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Infection and Immunity, August 2000, p. 4658-4665, Vol. 68, No. 8
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of Adherence of Nontypeable Haemophilus influenzae to Human Epithelial Cells

Muriel van Schilfgaarde,1,2 Peter van Ulsen,2 Paul Eijk,1 Michiel Brand,1 Martin Stam,1 Jamal Kouame,1 Loek van Alphen,1,2,* and Jacob Dankert1

Department of Medical Microbiology, University of Amsterdam, Academic Medical Center, 1105 AZ Amsterdam,1 and Laboratory for Vaccine Research, National Institute of Public Health and the Environment, 3720 BA Bilthoven,2 The Netherlands

Received 29 December 1999/Returned for modification 17 February 2000/Accepted 20 April 2000

The adherence of 58 nontypeable Haemophilus influenzae isolates obtained from patients with otitis media or chronic obstructive pulmonary disease (COPD) and obtained from the throats of healthy individuals to Chang and NCI-H292 epithelial cells was compared. Otitis media isolates, but not COPD isolates, adhered significantly more to both cell lines than did throat isolates. Since high-molecular-weight (HMW) proteins are major adhesins of nontypeable H. influenzae, the isolates were screened for HMW protein expression by Western blotting with two polyclonal sera and PCR with hmw-specific primers. Twenty-three of the 32 adhering isolates (72%) and only 1 of the 26 nonadherent strains were HMW protein or hmw gene positive. Among the 32 isolates adhering to either cell line, 5 different adherence patterns were distinguished based on the inhibiting effect of dextran sulfate. Using H. influenzae strain 12 expressing two well-defined HMW proteins (HMW1 and HMW2) and its isogenic mutants as a reference, we observed HMW1-like adherence to both cell lines for 16 of the 32 adherent isolates. Four others showed HMW2-like adherence to NCI-H292. Of the three other patterns of adherence, one probably also involved HMW protein. Screening of the isolates with six HMW-specific monoclonal antibodies in a whole-cell enzyme-linked immunosorbent assay showed that the HMW proteins of COPD isolates and carrier isolates were more distinct from the HMW proteins from H. influenzae strain 12 than those from otitis media isolates. Characterization of the HMW protein of a COPD isolate by adherence and DNA sequence analysis showed that despite large sequence diversity in the hmwA gene, probably resulting in the antigenic differences, the HMW protein mediated the HMW2-like adherence of this strain.

* Corresponding author. Mailing address: Laboratory for Vaccine Research, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA Bilthoven, The Netherlands. Phone: 31-30-2742701. Fax: 31-30-2744429. E-mail: loek.van.alphen{at}rivm.nl.

Infection and Immunity, August 2000, p. 4658-4665, Vol. 68, No. 8
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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