium tuberculosis has 10 universal stress proteins, whose function is unknown. However, proteomic and transcriptomic analyses have shown that a number of usp genes are significantly upregulated under hypoxic conditions and in response to nitric oxide and carbon monoxide, as well as during M. tuberculosis infection of macrophage cell lines. Six of these USPs are part of the DosR regulon and this, along with their expression pattern and the phenotypes of usp mutants in other bacterial spe ...[more]

PMID: 20541977

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Project description:ABSTRACT Triosephosphate isomerase (TPI) catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). This reaction is required for glycolysis and gluconeogenesis, and tpi has been predicted to be essential for growth of Mycobacterium tuberculosis. However, when studying a conditionally regulated tpi knockdown mutant, we noticed that depletion of TPI reduced growth of M. tuberculosis in media containing a single carbon source but not in media that contained both a glycolytic and a gluconeogenic carbon source. We used such two-carbon-source media to isolate a tpi deletion (?tpi) mutant. The ?tpi mutant did not survive with single carbon substrates but grew like wild-type (WT) M. tuberculosis in the presence of both a glycolytic and a gluconeogenic carbon source. (13)C metabolite tracing revealed the accumulation of TPI substrates in ?tpi and the absence of alternative triosephosphate isomerases and metabolic bypass reactions, which confirmed the requirement of TPI for glycolysis and gluconeogenesis in M. tuberculosis. The ?tpi strain was furthermore severely attenuated in the mouse model of tuberculosis, suggesting that M. tuberculosis cannot simultaneously access sufficient quantities of glycolytic and gluconeogenic carbon substrates to establish infection in mice. IMPORTANCE The importance of central carbon metabolism for the pathogenesis of M. tuberculosis has recently been recognized, but the consequences of depleting specific metabolic enzymes remain to be identified for many enzymes. We investigated triosephosphate isomerase (TPI) because it is central to both glycolysis and gluconeogenesis and had been predicted to be essential for growth of M. tuberculosis. This work identified metabolic conditions that make TPI dispensable for M. tuberculosis growth in culture and proved that M. tuberculosis relies on a single TPI enzyme and has no metabolic bypass for the TPI-dependent interconversion of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in glycolysis and gluconeogenesis. Finally, we demonstrate that TPI is essential for growth of the pathogen in mouse lungs.

Project description:We have previously shown that the Mycobacterium tuberculosis universal stress protein Rv2623 regulates mycobacterial growth and may be required for the establishment of tuberculous persistence. Here, yeast two-hybrid and affinity chromatography experiments have demonstrated that Rv2623 interacts with one of the two forkhead-associated domains (FHA I) of Rv1747, a putative ATP-binding cassette transporter annotated to export lipooligosaccharides. FHA domains are signaling protein modules that mediate protein-protein interactions to modulate a wide variety of biological processes via binding to conserved phosphorylated threonine (pT)-containing oligopeptides of the interactors. Biochemical, immunochemical and mass spectrometric studies have shown that Rv2623 harbors pT and specifically identified threonine 237 as a phosphorylated residue. Relative to wild-type Rv2623 (Rv2623WT), a mutant protein in which T237 has been replaced with a non-phosphorylatable alanine (Rv2623T237A) exhibits decreased interaction with the Rv1747 FHA I domain and diminished growth-regulatory capacity. Interestingly, compared to WT bacilli, an M. tuberculosis Rv2623 null mutant (?Rv2623) displays enhanced expression of phosphatidyl-myo-inositol mannosides (PIMs), while the ?Rv1747 mutant expresses decreased levels of PIMs. Animal studies have previously shown that ?Rv2623 is hypervirulent, while ?Rv1747 is growth-attenuated. Collectively, these data have provided evidence that Rv2623 interacts with Rv1747 to regulate mycobacterial growth; and this interaction is mediated via the recognition of the conserved Rv2623 pT237-containing FHA-binding motif by the Rv1747 FHA I domain. The divergent aberrant PIM profiles and the opposing in vivo growth phenotypes of ?Rv2623 and ?Rv1747, together with the annotated lipooligosaccharide exporter function of Rv1747, suggest that Rv2623 interacts with Rv1747 to modulate mycobacterial growth by negatively regulating the activity of Rv1747; and that Rv1747 might function as a transporter of PIMs. Because these glycolipids are major mycobacterial cell envelope components that can impact on the immune response, our findings raise the possibility that Rv2623 may regulate bacterial growth, virulence, and entry into persistence, at least in part, by modulating the levels of bacillary PIM expression, perhaps through negatively regulating the Rv1747-dependent export of the immunomodulatory PIMs to alter host-pathogen interaction, thereby influencing the fate of M. tuberculosis in vivo.

Dataset Information

Individual Mycobacterium tuberculosis universal stress protein homologues are dispensable in vitro.

Publications

Individual Mycobacterium tuberculosis universal stress protein homologues are dispensable in vitro.

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