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Microbiology 149 (2003), 1437-1445; DOI  10.1099/mic.0.26161-0
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Microbiology 149 (2003), 1437-1445; DOI  10.1099/mic.0.26161-0
© 2003 Society for General Microbiology

Structural and functional features of Rhodococcus ruber lipoarabinomannan

Kevin J. C. Gibson1,2, Martine Gilleron3, Patricia Constant3, Germain Puzo3, Jérôme Nigou1,3 and Gurdyal S. Besra2

1 Department of Microbiology and Immunology, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK
2 School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
3 Department of Molecular Mechanisms of Mycobacterial Infections, Institut de Pharmacologie et de Biologie Structurale, CNRS, UMR 5089, 205 route de Narbonne, 31077 Toulouse cedex 4, France

Correspondence
Gurdyal S. Besra
g.besra{at}bham.ac.uk

The genus Rhodococcus is part of the phylogenetic group nocardioform actinomycetes, which also includes the genus Mycobacterium. Members of this phylogenetic group have a characteristic cell envelope structure, which is dominated by various complex lipids. Among these, lipoglycans are of particular interest since mycobacterial lipoarabinomannans are important immunomodulatory molecules that are likely to be involved in the subsequent fate of mycobacterial bacilli once inside phagocytic cells. Rhodococcus ruber is a species closely related to an established opportunistic human pathogen, Rhodococcus equi. This paper reports the isolation and characterization of R. ruber lipoarabinomannan, designated as RruLAM. SDS-PAGE and gas chromatography analyses revealed that RruLAM was of an intermediate size between Mycobacterium tuberculosis lipoarabinomannan and lipomannan. Using a combination of chemical degradation and 1H, 13C-NMR experiments, the carbohydrate structure of RruLAM was unambiguously shown to be composed of a linear ({alpha}1->6)-Manp backbone substituted at some O-2 positions by a single t-{alpha}-Araf sugar unit. Integration of the anomeric proton signals provided an indication of the degree of branching as approximately 45 %. The RruLAM structure is much simpler than that established for M. tuberculosis lipoarabinomannan but is also different from that determined for the closely related species and opportunistic human pathogen, R. equi. RruLAM was unable to induce the production of TNF-{alpha} by either human or murine macrophage cell lines, suggesting that more sophisticated structures, such as phosphoinositol capping motifs, are required for such activity.

Abbreviations: 1D, one-dimensional; 2D, two-dimensional; APTS, 1-aminopyrene-3,6,8-trisulfonate; Araf, arabinofuranose; CE-LIF, capillary electrophoresis-laser-induced fluorescence; Gro, glycerol; HMQC, heteronuclear multiple quantum correlation spectroscopy; HOHAHA, homonuclear Hartmann–Hahn spectroscopy; Ins, inositol; IL, interleukin; LAM, lipoarabinomannan; LM, lipomannan; Manp, mannopyranose; ManLAM, LAM with mannosyl caps; NOESY, nuclear Overhauser and exchange spectroscopy; MPI, mannosyl-phosphatidyl-myo-inositol; PILAM, LAM with phosphoinositide caps; PIM, phosphatidyl-myo-inositol mannoside; ReqLAM, lipoglycan of R. equi; RruLAM, lipoglycan of R. ruber; t-, terminal; TFA, trifluoroacetic acid; TMS, trimethylsilate; TNF-{alpha}, tumour necrosis factor {alpha}

K. J. C. G. would like to dedicate this publication to the memory of a close friend, Jonathan D. T. Compton.




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