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Research Paper |
Institute of Infections and Immunity, Queens Medical Centre, University of Nottingham, Nottingham NG7 2UH, UK1
School of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK2
SmithKline Beecham Pharmaceuticals, Harlow CM19 5AW, UK3
MRC Molecular Pathogenesis Group, Department of Oral Microbiology, St Bartholomews and the Royal London School of Dentistry, 32 Newark St, London E1 2AA, UK4
Author for correspondence: Paul Williams. Tel: +44 115 9515047. Fax: +44 115 8466296. e-mail: paul.williams{at}nottingham.ac.uk
Porphyromonas gingivalis is a Gram-negative black-pigmented obligate anaerobe implicated in the aetiology of human periodontal disease. The virulence of P. gingivalis is associated with the elaboration of the cysteine proteases Arg-gingipain (Rgp) and Lys-gingipain (Kgp), which are produced at high bacterial cell densities. To determine whether quorum sensing plays a role in the regulation of Rgp and Kgp, biosensors capable of detecting either N-acylhomoserine lactone (AHLs) or the luxS-dependent autoinducer (AI-2) quorum-sensing signalling molecules in spent culture supernatants were first employed. While no AHLs could be detected, the Vibrio harveyi BB170 biosensor was activated by spent P. gingivalis W50 culture supernatants. The P. gingivalis luxS gene was cloned and demonstrated to restore AI-2 production in the Escherichia coli luxS mutant DH5
. Mutation of luxS abolished AI-2 production in P. gingivalis. Western blotting using antibodies raised against the recombinant protein revealed that LuxS levels increased throughout growth even though AI-2 activity was only maximally detected at the mid-exponential phase of growth and disappeared by the onset of stationary phase. Similar results were obtained with E. coli DH5 transformed with luxS, suggesting that AI-2 production is not limited by a lack of LuxS protein. Analysis of Rgp and Kgp protease activities revealed that the P. gingivalis luxS mutant produced around 45% less Rgp and 30% less Kgp activity than the parent strain. In addition, the luxS mutant exhibited a fourfold reduction in haemagglutinin titre. However, these reductions in virulence determinant levels were insufficient to attenuate the luxS mutant in a murine lesion model of P. gingivalis infection.
Keywords: Porphyromonas gingivalis, luxS, quorum sensing, gingipains, haemagglutinins
Abbreviations: AHLs, N-acylhomoserine lactones; AI-2, autoinducer 2
a Present address: Alizyme, Granta Park, Great Abington, Cambridge CB1 6GS, UK.
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