Mycobacterium abscessus Glycopeptidolipids Mask Underlying Cell Wall Phosphatidyl-myo-Inositol Mannosides Blocking Induction of Human Macrophage TNF-α by Preventing Interaction with TLR2

Abstract

Mycobacterium abscessus causes disease in patients with structural abnormalities of the lung, and it is an emerging pathogen in patients with cystic fibrosis. Colonization of the airways by nontuberculous mycobacteria is a harbinger of invasive lung disease. Colonization is facilitated by biofilm formation, with M. abscessus glycopeptidolipids playing an important role. M. abscessus can transition between a noninvasive, biofilm-forming, smooth colony phenotype that expresses glycopeptidolipid, and an invasive rough colony phenotype that expresses minimal amounts of glycopeptidolipid and is unable to form biofilms. The ability of this pathogen to transition between these phenotypes may have particular relevance to lung infection in cystic fibrosis patients since the altered pulmonary physiology of these patients makes them particularly susceptible to colonization by biofilm-forming bacteria. In this study we demonstrate that rough variants of M. abscessus stimulate the human macrophage innate immune response through TLR2, while smooth variants do not. Temperature-dependent loss or physical removal of glycopeptidolipid from the cell wall of one of the smooth variants leads to TLR2 stimulation. This response is stimulated in part through phosphatidyl-myo-inositol mannosides that are present in the cell wall of both rough and smooth variants. Mannose-binding lectins bind to rough variants, but lectin binding to an isogenic smooth variant is markedly reduced. This suggests that glycopeptidolipid in the outermost portion of the M. abscessus cell wall masks underlying cell wall lipids involved in stimulating the innate immune response, thereby facilitating colonization. Conversely spontaneous “unmasking” of cell wall lipids may promote airway inflammation.

Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response

“…We have postulated that this is due to GPL expression by 390S, which is supported by genetic studies demonstrating that GPL is necessary for both biofilm formation and sliding motility in Mycobacterium smegmatis, a non-pathogenic NTM (Recht et al, 2000). We have provided evidence that expression of GPL facilitates the M. abscessus colonization phenotype, but 'masks' underlying bioactive cell-wall lipids involved in virulence (Rhoades et al, 2009). In this study we have used genetic recombineering to delete the M. abscessus 390S mmpL4b gene, a gene coding for a membrane protein which has been found to play an essential role in GPL expression by NTM (Recht et al, 2000;Ripoll et al, 2007;Medjahed & Reyrat, 2009).…”

“…We have demonstrated that the smooth colony phenotype of M. abscessus is due to expression of glycopeptidolipid (GPL) in the bacterial cell wall; GPL is minimally expressed by rough variants (Howard et al, 2006). We have also demonstrated that the rough variants 390R and 390V are able to grow in human macrophages and stimulate macrophage Toll-like receptor 2 (TLR2), while the 390S variant lacks these capabilities (Byrd & Lyons, 1999;Howard et al, 2006;Rhoades et al, 2009). Unlike the rough variants, the 390S variant is able to form biofilms and exhibits sliding motility.…”

“…The M. abscessus 390S smooth colony variant was used as wild-type in these studies (Byrd & Lyons, 1999;Howard et al, 2002Howard et al, , 2006Rhoades et al, 2009). Bacteria were maintained as titrated frozen stocks stored at 270 uC with intermittent passage for 3 days on Middlebrook 7H11 agar plates supplemented with Middlebrook OADC (BD) followed by flash freezing.…”

Pathogen-Derived Oligosaccharides Improve Innate Immune Response to Intracellular Parasite Infection

“…1,31,32 Previous studies have shown that mannose-containing glycolipids induce IL-12 production in a manner different from classical TLR ligands, 33 whereas ManLAM was shown to inhibit the production of IL-12 in human dendritic cells via an MR-dependent mechanism that interfered with TLR-based responses. 23 We examined IL12p40 production after the addition of TLR2 and MR blocking antibodies.…”

“…Intracellular pathogens mediate their interactions through specific host cell receptors to facilitate host cell adherence and entry. [31][32][33] Use of particular host-cell receptors and mode of entry are determinative in specifying the subsequent intracellular fate of the organism and in directing the innate immune response. 31,36 The present novel pathogen-derived CSCBs 19 allowed investigation of how sugars themselves alter in vitro and in vivo innate immune responses.…”