Hag Directly Mediates the Adherence ofMoraxella catarrhalisto Human Middle Ear Cells

Abstract

Moraxella catarrhalis is a human pathogen that causes otitis media in young children and lung infections in patients with chronic obstructive pulmonary disease. In this study, the role of the surface protein Hag in the adherence of multiple M. catarrhalis strains was examined. The hag genes of four clinical isolates were disrupted with a spectinomycin resistance cassette, and the binding of isogenic mutants to primary cultures of human middle ear epithelial cells (HMEE), as well as A549 pneumocytes, was measured. These experiments revealed that the attachment of most mutants to both cell types was 10-fold less than that of their wild-type progenitors. To determine whether Hag directly mediates adherence to human cells, the hag genes from three M. catarrhalis isolates were cloned and expressed in a nonadherent Escherichia coli cloning strain. At least 17-fold more E. coli bacteria expressing Hag attached to HMEE cells than an adherence-negative control. Surprisingly, Hag expression did not increase the binding of recombinant E. coli to A549 monolayers. Our data demonstrate that the involvement of Hag in M. catarrhalis adherence to A549 and HMEE cells is conserved among isolates and that Hag directly mediates binding to HMEE cells.Moraxella catarrhalis is a gram-negative bacterium that causes respiratory tract infections in humans. In adults, this organism causes up to 35% of all infectious exacerbations in patients suffering from chronic obstructive pulmonary disease, contributing to the progression of this fourth leading cause of death in the United States (48, 49). M. catarrhalis also causes ϳ20% of all cases of otitis media (middle ear infection) in children of developed countries (6, 9, 12, 23-26, 36, 45). More than 80% of children have at least one ear infection by the age of 3 years, and these episodes can cause significant delays in development of language and learning skills (24-26). More than 90% of M. catarrhalis strains isolated from patients are ␤-lactam resistant (21, 27, 34), and there is currently no vaccine protective against this organism. Clearly, M. catarrhalis is a significant health concern and the development of a vaccine and novel therapeutic approaches is desirable.Adherence is a necessary step of pathogenesis by most infectious agents (4,20,51 Building on past research, we have generated isogenic hag mutants in four M. catarrhalis clinical isolates of various geographic and clinical origins to determine whether Hag expression is important for adherence to HMEE and A549 cells. We also report the successful cloning and expression of hag genes in E. coli in order to demonstrate that this protein directly mediates binding to middle ear cells. MATERIALS AND METHODSBacterial strains, plasmids, tissue culture cell lines, and culture conditions. A549 and primary HMEE cells were cultured as previously described (18,19,55). HMEE cells were kindly provided by Thomas DeMaria (Ohio State University). These cultures were obtained at passage 5, a stage at which they were shown to be free of fibroblast c...

Molecular Aspects ofMoraxella catarrhalisPathogenesis

“…1E) (56). Expression of MID/Hag is subject to translational phase variation via slipped-strand mispairing in a poly(G) tract located at the 5Ј end within the open reading frame (26,98). Möllenkvist et al showed that mid mRNA and MID were present in strains with 1, 2, or 3 triplets of G residues, whereas both were found to be absent in strains with 4, 7, 8, or 10 G residues.…”

“…For example, MIDexpressing isolates agglutinate human erythrocytes and adhere to A549 cells, and the extent of these actions has been found to correlate with MID expression levels (41). In addition, Hag mediates attachment to human middle ear epithelial cells (26,73), NCIH292 lung epithelial cells (25), and Chang cells (25,109). In a study by Bullard et al, isogenic hag mutants of M. catarrhalis V1171, O35E, McGHS1, and O12E were tested for their adhesion capacities (25).…”

Colonization of healthy children by Moraxella catarrhalis is characterized by genotype heterogeneity, virulence gene diversity and co-colonization with Haemophilus influenzae

“…Furthermore, there is increasing information regarding the biological mechanisms facilitating M. catarrhalis-mediated colonization and disease development, with most publications stressing the importance of bacterial adherence as an essential first step in this process. To date, several important M. catarrhalis adhesins have been described, including the ubiquitous surface proteins UspA1 and UspA2, and the haemagglutinin/ Moraxella IgD-binding protein Hag/MID (Bullard et al, 2005;Helminen et al, 1994). UspA1 and UspA2 acquire their adherence characteristics through multifunctional binding sites, which include domains that have the ability to attach to epithelial cells via cell-associated fibronectin, laminin and vitronectin (Tan et al, 2006b).…”

Hag Mediates Adherence ofMoraxella catarrhalisto Ciliated Human Airway Cells

“…Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20,21,59,60,77,98), UspA2H (59, 75), Hag (also designated MID) (22,23,37,42,66), OMPCD (4,41), McaP (61,100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7,79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells.…”