Temperature-dependent Regulation of Mycolic Acid Cyclopropanation in Saprophytic Mycobacteria

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 ␣-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, ␣-mycolic acid cyclopropanation was inducible in cultures grown at 25°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°C compared with 37°C. An MSMEG_1351 knock-out strain of M. smegmatis, generated by recombineering, exhibited a deficiency in cyclopropanation of ␣-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 ⌬pcaA 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.Mycolic acids are major components of the hydrophobic cell wall of Corynebacterineae (1-4). These ␣-branched ␤-hydroxylated long chain fatty acids are covalently linked to the arabinogalactan layer that is attached to the peptidoglycan backbone and are also present as extractable lipids conjugated with sugars such as trehalose, forming trehalose dimycolate (4). Mycolic acids from mycobacteria possess a long main carbon chain called meromycolate, which in its nascent form is interrupted by double bonds at two specific sites called the proximal (closer to the ␤-hydroxy) and the distal positions. Chemical modifications of these double bonds give rise to different types of mycolic acids, depending on the nature of the chemical groups introduced. 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). These enzymes share extensive sequence similarity, but genetic studies have revealed the distinct functional characteristics of each in the biosynthesis of mycolic acids....

Growth of <em>Mycobacterium tuberculosis</em> Biofilms

“…Most microbial species in their natural habitat spontaneously grow in self-assembled, surface attached multicellular communities called biofilms, which are highly tolerant to antibiotics 3 . Recently, it has been shown that several mycobacterial species, have strong propensity to grow in vitro as biofilms, which provide a microenvironment that promote development of drug tolerant persisters 9,[13][14][15] . While fast growing environmental species such as M. smegmatis can readily form biofilms in detergent-free media, the slow growing pathogenic species M. tuberculosis require specific conditions to form the multicellular structures 9 .…”

The Non-Essential Mycolic Acid Biosynthesis Genes hadA and hadC Contribute to the Physiology and Fitness of Mycobacterium smegmatis

“…This can be done by changing the relative amount and structures of various lipids [ 39 ]. Indeed there are several reports showing that MAs structures are regulated by temperature with the ratio of saturation/unsaturation and the relative presence of functional groups in the MAs as well as the chain lengths of MAs being critical determinants of the fluidity of the mycomembrane [ 38 , 60 , 61 , 62 , 63 ]. Our data indicated a change in the sensitivity of the hadC and hadAC mutants to either cold- or high-temperatures.…”

Rv0132c of Mycobacterium tuberculosis Encodes a Coenzyme F420-Dependent Hydroxymycolic Acid Dehydrogenase

“…Major mycolic acids produced by M. smegmatis lack cyclopropanation under normal growth conditions [23] , [50] . This modification occurs during growth at 25°C [51] and the growth temperature in our study was 37°C.…”