CHARACTERIZATION OF A MAJOR ADHESIN ALSO ACTING AS THE HEMAGGLUTININ IN A PATHOGENIC STRAIN OF MORAXELLA CATARRHALIS

Abstract

Moraxella catarrhalis has emerged as an important human pathogen over the past two decades. It has been responsible for up to 3.5 million episodes of otitis media (Klein, J.O. 1994; Murphy, T.F. 1996; Ruuskanen et al. 1994) and causes lower respiratory tract infections in adults with chronic obstructive pulmonary disease. The cost of treatment and the pain and suffering associated with these diseases prompted increased research of this organism. In this laboratory, prior research demonstrated a 100% correlation between hemagglutination (HA) and the presence of an outer membrane protein of 190 kD, OMP106. Most importantly, OMP106 was present in all pathogenic strains and absent from nonpathogenic strains that were screened. Because HA is a model of bacterial adherence in eukaryotic cells, OMP106 was further investigated as a possible adhesin. An adherence model using nasopharyngeal cells was optimized for a reference strain of M. catarrhalis, MC2926. Antiserum to OMP106-containing outer membrane preparations inhibited adherence and HA. Monoclonal antibodies (MAb) were generated and screened for their capacity to do the same. Results showed that a MAb (SRB-3) acted with the same efficiency as polyclonal sera, yet was incapable of recognizing denatured OMP106 in western blots. Furthermore, bacteria isolated from adherence assays after treatment with SRB-3 and grown overnight on agar plates were now OMP106 and HA negative. A second MAb (SRB-1) was isolated that reacted with denatured OMP106 in western blots, but this MAb neither blocked adherence nor inhibited HA. However, under native conditions, both SRB-1 and 3 reacted with OMP106 in western blots, recognizing a band migrating in the high-molecular weight region of the gel. These data indicate that SRB-3 possibly interacts with the epitope involved in adherence. This interaction may require that the epitope is present in a tertiary or perhaps quaternary conformation. To demonstrate that OMP106 is the adhesin and hemagglutinin of MC2926, an isogenic mutant was generated. The omp106 gene was reconstructed in a plasmid (pomp106K0) with the open reading frame disrupted by the insertion of a kanamycin resistance gene. By homologous recombination with pomp106K0 in MC2926, the wildtype gene was knocked out generating a mutant, MC2953. Western blot and SDS-PAGE analysis of MC2953 demonstrated that OMP106 was not present. Furthermore, the adherence assay showed binding was equal to MC2926 treated with anti-OMP106 sera or SRB-3 and the mutant was HA negative. These data clearly demonstrate that OMP106 is a novel adhesin and the hemagglutinin for M catarrhalis strain MC2926.