PimF, a Mannosyltransferase of Mycobacteria, Is Involved in the Biosynthesis of Phosphatidylinositol Mannosides and Lipoarabinomannan

Abstract

Phosphatidylinositol mannosides (PIMs) and their related molecules lipomannan (LM) and lipoarabinomannan (LAM) are important components of the mycobacterial cell wall. These molecules mediate host-pathogen interactions and exhibit immunomodulatory activities. The biosynthesis of these lipoglycans is not fully understood. In this study, we have identified a mycobacterial gene (Rv1500) that is involved in the synthesis of PIMs. We have named this gene pimF. Transposon mutagenesis of pimF of Mycobacterium marinum resulted in multiple phenotypes, including altered colony morphology, disappearance of tetracyl-PIM 7 , and accumulation of tetraacyl-PIM 5 . The syntheses of LAM and LM were also affected. In addition, the pimF mutant exhibited a defect during infection of cultured macrophage cells. Although the mutant was able to replicate and persist within macrophages, the initial cell entry step was inefficient. Transformation of the M. marinum mutant with the pimF homolog of Mycobacterium tuberculosis complemented all of the above mentioned phenotypes. These results provide evidence that PimF is a mannosyltransferase. However, sequence analysis indicates that PimF is distinct from mannosyltransferases involved in the early steps of PIM synthesis. PimF catalyzes the formation of high molecular weight PIMs, which are precursors for the synthesis of LAM and LM. As such, this work marks the first analysis of a mannosyltransferase involved in the later stages of PIM synthesis.The mycobacterial cell wall modulates interactions between the tubercle bacillus and its environment. The cell wall consists of a covalently bound structure, the mycolic acid-arabinogalactan-peptidoglycan complex, and a variety of free lipids that complement the mycolate residues to form an asymmetric bilayer (1-3). Among the cell wall-associated lipids, phosphatidylinositol mannosides (PIMs) 1 and their multiglycosylated counterparts lipomannan (LM) and lipoarabinomannan (LAM), have emerged as major factors in mediating host-pathogen interactions and are presumed to be necessary for the survival and persistence of the pathogen within the host (4). LAM has been implicated in various immunomodulatory effects, including the down-regulation of cell-mediated immunity (5). In addition, the mannose-capped LAM found in the slowly growing mycobacteria, including Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis, and Mycobacterium avium, has been shown to bind cell surface receptors of macrophages and dendritic cells and aid invasion of host cells (6 -8).PIMs are known to mediate the attachment of M. tuberculosis to nonphagocytic cells and have been implicated in the recruitment of natural killer T cells, which affects the granulomatous response (9, 10).Mycobacterial LAMs are lipoglycans composed of three structural components: the membrane anchor, which is a mannosyl-phosphatidyl-myo-inositol; the backbone, which consists of two homopolysaccharides, mannopyranose (Manp) and arabinofuranose (Araf); and the capping motif, which varies among...

PimE Is a Polyprenol-phosphate-mannose-dependent Mannosyltransferase That Transfers the Fifth Mannose of Phosphatidylinositol Mannoside in Mycobacteria

“…According to the nomenclature for mannosyltransferases involved in PIM biosynthesis, we have designated this enzyme PimE. Previously, PimF was reported as a mannosyltransferase involved in the later stage of PIM/LAM biosynthesis (27). However, a recent study suggests that this enzyme is involved in the biosynthesis of glycosylated acyltrehalose lipooligosaccharides rather than PIM (28).…”

“…PimC has been identified in M. tuberculosis as the third mannosyltransferase, but the gene is only present in some clinical strains of M. tuberculosis and absent from M. smegmatis, suggesting that there must be another gene involved in this step (26). PimF was reported as a gene involved in LM synthesis (27), but later redefined as a glycosyltransferase involved in the synthesis of lipooligosaccharide (28). The only other gene known to be involved in mycobacterial GPI biosynthesis is EmbC, which is an arabinosyltransferase involved in the synthesis of the arabinan side chain of LAM (29).…”

Function of Phosphatidylinositol in Mycobacteria

“…It remains unclear whether the loss of viability of inositol-starved cells is due to loss of specific polar PIM species or the global loss of all inositol lipids. M. smegmatis and M. marinum mutants with defects in apolar PIM biosynthesis have recently been generated (30). 3 These mutants display a relatively mild growth and colony phenotype but also accumulate high levels of PIM precursors (30).…”

“…M. smegmatis and M. marinum mutants with defects in apolar PIM biosynthesis have recently been generated (30). 3 These mutants display a relatively mild growth and colony phenotype but also accumulate high levels of PIM precursors (30). Thus, whereas polar PIMs may be important for cell wall biogenesis FIG.…”

LosA, a Key Glycosyltransferase Involved in the Biosynthesis of a Novel Family of Glycosylated Acyltrehalose Lipooligosaccharides from Mycobacterium marinum

“…It has been proposed that this is the direct precursor of lipomannan, characterized by a linear ␣(136)-linked mannan backbone linked with ␣(132) mannopyranose side chains and that lipomannan is further glycosylated with arabinan to produce lipoarabinomannan (22). Recently, Alexander and co-workers (23) reported that, in M. marinum, a glycosyltransferase, termed "PimF, " was involved in the biosynthesis of higher molecular weight PIMs, specifically PIM 7 from PIM 5 . However, the thin layer chromatographic (TLC) behavior of the putative PIM 7 was reminiscent of a highly antigenic polar phosphorus-free glycolipid isolated previously from M. marinum (24).…”