Article

Molecular Basis of Phosphatidyl-myo-inositol Mannoside Biosynthesis and Regulation in Mycobacteria

Unidad de Biofisica, Centro Mixto Consejo Superior de Investigaciones Cientificas-Universidad del País Vasco/Euskal Herriko Unibertsitatea, Leioa, Bizkaia 48940, Spain.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2010; 285(44):33577-83. DOI: 10.1074/jbc.R110.168328
Source: PubMed

ABSTRACT Phosphatidyl-myo-inositol mannosides (PIMs) are unique glycolipids found in abundant quantities in the inner and outer membranes of the cell envelope of all Mycobacterium species. They are based on a phosphatidyl-myo-inositol lipid anchor carrying one to six mannose residues and up to four acyl chains. PIMs are considered not only essential structural components of the cell envelope but also the structural basis of the lipoglycans (lipomannan and lipoarabinomannan), all important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy. Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis and regulation is still incomplete. Recent advances in the identification of key proteins involved in PIM biogenesis and the determination of the three-dimensional structures of the essential phosphatidyl-myo-inositol mannosyltransferase PimA and the lipoprotein LpqW have led to important insights into the molecular basis of this pathway.

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    • "Mycobacterium smegmatis mc 2 155 counterparts of both these mannosyltransferases have been successfully produced and purified from Escherichia coli hosts. This allowed thorough structural and mechanistic characterization of PimA protein [10] [11] [12] [13] and in-depth investigation of PimB enzyme activity [8]. In contrast, acyltransferase Rv2611c has been only partially characterized. "
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    ABSTRACT: Phosphatidylinositol mannosides are essential structural components of the mycobacterial cell envelope. They are implicated in host-pathogen interactions during infection and serve as a basis for biosynthesis of other unique molecules with immunomodulatory properties- mycobacterial lipopolysaccharides lipoarabinomannan and lipomannan. Acyltransferase Rv2611 is involved in one of the initial steps in the assembly of these molecules in Mycobacterium tuberculosis - the attachment of an acyl group to position-6 of the 2-linked mannosyl residue of the phosphatidylinositol mannoside anchor. Although the function of this enzyme was annotated 10 years ago, it has never been completely biochemically characterized due to lack of the pure protein. We have successfully overexpressed and purified MSMEG_2934, the ortholog of Rv2611c from the non-pathogenic model organism M. smegmatis mc(2)155 using mycobacterial pJAM2 expression system, which allowed confirmation of its in vitro acyltransferase activity, and establishment of its substrate specificity.
    Protein Expression and Purification 08/2014; 100:33-39. DOI:10.1016/j.pep.2014.04.014 · 1.51 Impact Factor
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    • "RvD2-ORF1 from M. tuberculosis CDC1551, designated as PimC and an unknown glycosyltransferase (PimD), catalyzes the addition of a Manp residue from GDP-mannose to 6-OH of mannose at the non-reducing end of Ac1/Ac2PIM2 to generate Ac1/Ac2PIM3 [14] and Ac1/Ac2PIM4, respectively. At this key junction point in the biosynthetic pathway, polyprenylphosphate-based mannose (PPM) donors are employed by the GT-C superfamily for elongation and branching of Ac1/Ac2PIM4 leading to Ac1/Ac2PIM6, LM and LAM [15]. "
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    ABSTRACT: The biosynthesis of mycobacterial mannose-containing lipoglycans, such as lipomannan (LM) and the immunomodulator lipoarabinomanan (LAM), is carried out by the GT-C superfamily of glycosyltransferases that require polyprenylphosphate-based mannose (PPM) as a sugar donor. The essentiality of lipoglycan synthesis for growth makes the glycosyltransferase that synthesizes PPM, a potential drug target in Mycobacterium tuberculosis, the causative agent of tuberculosis. In M. tuberculosis, PPM has been shown to be synthesized by Ppm1 in enzymatic assays. However, genetic evidence for its essentiality and in vivo role in LM/LAM and PPM biosynthesis is lacking. In this study, we demonstrate that MSMEG3859, a Mycobacterium smegmatis gene encoding the homologue of the catalytic domain of M. tuberculosis Ppm1, is essential for survival. Depletion of MSMEG3859 in a conditional mutant of M. smegmatis resulted in the loss of higher order phosphatidyl-myo-inositol mannosides (PIMs) and lipomannan. We were also able to demonstrate that two other M. tuberculosis genes encoding glycosyltransferases that either had been shown to possess PPM synthase activity (Rv3779), or were involved in synthesizing similar polyprenol-linked donors (ppgS), were unable to compensate for the loss of MSMEG3859 in the conditional mutant.
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    • "PIMs from M. bovis showed inhibitory activity similar to that of PIMs from H37Rv. This is consistent with the fact that both differently acylated isoforms, PIM2 and PIM6, are present in both H37Rv and M. bovis BCG [68]. "
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    ABSTRACT: Mycobacterium tuberculosis (Mtb) is able to evade the immune defenses and may persist for years, decades and even lifelong in the infected host. Mtb cell wall components may contribute to such persistence by modulating several pivotal types of immune cells. Dendritic cells (DCs) are the most potent antigen-presenting cells and hence play a crucial role in the initial immune response to infections by connecting the innate with the adaptive immune system. We investigated the effects of two of the major mycobacterial cell wall-associated types of glycolipids, mannose-capped lipoarabinomannan (ManLAM) and phosphatidylinositol mannosides (PIMs) purified from the Mtb strains H37Rv and Mycobacterium bovis, on the maturation and cytokine profiles of immature human monocyte-derived DCs. ManLAM from Mtb H37Rv stimulated the release of pro-inflammatory cytokines TNF, IL-12, and IL-6 and expression of co-stimulatory (CD80, CD86) and antigen-presenting molecules (MHC class II). ManLAM from M. bovis also induced TNF, IL-12 and IL-6 but at significantly lower levels. Importantly, while ManLAM was found to augment LPS-induced DC maturation and pro-inflammatory cytokine production, addition of PIMs from both Mtb H37Rv and M. bovis strongly reduced this stimulatory effect. These results indicate that the mycobacterial cell wall contains macromolecules of glycolipid nature which are able to induce strong and divergent effects on human DCs; i.e while ManLAM is immune-stimulatory, PIMs act as powerful inhibitors of DC cytokine responses. Thus PIMs may be important Mtb-associated virulence factors contributing to the pathogenesis of tuberculosis disease. These findings may also aid in the understanding of some earlier conflicting reports on the immunomodulatory effects exerted by different ManLAM preparations.
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