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Infection and Immunity, May 2007, p. 2484-2492, Vol. 75, No. 5
0019-9567/07/$08.00+0     doi:10.1128/IAI.02004-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Flow Cytometric Analysis of Adherence of Porphyromonas gingivalis to Oral Epithelial Cells{triangledown}

Rishi D. Pathirana,1 Neil M. O'Brien-Simpson,1 Kumar Visvanathan,2 John A. Hamilton,2 and Eric C. Reynolds1*

Cooperative Research Centre for Oral Health Science, School of Dental Science, and the Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne,1 Cooperative Research Centre for Chronic Inflammatory Diseases, Department of Medicine, Royal Melbourne Hospital, The University of Melbourne, Melbourne, Victoria, Australia2

Received 21 December 2006/ Returned for modification 24 January 2007/ Accepted 27 February 2007

By using fluorescence microscopy, fluorescently labeled Porphyromonas gingivalis W50 was shown to adhere to oral epithelial (KB) cells as discrete cells or small cell aggregates, whereas P. gingivalis ATCC 33277 bound as large cell aggregates. Flow cytometric analysis showed that for P. gingivalis W50 there was a logarithmic relationship between the bacterial cell ratio (BCR), that is the number of bacterial cells to KB cells, and the percentage of KB cells with W50 cells attached. This percentage of KB cells with W50 attached reached a plateau of ~84% cells at a BCR of 500:1. In contrast, a quadratic relationship was observed between BCR and the percentage of KB cells with P. gingivalis ATCC 33277 attached, reaching a maximum of 47% at a BCR of 100:1 but decreasing to 7% at a BCR of 1,000:1. The lower binding of ATCC 33277 at high cell concentrations was attributed to autoaggregation. P. gingivalis W50 cells treated with an inhibitor (N{alpha}-p-tosyl-L-lysine chloromethyl ketone [TLCK]) of its RgpA-Kgp proteinase-adhesin complex exhibited significantly reduced binding to KB cells than to untreated cells, suggesting a role for proteinase activity in binding to KB cells. Competitive inhibition with purified proteinase-active and TLCK-inactivated RgpA-Kgp complex significantly decreased the adherence of P. gingivalis W50 cells to KB cells. Furthermore, isogenic mutants of P. gingivalis W50 lacking the kgp gene product, but not the rgpA or rgpB gene products, exhibited significantly decreased adherence to KB cells compared to the wild type.

* Corresponding author. Mailing address: Centre for Oral Health Science, School of Dental Science, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, 720 Swanston Street, Melbourne, Victoria 3010, Australia. Phone: 61 3 9341 1547. Fax: 61 3 9341 1596. E-mail: e.reynolds{at}unimelb.edu.au

{triangledown} Published ahead of print on 5 March 2007.

Editor: A. D. O'Brien

Infection and Immunity, May 2007, p. 2484-2492, Vol. 75, No. 5
0019-9567/07/$08.00+0     doi:10.1128/IAI.02004-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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