The UspA1 and Hag proteins have previously been shown to be involved in the ability of the Moraxella catarrhalis wild-type strain O35E to bind to human Chang and A549 cells, respectively. In an effort to identify novel adhesins, we generated a plasmid library of M. catarrhalis DNA fragments, which was introduced into a nonadherent Escherichia coli strain. Recombinant E. coli bacteria were subsequently enriched for clones that gained the ability to bind to Chang and A549 cells, yielding the plasmid pELFOS190. The unencapsulated, gram-negative bacterium Moraxella catarrhalis is one of the leading causes of otitis media in young children and of lower respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD). In developed countries, more than 80% of children under the age of 3 years will be diagnosed at least once with otitis media, and M. catarrhalis is responsible for 15 to 25% of all of these cases (6, 7, 10, 12, 26-29, 41, 51, 60). Lower respiratory tract infections (exacerbations) have been shown to contribute to the progression of COPD, and of the approximately 20 million cases of exacerbations documented each year in the United States, up to 35% result from M. catarrhalis infections (3,41,43,54,55).A vaccine that provides protection against M. catarrhalis is highly desirable due to this substantial morbidity as well as the growing concern over antibiotic resistance observed in clinical isolates (30). Toward this end, current research is focused on the identification of potential antigens with emphasis on the outer membrane proteins (OMPs) (26,34,35). While M. catarrhalis OMPs with a wide variety of functions have been identified, adhesins are particularly attractive vaccine candidates because they are surface-exposed antigens. Furthermore, adhesins generally play a crucial role in colonization and pathogenesis. For many bacteria, adherence to epithelial surfaces contributes to the evasion of host clearance mechanisms (4,25,59). Previous studies with M. catarrhalis have identified UspA1 (32, 36), UspA2 (2, 8, 36), Hag (14,15,17,19,24,45,47; M. M. Holm and E. R. Lafontaine, unpublished data), lipooligosaccharide (24), OMPCD (49), and UspA2H (32) as potential adhesins. Of these, only UspA1 and UspA2H have been directly shown to mediate adherence to human cells (32,36). However, while UspA1 is expressed by virtually all strains tested to date, only ϳ20% of clinical isolates contain an uspA2H gene (32, 37).The present study describes the identification of the novel M. catarrhalis OMP McaP. While this protein was identified based on its ability to function as an adhesin, we report that it also displays the enzymatic activity of an esterase and a phospholipase B (PLB). MATERIALS AND METHODSStrains, plasmids, tissue culture (TC) cell lines, and growth conditions. The bacterial strains and plasmids described in this study are listed in Table 1. M. catarrhalis was routinely cultured at 37°C on Todd-Hewitt medium (Difco). When cultured on agar plates, M. catarrhalis was incubated in an at...

Previous studies have demonstrated that the Moraxella catarrhalis surface antigen UspA1 is an adhesin for Chang human conjunctival cells. The present report demonstrates that lack of UspA1 expression does not affect the adherence of strain O35E to A549 human lung cells or primary cultures of human middle ear epithelial (HMEE) cells. These results imply that another molecule mediates the adherence of M. catarrhalis to these two cell lines. To identify this adhesin, strain O35E was mutagenized with a transposon and 1,000 mutants were screened in a microcolony formation assay using A549 cells. Nine independent isolates exhibited an 8-to 19-fold reduction in adherence and contained a transposon in the same locus. Nucleotide sequence data and PCR analysis indicated that the transposons were inserted in different locations in the gene encoding the surface protein Hag. Quantitative assays using one representative transposon mutant, O35E.TN2, showed considerably decreased binding to A549 as well as HMEE cells. However, this mutant adhered at wild-type levels to Chang conjunctival cells. These findings suggest that the M. catarrhalis Hag protein is an adhesin for cell lines derived from human lung and middle ear tissues.Moraxella catarrhalis is a significant pathogen of the human respiratory tract. This gram-negative bacterium is now recognized as a leading cause of otitis media (middle ear infection), together with Streptococcus pneumoniae and nontypeable isolates of Haemophilus influenzae (11,15,28,39). More than 80% of infants have at least one episode of ear infection by the age of 3 years, and M. catarrhalis causes 15 to 20% of all cases (9,14,28,29,31,39,50,59). In the United States, ϳ24 million office visits are made annually for the treatment of otitis media (29,30,39,59) and of these, roughly a sixth are caused by M. catarrhalis. This organism also causes 10 to 35% of all cases of lower respiratory tract infections in adults that are suffering from chronic obstructive pulmonary disease (39,52,53). This disease is the fourth leading cause of death in the United States, surpassed only by heart attacks, cancer, and stroke (3, 43). Lower respiratory tract infections contribute substantially to the progression of chronic obstructive pulmonary disease; thus, infectious exacerbations due to M. catarrhalis constitute an important health problem.M. catarrhalis has also been associated with diseases such as wound infections, bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (8,10,11,28,39,44,55,60,63,64). Patients with underlying conditions are particularly susceptible. Long considered to be a nonpathogenic inhabitant of the human nasopharynx, M. catarrhalis has clearly emerged as an important cause of infectious diseases, and under unpredictable circumstances (e.g., immunosuppressive conditions or chronic disease), the organism can be virulent and cause serious organ complications.M. catarrhalis infections are a matter of concern due to the lack of a vaccine and the ...

The outer membrane protein CD (OMPCD) of Moraxella catarrhalis is an outer membrane protein with several attributes of a potential vaccine antigen. We isolated four transposon mutants of strain O35E on the basis of their reduced binding to A549 human lung cells in microcolony formation assays, and we determined that they contain a transposon in ompCD. We also found that these transposon insertions had pleiotropic effects: mutants grew slower, became serum sensitive, bound ϳ10-fold less to A549 cells, and appeared transparent when grown on solid medium. We confirmed that these various phenotypes could be attributed solely to disruption of ompCD by constructing the isogenic strain O35E.CD1. O35E-ompCD was cloned, and recombinant Escherichia coli bacteria expressing the gene product exhibited a 10-fold increase in adherence to A549 cells. This is the first report of M. catarrhalis ompCD mutants, and our findings demonstrate that this gene product is an adhesin for human lung cells.The gram-negative bacterium Moraxella catarrhalis is a pathogen of the human respiratory tract that causes otitis media in young children (17,20,33,44) and lower respiratory tract infections in adults with chronic obstructive pulmonary disease (44,62,63). Patients with underlying conditions appear to be particularly susceptible, as illustrated by the increasing number of cases of M. catarrhalis-caused wound infections, bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (8,14,17,33,44,51,65,68,70,71).Little is known about pathogenesis by M. catarrhalis. Most research has thus far focused on the identification and characterization of a few outer membrane proteins for vaccine development purposes. These include the adhesins UspA1 (30,36,37,40,41), UspA2H (36,41), Hag (21,22,31,54), McaP (69), and MID (23,24,26,43,52), the serum resistance factor UspA2 (3,4,15,18,36,40,41), the iron acquisition proteins CopB (2,5,11,28,29,64), LbpA/LbpB (11,13,19,74), TbpA/ TbpB (13,16,50,74), and OmpB1 (12,13,38,39), and the highly conserved proteins outer membrane protein E (OMPE) (9, 10, 45) and OMPCD (32,46,47,61). Although no specific biological function has been attributed to OMPCD, this molecule is predicted to be structurally similar to bacterial porins (47) and binds middle ear mucin (59). Thus, OMPCD may be involved in nutrient acquisition and/or adherence to mucosal surfaces.The present study describes the isolation and characterization of ompCD mutants of the M. catarrhalis wild-type strain O35E, and the data demonstrate that OMPCD is an adhesin for A549 human lung epithelial cells. MATERIALS AND METHODSStrains, plasmids, tissue culture cell lines, and growth conditions. Strains and plasmids are described in Table 1. M. catarrhalis strains were grown at 37°C in Todd Hewitt (TH) broth (Difco) or on TH agar plates in an atmosphere of 92.5% air-7.5% CO 2 . M. catarrhalis transposon mutants were selected with 20 g of kanamycin (KAN)/ml. Escherichia coli strains were grown in Luria-Bertani (LB) bro...

Moraxella catarrhalis is a pathogen of the human airways. We found that expression of the M. catarrhalis gene mcmA by Escherichia coli increases adherence to epithelial cells 100-fold. Furthermore, we discovered that disrupting mcmA decreases M. catarrhalis adherence to laryngeal and lung cells, which are relevant to pathogenesis by the bacterium.

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