Identification of a Novel Protein with a Role in Lipoarabinomannan Biosynthesis in Mycobacteria

Dianne Anderson,Helen Billman-Jacobe,John Patterson,Ross Coppel,Svetozar Kovacevic,Malcolm J. McConville,Yasu S. Morita,Ruth Haites,Benjamin N.I. McMillan

doi:10.1074/jbc.m511709200

Dianne Anderson, Helen Billman-Jacobe

Open Access

https://doi.org/10.1074/jbc.m511709200

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Abstract

All species of Mycobacteria synthesize distinctive cell walls that are rich in phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). PIM glycolipids, having 2-4 mannose residues, can either be channeled into polar PIM species (with 6 Man residues) or hypermannosylated to form LM and LAM. In this study, we have identified a Mycobacterium smegmatis gene, termed lpqW, that is required for the conversion of PIMs to LAM and is highly conserved in all mycobacteria. A transposon mutant, Myco481, containing an insertion near the 3' end of lpqW exhibited altered colony morphology on complex agar medium. This mutant was unstable and was consistently overgrown by a second mutant, represented by Myco481.1, that had normal growth and colony characteristics. Biochemical analysis and metabolic labeling studies showed that Myco481 synthesized the complete spectrum of apolar and polar PIMs but was unable to make LAM. LAM biosynthesis was restored to near wild type levels in Myco481.1. However, this mutant was unable to synthesize the major polar PIM (AcPIM6) and accumulated a smaller intermediate, AcPIM4. Targeted disruption of the lpqW gene and complementation of the initial Myco481 mutant with the wild type gene confirmed that the phenotype of this mutant was due to loss of LpqW. These studies suggest that LpqW has a role in regulating the flux of early PIM intermediates into polar PIM or LAM biosynthesis. They also suggest that AcPIM4 is the likely branch point intermediate in polar PIM and LAM biosynthesis.

Highlights

Phages and to their innate resistance to many antibiotics
AcPIM4 appears to represent a possible branch point intermediate in polar phosphatidylinositol mannosides (PIMs) and LM/LAM biosynthesis, very little is known about the enzymes or other proteins that regulate the flux of early PIM intermediates into polar PIMs or LM/LAM
We show that disruption of the lpqW gene has a dramatic affect on the ability of M. smegmatis to grow on complex medium and influences the flux of PIM intermediates into LAM

Summary

Introduction

Phages and to their innate resistance to many antibiotics. The mycobacterial cell wall has a multilaminate structure, comprising an asymmetric outer membrane and an inner layer of arabinogalactan polysaccharide and peptidoglycan [2, 3]. The PIMs, LM and LAM, may be located in the plasma membrane as well as being exposed on the surface and shed into the extracellular milieu [6, 7] Both PIMs and LAM are thought to be important virulence factors in pathogenic species [5, 8], and recent gene disruption studies have shown that phosphatidylinositol (PI) and early PIM species are essential for growth of the fast-growing saprophytic species Mycobacterium smegmatis [9, 10]. These studies indicate that PIMs and/or LAM fulfill fundamental roles in cell wall biogenesis and cell division. This study demonstrates that LAM and polar PIM biosynthesis can be independently regulated

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