Biosynthesis of mycobacterial lipoarabinomannan: Role of a branching mannosyltransferase

Abstract

Mycobacterial diseases, such as tuberculosis and leprosy, remain serious human public health problems. One critical feature that contributes to the particular pathogenicity and physiology of mycobacteria is the unique, highly hydrophobic and impermeable cell wall (1). Beyond the cytoplasmic membrane, the cell wall of Mycobacterium tuberculosis consists of a core of mycolic acids, arabinogalactan and peptidoglycan, providing the template for insertion of products such as the phthiocerol-containing lipids, the trehalose mycolates, phosphatidylinositol mannosides (PIMs), and their more glycosylated derivatives, lipomannan (LM) and lipoarabinomannan (LAM) (2), all of which contribute to the particular physiology and disease induction capacity of Mycobacterium species. LAM, in its various forms, including those with mannose (Man) “caps” (ManLAM), has been implicated in many of the key aspects of the pathogenesis of tuberculosis and leprosy, such as induction of phagocytosis, phagosomal alteration and inhibition of fusion with lysosomes, and induction of innate, humoral, and acquired T cell-mediated immunity (3, 4). However, all of these studies (often conflicting) were conducted with the isolated molecule. Mutants devoid of LAM are crucial to fully resolve its role in disease pathogenesis and bacterial physiology.

Although structurally well defined (5), the underlying enzymology and genetics of LAM biosynthesis are unknown. It has been believed, although not empirically demonstrated, that both LM and LAM have their origins in the PIMs, because all contain a phosphatidylinositol (PI) moiety (5, 6). The first step in PIM synthesis involves the transfer of a Manp residue to the 2 position of the myo-inositol ring of PI to form PIM₁, catalyzed by PimA (7, 8). Biosynthesis proceeds by means of the sequential addition of Manp residues to PIM₁ or its acylated counterpart (AcPIM₁),^§ catalyzed in part by PimB and PimC, to produce PIM species having from two (PIM₂) to three (PIM₃) Manp residues (9, 10). The Manp units at position 6 of the inositol of PIM₃ are further elongated with Manp to generate higher PIMs (PIM₄, PIM₅, and PIM₆) (11, 12). However, the mannosyltransferases (ManTs) involved in these biosynthetic steps have not been identified. PIM₆ is likely a “dead-end” product, because it contains 2-linked Manp, a combination not found in the mannan core of LM/LAM (13); PIM₄ is the likely precursor for the subsequent extension of the mannan chain, giving rise to “linear LM” (12), which is further mannosylated at the C-2 positions, resulting in mature, branched LM. LAM itself retains the PI end and mannan backbone of LM; the Araf (arabinofuranose) residues originate in decaprenyl-P-arabinofuranose (C₅₀-P-Araf), and addition is partially catalyzed by EmbC (14).

The only well characterized glycosyltransferases (GTs) implicated in PIM/LM/LAM biosynthesis are the three initial ManTs, PimA, PimB, and PimC, and EmbC (8–10, 14). Apparently, the ManTs that use GDP-Man as sugar donors occur on the cytoplasmic face of the plasma membrane (15), whereas those responsible for further elongation, requiring C₅₀-P-Man as the sugar donor, are likely located in the cytoplasmic membrane or extracytoplasmic space (12, 15, 16). The lumen-associated ManTs of the eukaryotic protein glycosylation pathway, relying on polyprenyl-linked sugar as donors and members of the GT-C superfamily of enzymes for glycosylation, are also invariably integral membrane proteins, sharing similar hydropathy plots and the presence of a conserved motif in an extracytoplasmic loop (17). Recent iterative BLAST searches of sequence databases advanced a list of 11 ORFs from the genome of M. tuberculosis with multiple transmembrane domains (17) (Table 2, which is published as supporting information on the PNAS web site). This development prompted us to examine these putative GTs through bioinformatics-informed mutagenesis as being responsible for the latter steps in LM/LAM biosynthesis.

Signal volumes were integrated and normalized to t-β-Araf, which was taken as the reference signal. Chemical shifts were measured in ^H₂O at 500 MHz and 295 K. —, not detected.

Acknowledgments

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