Temperature-dependent regulation of mycolic acid cyclopropanation in saprophytic mycobacteria: role of the Mycobacterium smegmatis 1351 gene (MSMEG_1351) in CIS-cyclopropanation of alpha-mycolates

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.

FIGURE 1.

Regulation of mycolic acid cyclopropanation by growth temperature in M. smegmatis. Mycobacterial cultures grown at the indicated temperatures were processed for either in vivo relative quantification of cis-cyclopropane by ¹H HR-MAS NMR (A) or for ¹⁴C-radiolabeling and extraction of mycolic acid (B and C). Mycolates were resolved on a normal TLC plate (B, left panel), a silver nitrate-impregnated TLC plate (B, right panel), or by two-dimensional TLC (C). FAME, fatty acid methyl ester. The new spot accumulating at low growth temperatures, termed lipid Z in B is indicated by an arrow. α₁ and α₂ correspond to the cis/cis and cis/trans di-unsaturated α-mycolic acids, respectively. e, epoxy mycolic acids.

FIGURE 2.

Identification of the genes involved in the production of the cyclopropanated mycolates. A, schematic representation and genomic organization of the potential methyltransferases in M. smegmatis and comparison with M. tuberculosis. Black boxes indicate the mycolic acid methyltransferases; gray boxes indicate open reading frames encoding conserved proteins between the two species, and white boxes represent open reading frames unique to either M. smegmatis or M. tuberculosis. Coordinates of each gene cluster are indicated in kb. B, alignment of the M. smegmatis putative methyltransferases. The AdoMet-binding site as described previously (14) is underlined. C, argentation TLC of ¹⁴C-radiolabeled mycolic acids from the parental strain (1st lane), the ΔMSMEG_1351 mutant carrying either the empty pMV306 (2nd lane) or pMV306_1351 (3rd lane), all grown at 25 °C. The arrowhead indicates the position of lipid Z that is lacking in the ΔMSMEG_1351 mutant. D, temperature-regulated expression of MSMEG_1351. Wild-type M. smegmatis was grown at the indicated temperature, and the levels of MSMEG_1351 transcripts relative to those of the RNA polymerase σ-factor mysA gene (MSMEG_2758) were measured by quantitative reverse transcription-PCR. MSMEG_1351 transcript levels at the different temperatures were normalized to those from cells growing at 37 °C. Results are expressed as means ± S.D. from four independent experiments.

FIGURE 3.

Structural analysis of lipid Z. A, MALDI-MS analysis of intact carboxymethylated mycolic acids from α-mycolic acids devoid of lipid Z (upper panel) and lipid Z (lower panel). B, EI-MS analysis of in-source pyrolysis fragment generated from a mixture of α-mycolates lacking lipid Z (upper panel) or containing lipid Z (lower panel). C, EI-MS analysis of fragment ions generated from carboxymethylated lipid Z. Cis- and trans-conformation of functional groups were omitted in schematic representations of mycolates for the sake of clarity.

FIGURE 4.

Complementation of M. bovis BCG ΔpcaA M. smegmatis ΔMSMEG_1351 mutants. Complementation of M. bovis BCG ΔpcaA by MSMEG_1351. One-dimensional (A) and two-dimensional (B) argentation TLC of ¹⁴C-radiolabeled mycolic acids from M. bovis BCG Pasteur and BCG mc²2801 (BCG Pasteur pcaA::Tn5370) transformed with various vectors and grown at 37 °C. The arrowhead indicates the position of the hybrid mycolate accumulating in the ΔpcaA mutant, with a cis double bond at the proximal position in place of a cis-cyclopropane present in the wild-type α-mycolate as determined previously (5). α, α-mycolates; K, keto-mycolates; WT, wild type. C, single BCG colonies grown on cord reading agar and photographed after 15 days of incubation at 37 °C. Magnification, ×100. D, complementation of M. smegmatis ΔMSMEG_1351 by pcaA. Two-dimensional TLC analysis of ¹⁴C-radiolabeled mycolic acids from M. smegmatis ΔMSMEG_1351 transformed either with pMV261, pMV306_1351, or pMV261_pcaA grown at 25 °C. Arrows indicate the position of the cyclopropanated mycolate missing in the ΔMSMEG_1351 mutant.