The UspA2 Protein of Moraxella catarrhalis Is Directly Involved in the Expression of Serum Resistance

Abstract

Many strains of Moraxella catarrhalis are resistant to the bactericidal activity of normal human serum. Previous studies have shown that mutations involving the insertion of an antibiotic resistance cartridge into the M. catarrhalis uspA2 gene resulted in the conversion of a serum-resistant strain to a serum-sensitive phenotype. In the present study, the deletion of the entire uspA2 gene from the serum-resistant M. catarrhalis strain O35E resulted in a serum-sensitive phenotype and did not affect either the rate of growth or the lipooligosaccharide expression profile of this mutant. Inactivation of the classical complement pathway in normal human serum with Mg 2؉ and EGTA resulted in the survival of this uspA2 mutant. In contrast, blocking of the alternative complement pathway did not protect this uspA2 mutant from complement-mediated killing. To determine whether the UspA2 protein is directly involved in serum resistance, transformation and allelic exchange were used to replace the uspA2 gene in the serum-resistant strain O35E with the uspA2 gene from the serum-sensitive M. catarrhalis strain MC317. The resultant O35E transformant exhibited a serum-sensitive phenotype. Similarly, when the uspA2 gene from the serum-resistant strain O35E was used to replace the uspA2 gene in the serum-sensitive strain MC317, the MC317 transformant acquired serum resistance. The use of hybrid O35E-MC317 uspA2 genes showed that the N-terminal half of the O35E protein contained a 102-aminoacid region that was involved in the expression of serum resistance. In addition, when the uspA2 genes from strains O35E and MC317 were cloned and expressed in Haemophilus influenzae DB117, only the O35E UspA2 protein caused a significant increase in the serum resistance of the H. influenzae recombinant strain. These results prove that the UspA2 protein is directly involved in the expression of serum resistance by certain M. catarrhalis strains.Moraxella catarrhalis has gone from being regarded as a relatively harmless commensal organism found in the human nasopharynx to a pathogen which can cause a significant amount of disease (27,35,47,53). In the upper respiratory tract, M. catarrhalis is an important cause of otitis media in infants and young children (27). This unencapsulated, gramnegative bacterium can also cause infectious exacerbations of chronic obstructive pulmonary disease (46,47) and is an infrequent cause of other diseases, including pneumonia and sinusitis (reviewed in reference 35).Virtually nothing is known about the virulence mechanisms used by M. catarrhalis to produce disease. The lack of a relevant animal model for otitis media caused by this organism (27) has precluded direct investigation of this process at the experimental level. Numerous M. catarrhalis gene products that could be involved in the colonization of the human nasopharynx by this organism or in its ability to spread into other anatomic niches in the human body have been identified (reviewed in references 27 and 53), and recent studies have highlighted additional ...

Molecular Aspects ofMoraxella catarrhalisPathogenesis

“…However, it is conceivable that uspA2 expression in the serosensitive isolates is below the threshold required to become functionally relevant or may even be completely abolished. In addition, Attia et al have demonstrated that serum resistance is linked to UspA2, but it is not an inherited property of all UspA2 variants (11). The gene coding for the multifunctional Hag protein was found to be absent in almost all isolates belonging to 16S rRNA types 2/3 (17 out of 18), whereas hag was detected in almost all (175 out of 176) 16S rRNA type 1 isolates (150).…”

“…The multifunctional aspects of M. catarrhalis UspA proteins are illustrated by their roles in adhesion, invasion, and protection against the human complement system. The role of UspA proteins in serum resistance has been described by several authors (2,11,12,16,147). UspA2 is capable of binding several complement proteins: the C4-binding protein (C4bp) (106), C3 (107), and vitronectin (12,92).…”

Differential regulation of Moraxella catarrhalis-induced interleukin-8 response by protein kinase C isoforms

“…The UspA2 protein is a putative autotransporter macromolecule that forms relatively short, filamentous projections on the surface of M. catarrhalis cells and is a target for biologically active antibodies [6,37]. In contrast to UspA1, it is not functioning as adhesin but is directly involved in the expression of serum resistance found in UspA2-positive Moraxella strains [5,38,39]. In addition, the results of the present study suggest that the UspA2-mediated activation of PKC controlling the NF-kB-dependent IL-8 release may represent a new virulence mechanism of Moraxella UspA2 in respiratory epithelial cells.…”

Identification and Characterization of a Novel Outer Membrane Protein (OMP J) of Moraxella catarrhalis That Exists in Two Major Forms

“…Serum bactericidal survival testing of M. catarrhalis isolates 3.9, 3.18, and their isogenic ⌬ompJ2 mutants was based on a protocol described by Attia et al (1). Briefly, bacterial cultures were grown to mid-log phase (approximately 5 ϫ 10 8 CFU/ml) in MH broth and diluted 1/1,000 in Veronal-buffered saline containing 0.1% (wt/vol) gelatin.…”

“…Of particular importance is the UspA2 protein, a vitronectin binding protein whose N-terminal half may confer complement resistance on certain isolates (1,33,41). Other OMPs associated with virulence include the iron acquisition protein CopB (20), a hemagglutinin (28), and the lipooligosaccharide (44).…”