Abstract
The cell envelope is a crucial determinant of virulence and drug resistance in Mycobacterium tuberculosis. Several features of pathogenesis and immunomodulation of host responses are attributable to the structural diversity in cell wall lipids, particularly in the mycolic acids. Structural modification of the alpha-mycolic acid by introduction of cyclopropane rings as catalyzed by the methyltransferase, PcaA, is essential for a lethal, persistent infection and the cording phenotype in M. tuberculosis. Here, we demonstrate the presence of cyclopropanated cell wall mycolates in the nonpathogenic strain Mycobacterium smegmatis and identify MSMEG_1351 as a gene encoding a PcaA homologue. Interestingly, alpha-mycolic acid cyclopropanation was inducible in cultures grown at 25 degrees C. The growth temperature modulation of the cyclopropanating activity was determined by high resolution magic angle spinning NMR analyses on whole cells. In parallel, quantitative reverse transcription-PCR analysis showed that MSMEG_1351 gene expression is up-regulated at 25 degrees C compared with 37 degrees C. An MSMEG_1351 knock-out strain of M. smegmatis, generated by recombineering, exhibited a deficiency in cyclopropanation of alpha-mycolates. The functional equivalence of PcaA and MSMEG_1351 was established by cross-complementation in the MSMEG_1351 knock-out mutant and also in a DeltapcaA strain of Mycobacterium bovis BCG. Overexpression of MSMEG_1351 restored the wild-type mycolic acid profile and the cording phenotype in BCG. Although the biological significance of mycolic acid cyclopropanation in nonpathogenic mycobacteria remains unclear, it likely represents a mechanism of adaptation of cell wall structure and composition to cope with environmental factors.
Highlights
Mycolic acids are major components of the hydrophobic cell wall of Corynebacterineae [1,2,3,4]
In M. tuberculosis, PcaA is required for cis-cyclopropanation at the proximal site of ␣-mycolates [5], whereas MmaA2 converts double bonds to cis-cyclopropane rings at the distal and proximal positions of ␣- and oxygenated mycolates, respectively [6], and CmaA2 introduces a trans-cyclopropane ring in oxygenated mycolates [7]
The overexpression of M. tuberculosis cmaA1 leads to the conversion of the distal cis double bond into a cis-cyclopropane ring in the ␣-mycolate of M. smegmatis [15]
Summary
ROLE OF THE MYCOBACTERIUM SMEGMATIS 1351 GENE (MSMEG_1351) IN CIS-CYCLOPROPANATION OF ␣-MYCOLATES*□S. We demonstrate the presence of cyclopropanated cell wall mycolates in the nonpathogenic strain Mycobacterium smegmatis and identify MSMEG_1351 as a gene encoding a PcaA homologue. Mycobacterium tuberculosis carries the following three types of mycolic acids: ␣-subtypes, containing two cis-cyclopropane rings, and two oxygenated subtypes, harboring methoxy and keto functions on the distal position, with a cis- or trans-cyclopropane ring on the proximal site [1, 3]. These chemical modifications are performed by a group of S-adenosylmethionine (AdoMet)3-dependent methyltransferases [3]. Functional complementation of a Mycobacterium bovis BCG ⌬pcaA mutant with MSMEG_1351 restores both the cis-cyclopropanation of ␣-mycolates and the cording phenotype characterizing M. bovis BCG or M. tuberculosis, strongly indicating that MSMEG_1351 is a functional orthologue of PcaA
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