Biofilm Formation by Moraxella catarrhalis In Vitro: Roles of the UspA1 Adhesin and the Hag Hemagglutinin

Abstract

Mutant analysis was used to identify Moraxella catarrhalis gene products necessary for biofilm development in a crystal violet-based assay involving 24-well tissue culture plates. The wild-type M. catarrhalis strains that formed the most extensive biofilms in this system proved to be refractory to transposon mutagenesis, so an M. catarrhalis strain was constructed that was both able to form biofilms in vitro and amenable to transposon mutagenesis. Chromosomal DNA from the biofilm-positive strain O46E was used to transform the biofilm-negative strain O35E; transformants able to form biofilms were identified and subjected to transposon-mediated mutagenesis. Biofilmnegative mutants of these transformants were shown to have a transposon insertion in the uspA1 gene. Nucleotide sequence analysis revealed that the biofilm-positive transformant T14 contained a hybrid O46E-O35E uspA1 gene, with the N-terminal 155 amino acids being derived from the O46E UspA1 protein. Transformant T14 was also shown to be unable to express the Hag protein, which normally extends from the surface of the M. catarrhalis cell. Introduction of a wild-type O35E hag gene into T14 eliminated its ability to form a biofilm. When the hybrid O46E-O35E uspA1 gene from T14 was used to replace the uspA1 gene of O35E, this transformant strain did not form a biofilm. However, inactivation of the hag gene did allow biofilm formation by strain O35E expressing the hybrid O46E-O35E uspA1 gene product. The Hag protein was shown to have an inhibitory or negative effect on biofilm formation by these M. catarrhalis strains in the crystal violet-based assay.Moraxella catarrhalis is an important cause of otitis media in infants and very young children (9, 21). In adults with chronic obstructive pulmonary disease, M. catarrhalis is known to be a significant cause of infectious exacerbations (28, 38). In addition, M. catarrhalis has been shown to be an infrequent cause of several other diseases including pneumonia and sinusitis (for a review, see reference 28).The ability of this organism to colonize the mucosal surface of the nasopharynx is key to its ability to cause disease in other anatomic regions, because this colonization event provides a foothold for M. catarrhalis in its human host. In fact, nasopharyngeal colonization with M. catarrhalis is common throughout infancy, and a high rate of colonization with this organism is associated with an increased risk of otitis media (10). The mechanism(s) essential for colonization of the nasopharyngeal mucosa by M. catarrhalis has not been determined conclusively, although a number of M. catarrhalis gene products that may be involved in this process have been identified in the past few years. The UspA1 and UspA2H proteins have both been shown to function as adhesins for human epithelial cells in vitro (22); more recently, the Hag (MID) protein was shown to bind to both A549 human lung cells (11,18) and primary cultures of human middle ear epithelial cells (18). The M. catarrhalis OmpCD protein has been shown to bind bot...

Hag Mediates Adherence ofMoraxella catarrhalisto Ciliated Human Airway Cells

“…Most isolates tested to date contain a hag gene product (23,37,66,76,77,105), and studies have shown that Hag is a major target of new immunoglobulin A antibodies purified from the sputum of COPD patients who have successfully cleared M. catarrhalis infections (68). Taken together, these data support a key role for the Hag protein in the pathogenesis of M. catarrhalis in the human airway.…”

“…However, the mutated Hag protein did not complement VOL. 77,2009 Hag MEDIATES ADHERENCE TO CILIA 4601…”

“…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.…”

Molecular Aspects ofMoraxella catarrhalisPathogenesis

“…Indeed, the capacity of M. catarrhalis to form biofilms has been confirmed by in vitro assays (108,110), and M. catarrhalis has been found to be present in biofilms in vivo in the middle ears of OM patients (55). Pearson et al attempted to identify the genes required for biofilm formation by use of a transposon mutagenesis approach.…”

“…Pearson et al attempted to identify the genes required for biofilm formation by use of a transposon mutagenesis approach. This led to the recognition that the presence of UspA1 positively affects biofilm formation, while the presence of Hag has a negative effect (110). Later, however, it became apparent that both UspA1 and UspA2H play a role in biofilm formation (108).…”