The mmaA2 Gene of Mycobacterium tuberculosis Encodes the Distal Cyclopropane Synthase of the α-Mycolic Acid

Abstract

Infection with Mycobacterium tuberculosis (Mtb) remains a severe global health problem that has prompted an aggressive search for new antibiotic targets and vaccine strategies for this persistent pathogen. Recently, a wide variety of genetic determinants of Mtb pathogenicity have been identified, including several genes involved in the biogenesis of the complex Mtb cell envelope. Among these are the mycolic acid cyclopropane synthases, a family of proteins that modify the major cell envelope lipids of Mtb with a diversity of cyclopropane rings. Despite substantial sequence identity, these proteins catalyze highly specific cyclopropane modifications, including proximal modification of the ␣-mycolate (pcaA) and trans-cyclopropane modification (cmaA2). Here we report the mycolic acid modification function of a third cyclopropane synthase, mmaA2, through the creation and analysis of an M. tuberculosis mmaA2 null mutant. Unexpectedly, mmaA2 is essential for the distal cyclopropane modification of the ␣-mycolate, a function previously attributed to cmaA1. ␣-Mycolates of a cmaA1 null mutant were unaffected, demonstrating that cmaA1 is not required for ␣-mycolate modification. Although fully cyclopropanated methoxymycolates are produced in the mmaA2 mutant, cis-cyclopropanation is impaired, leading to accumulation of unsaturated methoxymycolate derivatives. This study establishes mmaA2 as a distal cyclopropane synthase of the ␣-mycolates of M. tuberculosis and the first cyclopropane synthase to modify both ␣-and oxygenated mycolates. These results expand our knowledge of the biosynthesis of the Mtb cell envelope and will allow further elucidation of the relationship between Mtb pathogenesis and the fine structure of mycolic acids.

Towards understanding the functional diversity of cell wall mycolic acids of Mycobacterium tuberculosis

“…Examples of these are PcaA and MmaA2 mutants that express mycolic acids without α-mycolate cyclopropanation [10,11], CmaA2 mutants that are unable to effect trans-cyclopropanation in the oxygenated mycolates [12], or MmaA4 and MmaA3 mutants that produce mycolic acids without distal functionality of the oxygenated mycolates [13,14]. The significance of secreted free mycolic acids as potential role players in the manifestation of tuberculosis was recently noted when Ojha et al (2008) [15] demonstrated their role in biofilm formation during in vitro growth of M. tuberculosis.…”

Biochemical and Genetic Analysis of the Four DNA Ligases of Mycobacteria

“…Ligase Gene Disruptions in M. tuberculosis-Gene disruption was performed by specialized transduction of lig::hyg R cassettes using temperature-sensitive mycobacteriophages as described previously (19,20). The lig⌬::hyg R gene disruption cassettes were constructed by PCR amplifying genomic DNA segments flanking each ligase gene and inserting them on either side of the hygromycin resistance gene in plasmid pJSC407, a cloning vector containing the hyg R marker, a cos site, and a unique PacI restriction site for packaging into phAE87.…”

“…The lig⌬ knockout cassettes containing the hyg R marker flanked by upstream and downstream M. tuberculosis genomic DNA for each lig locus were packaged into a temperature-sensitive derivative of the mycobacteriophage TM4 and the recombinant phages were then used to transduce M. tuberculosis Erdman to hygromycin resistance (19,20). Southern hybridization of restriction endonuclease-digested genomic DNA from hygromycin-resistant transductants revealed the predicted fragment sizes for correctly targeted allelic exchange at the ligB, ligC, and ligD loci (data not shown).…”

Characterization of Mycobacterium tuberculosis complex isolated from iranian and afghani patients by spoligotyping method

“…The cyclopropane moiety of mycolic acids is a unique lipid structure that greatly affects the pathogenesis of M. tuberculosis (15). The bacterium uses a family of Sadenosylmethionine-dependent methyltransferases to modify the mycolic acids of its cell envelope with a variety of stereochemistries and positions for cyclopropyl groups (13). There are at least three mycolic acid cyclopropane synthases (PcaA, CmaA1, and CmaA2) that are responsible for these sitespecific modifications of mycolic acids (17).…”