The Mycobacterium tuberculosis cmaA2 Gene Encodes a Mycolic Acid trans-Cyclopropane Synthetase

Abstract

Infection with Mycobacterium tuberculosis remains a major global health emergency. Although detailed understanding of the molecular events of M. tuberculosis pathogenesis is still limited, recent genetic analyses have implicated specific lipids of the cell envelope as important effectors in M. tuberculosis pathogenesis. We have shown that pcaA, a novel member of a family of M. tuberculosis S-adenosyl methionine (SAM)-dependent methyl transferases, is required for ␣-mycolic acid cyclopropanation and lethal chronic persistent M. tuberculosis infection. To examine the apparent redundancy between pcaA and cmaA2, another cyclopropane synthetase of M. tuberculosis thought to be involved in ␣-mycolate synthesis, we have disrupted the cmaA2 gene in virulent M. tuberculosis by specialized transduction. Inactivation of cmaA2 causes accumulation of unsaturated derivatives of both the methoxy-and ketomycolates. Analysis by proton NMR indicates that the mycolic acids of the cmaA2 mutant lack trans-cyclopropane rings but are otherwise intact with respect to cyclopropane and methyl branch content. Thus, cmaA2 is required for the synthesis of the trans cyclopropane rings of both the methoxymycolates and ketomycolates. These results define cmaA2 as a trans-cyclopropane synthetase and expand our knowledge of the substrate specificity of a large family of highly homologous mycolic acid methyl transferases recently shown to be critical to M. tuberculosis pathogenesis.Mycobacterium tuberculosis infection continues to overwhelm the populations of the developing world. It has been estimated that in 1997 there were 8 million new cases of active tuberculosis that were added to the already existing 16 million cases (1). In the same year, 2 million people died of tuberculosis as a result of an astonishing case fatality rate of 23-50% (1). This high death rate for a disease treatable with available antibiotics reflects the geographic superimposition of HIV 1 and M. tuberculosis infection, and the logistical and economic burden of at least 6 months of multidrug therapy required to treat the disease. New drugs to shorten therapy and vaccine candidates to prevent M. tuberculosis infection are badly needed but will only come with a more thorough understanding of the mechanisms of M. tuberculosis pathogenesis. The cell envelope of M. tuberculosis is a highly complex array of distinctive lipids and glycolipids that has been intensely scrutinized as a potential effector in the interaction of M. tuberculosis with the human host (2-4). Investigation into the role of the cell envelope in virulence has been hampered by a lack of defined mutants of M. tuberculosis that fail to synthesize specific components of the cell surface. Recently, advances in the genetic manipulation of M. tuberculosis have allowed isolation of several mutants with defined cell envelope deficiencies and altered virulence (5-7). M. tuberculosis synthesizes three classes of mycolic acids, very long chain ␣-alkyl, ␤-hydroxyl fatty acids (Fig. 1) in its cell envelope. These three c...

Comparative proteome analysis of Mycobacterium tuberculosis grown under aerobic and anaerobic conditions

“…Another mycolic acid synthase, PcaA, was shown to be important for dormancy since pcaA knock-out strains lost their ability to persist, although not replicated in a mouse model (Glickman et al, 2000). Studies in M. tuberculosis have shown that Rv0503c (CmaA2), found in this study to be fourfold upregulated during anaerobisis, is required for the synthesis of the trans-cyclopropane rings of keto-and methoxymycolates (Glickman et al, 2001). Cyclopropanation of mycolic acids is a modification that is associated with pathogenic bacteria and is not common in the cell wall of saprophytic species such as M. smegmatis (Minnikin et al, 1982).…”

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.…”

“…DNA Ligation Assay-A 36-bp DNA duplex containing a centrally placed 3Ј-OH/5Ј-PO 4 nick was formed by annealing two 18-mer oligodeoxyribonucleotides to a complementary 36-mer strand as described previously (18,19). The 18-mer constituting the 5Ј phosphate-terminated strand d(ATTCCGATAGTGACTACA) was 5Ј 32 P-labeled and gelpurified, then annealed to the complementary 36-mer DNA (the template strand) in the presence of a 3Ј-OH 18-mer strand d(CATAT-CCGTGTCGCCCTT).…”

“…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).…”

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.…”