Disruption of Cg-Ppm1, a Polyprenyl Monophosphomannose Synthase, and the Generation of Lipoglycan-less Mutants in Corynebacterium glutamicum

Kevin J.C Gibson,Lothar Eggeling,William N Maughan,Karin Krumbach,Sudagar S Gurcha,Jérôme Nigou,Germain Puzo,Hermann Sahm,Gurdyal S Besra

doi:10.1074/jbc.m307988200

Abstract

The glycosyl donor, polyprenyl monophosphomannose (PPM), has been shown to be involved in the biosynthesis of the mycobacterial lipoglycans: lipomannan and lipoarabinomannan. The mycobacterial PPM synthase (Mt-ppm1) catalyzes the transfer of mannose from GDP-mannose to polyprenyl phosphates. Based on sequence homology to Mt-ppm1, we have identified the PPM synthase from Corynebacterium glutamicum. In the present study, we demonstrate that the corynebacterial synthase is composed of two distinct domains; a catalytic domain (Cg-ppm1) and a membrane domain (Cg-ppm2). Through the inactivation of Cg-ppm1, we observed a complex phenotype that included altered cell growth rate and inability to synthesize PPM molecules and lipoglycans. When Cg-ppm2 was deleted, no observable phenotype was noted, indicating the clear organization of the two domains. The complementation of the inactivated Cg-ppm1 strain with the corresponding mycobacterial enzyme (Mt-Ppm1/D2) led to the restoration of a wild type phenotype. The present study illustrates, for the first time, the generation of a lipoglycan-less mutant based on a molecular strategy in a member of the Corynebacterianeae family. Lipoglycans are important immunomodulatory molecules involved in determining the outcome of infection, and so the generation of defined mutants and their subsequent immunological characterization is timely.

Highlights

Corynebacterial strains are widely distributed throughout nature and represent an important branch of the Actinomycetales family
In this study we have previously demonstrated, through the indirect use of amphomycin and the direct use C. glutamicum::ppm1, that the formation of higher phosphatidyl-myo-inositol mannosides (PIMs) and linear LM is dependent on the transfer of mannose from GDP-Man to undecaprenol phosphate, which acts as the sugar donor for the growing lipoglycan [31, 35]
Mycobacterial LAM is well accepted as an important immunomodulatory molecule, and plays a significant role in the pathogenesis of tuberculosis

Summary

Introduction

Corynebacterial strains are widely distributed throughout nature and represent an important branch of the Actinomycetales family. When the plasmid-encoded paralogue was introduced to C. glutamicum::ppm1, no restoration of growth rate was observed (data not shown), which might indicate, together with the apparent recent origin, that this gene is a non-functional copy.

Results

Conclusion