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. 1999 Aug;67(8):4153-60.
doi: 10.1128/IAI.67.8.4153-4160.1999.

Opacity-associated protein A contributes to the binding of Haemophilus influenzae to chang epithelial cells

N V Prasadarao  1 , E Lysenko, C A Wass, K S Kim, J N Weiser
Affiliations

Opacity-associated protein A contributes to the binding of Haemophilus influenzae to chang epithelial cells

N V Prasadarao et al. Infect Immun. 1999 Aug.

Abstract

Opacity-associated protein A (OapA), which is responsible for the transparent-colony phenotype of Haemophilus influenzae, has been implicated in the colonization of the nasopharynx in an infant rat model of carriage. In this report, we show that OapA mediates attachment to Chang epithelial cells examined by using genetically defined type b and nontypeable H. influenzae strains with or without OapA. We also showed that OapA was conserved among H. influenzae strains by comparing deduced amino acid sequences. Both recombinant OapA and polyclonal anti-OapA antiserum blocked the binding of H. influenzae to Chang epithelial cells, suggesting that the interaction of H. influenzae is specific to OapA. Moreover, the binding of recombinant OapA to epithelial cells further provided evidence that OapA can promote attachment of H. influenzae. Expression of oapA gene in a nonadherent Escherichia coli strain significantly increased the binding to Chang epithelial cells, and disruption of the oapA gene with kanamycin resistance cassette insertion resulted in a significant loss of binding. These findings demonstrate that OapA plays a role in H. influenzae binding to human conjunctival epithelial cells.

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Figures

FIG. 1
FIG. 1
Light micrographs demonstrating the binding of H. influenzae strains having or lacking OapA to Chang epithelial cells. The epithelial cell monolayers were infected with the bacterial strains Eagan (A), H229 (B), Rd (C), H209 (D), H233 (E), and H487 (F) as described in the Materials and Methods section, rinsed with RPMI, and stained with Giemsa stain. Prior to inoculation of bacteria, in some experiments epithelial cell monolayers were incubated with 10 μg of rOapA/ml (G). For antibody inhibition, the anti-OapA antibody generated in guinea pig was incubated with the bacteria on ice for 1 h before infection of the epithelial cell monolayers (H). Original magnification; ×400.
FIG. 2
FIG. 2
Sequence conservation of OapA. The deduced amino acid sequence of OapA from strain Rd is given with differences in the type b strain Eagan and two nontypeable isolates (H135 and H142) indicated below. Strain H142 contains a tandem repeat of 16 amino acids followed by a repeat of the sequence AKPV (underlined). Strain Eagan contains an insertion of 12 amino acids ending in a repeat of the sequence QAEQP (underlined).
FIG. 3
FIG. 3
Western blot showing expression of OapA in whole-cell lysates. (A) H. influenzae strains. Lanes: 1, Eagan (type b); 2, H229 (Eagan oapA mutant); 3, Rd; 4, H209 (Rd oapA mutant); 5, H217 (H209 corrected oapA mutant); 6, H135 (nontypeable); 7, H142 (nontypeable); 8, H233 (nontypeable); and 9, H487 (H233 oapA mutant). (B) E. coli strains. Lane: 1, OapA-positive DH5α; 2, OapA-negative DH5α. Molecular size markers are indicated at the left and expressed in kilodaltons.
FIG. 4
FIG. 4
Effects of rOapA and anti-OapA antibody on the binding of H. influenzae strains to Chang epithelial cells. The epithelial cell monolayers were incubated with 10 μg of either rOapA or BSA per ml for 1 h at 37°C before the addition of bacteria. In some experiments the H. influenzae strains were incubated with either anti-OapA antiserum (diluted 1:100) (OapA Ab), control antibody (Cont.Ab), or PBS before they were added to the epithelial cell monolayers. The results were expressed as relative binding, with the H233 binding taken as 100%, and the bars indicate standard deviations from the means for triplicate wells and from at least three separate experiments. The binding of H. influenzae was significantly reduced after incubation with either rOapA or anti-OapA antibody compared to the control protein or antibody (P < 0.01) by two-tailed unpaired t test.
FIG. 5
FIG. 5
Immunocytochemistry of the binding of rOapA to Chang epithelial cells. Confluent epithelial cell layers were incubated with 20 μg of biotinylated BSA (A) or rOapA (B) per ml for 1 h at 37°C. The bound proteins were identified with streptavidin peroxidase as described in the Materials and Methods section. Original magnification, ×200.
FIG. 6
FIG. 6
Binding of E. coli strains expressing OapA to Chang epithelial cells. E. coli strains were incubated with epithelial cell monolayers for 2 h, washed, and stained with Giemsa stain as described in the Materials and Methods section. (A) DH5α containing vector only (control); (B) DH5α containing pE214 expressing OapA; (C) DH5α containing pE219 expressing no OapA; and (D) DH5α containing pEL1 with deleted oapB sequence. Original magnification, ×600.
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