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Transcriptome of phosphomimetic and phosphoablative kasB mutants of Mycobacterium tuberculosis

ABSTRACT: Mechanisms governing Mycobacterium tuberculosis acid-fastness and its capacity to induce long-term infections remain unknown. Serine/Threonine phosphorylation represents an emerging theme allowing mycobacteria to adapt their cell envelope structure/composition in response to environmental changes. We addressed whether phosphorylation of KasB, a mycolic acid biosynthetic enzyme, modulates M. tuberculosis pathogenicity. Phosphorylation of KasB occurred at Thr334 and Thr336 in vitro and in mycobacteria. A mutant strain bearing an kasB_T334D/T336D allele, mimicking constitutive KasB phosphorylation, was generated by specialized linkage transduction. This resulted in shortened mycolic acids and the lack of trans-cyclopropanation. Structural/modeling analyses revealed Thr334 and Thr336 in the vicinity of the catalytic triad, implying that phosphorylation of these residues impaired KasB activity. Importantly, the phosphomimetic strain lost acid-fast staining and was more attenuated than a kasB deletion mutant in immunocompetent and immunodeficient mice. The absence of lung pathology and mortality infers this mutant to represent a valuable vaccine candidate. This work emphasizes the critical role of Ser/Thr kinase-dependent signaling in controlling mycolic acid elongation, acid-fastness, virulence and has important clinical implications for diagnosis of latent infections.

ORGANISM(S): Mycobacterium tuberculosis

PROVIDER: GSE47640 | GEO | 2014/04/01

SECONDARY ACCESSION(S): PRJNA208269

REPOSITORIES: GEO

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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

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Transcriptome of phosphomimetic and phosphoablative kasB mutants of Mycobacterium tuberculosis

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