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Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis.

ABSTRACT:

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Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information is available on the expression and regulation of the biosynthesis genes. In this study, we report that the hadA, hadB, and hadC genes, which code for the mycolate biosynthesis dehydratase enzymes, are coexpressed with three genes that encode proteins of the translational apparatus. Consistent with the well-established control of the translation potential by nutrient availability, starvation leads to downregulation of the hadABC genes along with most of the genes required for the synthesis, modification, and transport of mycolates. The downregulation of a subset of the biosynthesis genes is partially dependent on RelMtb, the key enzyme of the stringent response. We also report the phylogenetic evolution scenario that has shaped the current genetic organization, characterized by the coregulation of the hadABC operon with genes of the translational apparatus and with genes required for the modification of the mycolates.

Importance

Mycobacterium tuberculosis infects one-third of the human population worldwide, and despite the available therapeutic arsenal, it continues to kill millions of people each year. There is therefore an urgent need to identify new targets and develop a better understanding of how the bacterium is adapting itself to host defenses during infection. A prerequisite of this understanding is knowledge of how this adaptive skill has been implanted by evolution. Nutrient scarcity is an environmental condition the bacterium has to cope with during infection. In many bacteria, adaptation to starvation relies partly on the stringent response. M. tuberculosis's unique outer membrane layer, the mycomembrane, is crucial for its viability and virulence. Despite its being the target of the major antituberculosis drugs, only scattered information exists on how the genes required for biosynthesis of the mycomembrane are expressed and regulated during starvation. This work has addressed this issue as a step toward the identification of new targets in the fight against M. tuberculosis.

SUBMITTER: Jamet S

PROVIDER: S-EPMC4652049 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis.

Jamet Stevie S Quentin Yves Y Coudray Coralie C Texier Pauline P Laval Françoise F Daffé Mamadou M Fichant Gwennaele G Cam Kaymeuang K

Journal of bacteriology 20150928 24

<h4>Unlabelled</h4>Mycobacterium tuberculosis, the etiological agent of tuberculosis, is a Gram-positive bacterium with a unique cell envelope composed of an essential outer membrane. Mycolic acids, which are very-long-chain (up to C100) fatty acids, are the major components of this mycomembrane. The enzymatic pathways involved in the biosynthesis and transport of mycolates are fairly well documented and are the targets of the major antituberculous drugs. In contrast, only fragmented information ...[more]

PMID: 26416833

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Project description:Mycolic acids are key cell wall components for the survival, pathogenicity, and antibiotic resistance of the human tubercle bacillus. Although it was thought that Mycobacterium tuberculosis tightly regulates their production to adapt to prevailing environmental conditions, the molecular mechanisms governing mycolic acid biosynthesis remained extremely obscure. Meromycolic acids, the direct precursors of mycolic acids, are synthesized by a type II fatty acid synthase from acyl carrier protein-bound substrates that are extended iteratively, with a reductive cycle in each round of extension, the second step of which is catalyzed by the essential beta-ketoacyl-acyl carrier protein reductase, MabA. In this study, we investigated whether post-translational modifications of MabA might represent a strategy employed by M. tuberculosis to regulate mycolic acid biosynthesis. Indeed, we show here that MabA was efficiently phosphorylated in vitro by several M. tuberculosis Ser/Thr protein kinases, including PknB, as well as in vivo in mycobacteria. Mass spectrometric analyses using LC-ESI/MS/MS and site-directed mutagenesis identified three phosphothreonines, with Thr(191) being the primary phosphor-acceptor. A MabA_T191D mutant, designed to mimic constitutive phosphorylation, exhibited markedly decreased ketoacyl reductase activity compared with the wild-type protein, as well as impaired binding of the NADPH cofactor, as demonstrated by fluorescence spectroscopy. The hypothesis that phosphorylation of Thr(191) alters the enzymatic activity of MabA, and subsequently mycolic acid biosynthesis, was further supported by the fact that constitutive overexpression of the mabA_T191D allele in Mycobacterium bovis BCG strongly impaired mycobacterial growth. Importantly, conditional expression of the phosphomimetic MabA_T191D led to a significant inhibition of de novo biosynthesis of mycolic acids. This study provides the first information on the molecular mechanism(s) involved in mycolic acid regulation through Ser/Thr protein kinase-dependent phosphorylation of a type II fatty acid synthase enzyme.

Project description:Mycobacterium tuberculosis has a complex cell envelope that is remodelled throughout infection to respond and survive the hostile and variable intracellular conditions within the host. Despite the importance of cell wall homeostasis in pathogenicity, little is known about the environmental signals and regulatory networks controlling cell wall biogenesis in mycobacteria. The mycolic acid desaturase regulator (MadR) is a transcriptional repressor responsible for regulation of the essential aerobic desaturases desA1 and desA2 that are differentially regulated throughout infection along with mycolate modification genes and thus, likely involved in mycolic acid remodelling. Here we generated a madR null mutant in M. smegmatis that exhibited traits of an impaired cell wall with increased permeability, susceptibility to rifampicin and cell surface disruption as a consequence of desA1/desA2 dysregulation. Analysis of mycolic acids revealed the presence of a highly desaturated mycolate in the null mutant that exists in relative trace amounts in the wildtype, but increases in abundance upon cell surface disruption as a result of relieved repression on the desA1/desA2 promoters. Transcriptomic profiling confirmed MadR as a cell surface disruption responsive regulator of desA1/desA2 and further implicating it in the control of bespoke β-oxidation pathways and transport evolutionarily diversified subnetworks associated with virulence. In vitro characterisation of MadR using electromobility shift assays and analysis of binding affinities is suggestive of a unique acyl-CoA pool sensing mechanism, whereby MadR is able to bind a range of acyl-CoA but MadR repression of desA1/desA2 promoters is only relieved upon binding of saturated acyl-CoA of chain length C16-C24. We propose this acyl effector ligand mechanism as distinct to other regulators of mycolic acid biosynthesis or fatty acid desaturases and places MadR as the key regulatory checkpoint that coordinates mycolic acid remodelling in response to host derived cell surface perturbation

Dataset Information

Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis.

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Importance

Publications

Evolution of Mycolic Acid Biosynthesis Genes and Their Regulation during Starvation in Mycobacterium tuberculosis.

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