A dTDP-L-rhamnose 4-epimerase required for glycopeptidolipid biosynthesis in Mycobacterium abscessus.

Mycobacterium abscessus causes severe lung infections in cystic fibrosis patients and exhibits smooth (S) or rough (R) morphotypes. Disruption of glycopeptidolipid (GPL) production results in the S-to-R transition but the underlying molecular mechanisms of this transition remain incompletely understood. Herein, we characterized MAB_4111c in relation to GPL synthesis and investigated the effects of MAB_4111c deletion in M. abscessus pathogenicity. An enzymatic assay indicated that MAB_4111c, also designated Tle for Talose epimerase, is converting dTDP-L-Rhamnose into dTDP-6-deoxy-L-Talose. A tle deletion mutant was constructed in the S variant of M. abscessus and relative areas of Rhamnose and 6-deoxy-Talose and their methylated forms expressed as ratios of total monosaccharides, showed an altered GPL profile lacking 6-deoxy-Talose. Thus, Tle provides dTDP-6-deoxy-L-Talose, subsequently used by the glycosyltransferase Gtf1 to transfer 6-deoxy-Talose to the GPL backbone. Strikingly, the tle mutant exhibited an R morphotype, showed impaired sliding motility and biofilm formation, and these phenotypes were rescued upon functional complementation. Moreover, deletion of tle in M. abscessus results in increased pathogenicity and killing in zebrafish embryos. Together, our results underscore the importance of the dTDP-L-Rhamnose 4-epimerase activity in GPL biosynthesis and in influencing M. abscessus virulence.

Multiple Mycobacterium abscessus O-acetyltransferases influence glycopeptidolipid structure and colony morphotype.

Mycobacterium abscessus causes severe lung infections. Clinical isolates can have either smooth (S) or rough (R) colony morphotypes; of these, S but not R variants have abundant cell wall glycopeptidolipids (GPL) consisting of a peptidolipid core substituted by a 6-deoxy-α-L-talose (6-dTal) and rhamnose residues. Deletion of gtf1, encoding the 6-dTal transferase, results in the S-to-R transition, mycobacterial cord formation, and increased virulence, underscoring the importance of 6-dTal in infection outcomes. However, since 6-dTal is di-O-acetylated, it is unclear whether the gtf1 mutant phenotypes are related to the loss of the 6-dTal or the result of the absence of acetylation. Here, we addressed whether M. abscessus atf1 and atf2, encoding two putative O-acetyltransferases located within the gpl biosynthetic locus, transfer acetyl groups to 6-dTal. We found deletion of atf1 and/or atf2 did not drastically alter the GPL acetylation profile, suggesting there are additional enzymes with redundant functions. We subsequently identified two paralogs of atf1 and atf2, MAB_1725c and MAB_3448. While deletion of MAB_1725c and MAB_3448 had no effect on GPL acetylation, the triple atf1-atf2-MAB_1725c mutant did not synthetize fully acetylated GPL, and the quadruple mutant was totally devoid of acetylated GPL. Moreover, both triple and quadruple mutants accumulated hyper-methylated GPL. Finally, we show deletion of atf genes resulted in subtle changes in colony morphology but had no effect on M. abscessus internalization by macrophages. Overall, these findings reveal the existence of functionally redundant O-acetyltransferases and suggest that O-acetylation influences the glycan moiety of GPL by deflecting biosynthetic flux in M. abscessus.

Glycopeptidolipid glycosylation controls surface properties and pathogenicity in Mycobacterium abscessus.

Mycobacterium abscessus is an emerging and difficult-to-manage mycobacterial species that exhibits smooth (S) or rough (R) morphotypes. Disruption of glycopeptidolipid (GPL) production results in transition from S to R and severe lung disease. A structure-activity relationship study was undertaken to decipher the role of GPL glycosylation in morphotype transition and pathogenesis. Deletion of gtf3 uncovered the prominent role of the extra rhamnose in enhancing mannose receptor-mediated internalization of M. abscessus by macrophages. In contrast, the absence of the 6-deoxy-talose and the first rhamnose in mutants lacking gtf1 and gtf2, respectively, affected M abscessus phagocytosis but also resulted in the S-to-R transition. Strikingly, gtf1 and gtf2 mutants displayed a strong propensity to form cords and abscesses in zebrafish, leading to robust and lethal infection. Together, these results underscore the importance and differential contribution of GPL monosaccharides in promoting virulence and infection outcomes.

O-Methylation of the Glycopeptidolipid Acyl Chain Defines Surface Hydrophobicity of Mycobacterium abscessus and Macrophage Invasion.

Mycobacterium abscessus, an emerging pathogen responsible for severe lung infections in cystic fibrosis patients, displays either smooth (S) or rough (R) morphotypes. The S-to-R transition is associated with reduced levels of glycopeptidolipid (GPL) production and is correlated with increased pathogenicity in animal and human hosts. While the structure of GPL is well established, its biosynthetic pathway is incomplete. In addition, the biological functions of the distinct structural parts of this complex lipid remain elusive. Herein, the fmt gene encoding a putative O-methyltransferase was deleted in the M. abscessus S variant. Subsequent biochemical and structural analyses demonstrated that methoxylation of the fatty acyl chain of GPL was abrogated in the Δfmt mutant, and this defect was rescued upon complementation with a functional fmt gene. In contrast, the introduction of fmt derivatives mutated at residues essential for methyltransferase activity failed to complement GPL defects, indicating that fmt encodes an O-methyltransferase. Unexpectedly, phenotypic analyses showed that Δfmt was more hydrophilic than its parental progenitor, as demonstrated by hexadecane-aqueous buffer partitioning and atomic force microscopy experiments with hydrophobic probes. Importantly, the invasion rate of THP-1 macrophages by Δfmt was reduced by 50% when compared to the wild-type strain. Together, these results indicate that Fmt O-methylates the lipid moiety of GPL and plays a substantial role in conditioning the surface hydrophobicity of M. abscessus as well as in the early steps of the interaction between the bacilli and macrophages.