Young adult chinchillas were atraumatically inoculated with Moraxella catarrhalis via the nasal route. Detailed histopathologic examination of nasopharyngeal tissues isolated from these M. catarrhalis-infected animals revealed the presence of significant inflammation within the epithelium. Absence of similar histopathologic findings in sham-inoculated animals confirmed that M. catarrhalis was exposed to significant host-derived factors in this environment. Twenty-four hours after inoculation, viable M. catarrhalis organisms were recovered from the nasal cavity and nasopharynx of the animals in numbers sufficient for DNA microarray analysis. More than 100 M. catarrhalis genes were upregulated in vivo, including open reading frames ( M oraxella catarrhalis is a Gram-negative mucosal pathogen that has attracted increased interest within the scientific and medical communities for its role in several clinically significant human infections. The bacterium is a cause of upper respiratory tract infections including sinusitis and otitis media in healthy children (10, 17, 62). More recently, M. catarrhalis has been shown to be involved in conjunctivitis in children (9) and in acute exacerbations of chronic sinusitis in adults (11). Additionally, in adults, it is an important etiologic agent of exacerbations of chronic obstructive pulmonary disease (COPD) (54,55,62). It has been estimated that M. catarrhalis is responsible for up to 10% of exacerbations of COPD in the United States, a finding which translates into as many as 4 million infections per year (43).For M. catarrhalis to cause clinical disease, it typically must spread from its initial site of colonization in the nasopharynx into either the middle ear or the lower respiratory tract. It is believed that biofilm formation is an important event involved in colonization of the nasopharynx, and a recent study demonstrated that M. catarrhalis was present in a biofilm in the middle ear of children with chronic otitis media (25). It is likely that M. catarrhalis exists in a biofilm together with other normal flora in the nasopharynx. Until relatively recently, no studies had been performed in an in vivo environment to identify and better characterize the bacterial factors involved with colonization of the nasopharynx by M. catarrhalis. However, utilizing a chinchilla model, Luke et al. (36) demonstrated that type IV pili are important for colonization by M. catarrhalis in this animal model.Previous studies have examined the human antibody response to known surface proteins of M. catarrhalis as a surrogate for identification of bacterial genes expressed in vivo (for a representative example, see reference 42), and one study was able to detect mRNA from a small number of selected M. catarrhalis genes in nasopharyngeal secretions from young children with acute respiratory tract illness (39). The demonstration that the chinchilla nasopharynx can be colonized by M. catarrhalis (5, 36), together with the development of M. catarrhalis DNA microarrays (19,65), presented the op...

Moraxella catarrhalis is subjected to oxidative stress from both internal and environmental sources. A previous study (C. D. Pericone, K. Overweg, P. W. Hermans, and J. N. Weiser, Infect. Immun. 68:3990-3997, 2000) indicated that a wild-type strain of M. catarrhalis was very resistant to killing by exogenous hydrogen peroxide (H 2 O 2 ). The gene encoding OxyR, a LysR family transcriptional regulator, was identified and inactivated in M. catarrhalis strain O35E, resulting in an increase in sensitivity to killing by H 2 O 2 in disk diffusion assays and a concomitant aerobic serial dilution effect. Genes encoding a predicted catalase (KatA) and an alkyl hydroperoxidase (AhpCF) showed dose-dependent upregulation in wild-type cells exposed to H 2 O 2 . DNA microarray and real-time reverse transcription-PCR (RT-PCR) analyses identified M. catarrhalis genes whose expression was affected by oxidative stress in an OxyR-dependent manner. Testing of M. catarrhalis O35E katA and ahpC mutants for their abilities to scavenge exogenous H 2 O 2 showed that the KatA catalase was responsible for most of this activity in the wild-type parent strain. The introduction of the same mutations into M. catarrhalis strain ETSU-4 showed that the growth of a ETSU-4 katA mutant was markedly inhibited by the addition of 50 mM H 2 O 2 but that this mutant could still form a biofilm equivalent to that produced by its wild-type parent strain.

Moraxella catarrhalis O35E was shown to synthesize a 105-kDa protein that has similarity to both acid phosphatases and autotransporters. The N-terminal portion of the M. catarrhalis acid phosphatase A (MapA) was most similar (the BLAST probability score was 10 Initially thought to be a harmless commensal organism, Moraxella catarrhalis has gradually gained repute as an etiologic agent of at least two significant diseases in humans. This gram-negative, unencapsulated bacterium has been shown to colonize the upper airways of infants and very young children (14,15) and is one of the three most prominent causes of otitis media (45). Additionally, adults with chronic obstructive pulmonary disease are at risk for infectious exacerbations caused by M. catarrhalis (45,66). A recent study indicates that each year in the United States, as many as four million chronic obstructive pulmonary disease exacerbations may be attributed to M. catarrhalis (46).The secretion of proteins by gram-negative bacteria is a function necessary for numerous metabolic and physiologic processes. Five different secretion systems have been well characterized in bacteria (13), and a sixth has recently been described (44, 56). The type V secretion system has received increased attention in recent years (11,27,35,38). The absence of a requirement for energy coupling or accessory factors for successful protein secretion has resulted in this class of proteins being described as autotransporters. In gram-negative bacteria, autotransporters make up the largest family of outer membrane porins involved in protein translocation (12). The autotransporter secretion system was first described for the immunoglobulin A1 protease of Neisseria gonorrhoeae (54, 55), and subsequently, numerous autotransporters have been described for other gram-negative bacteria (25,27). A threedomain model for type V secretion systems has emerged, comprising (i) an amino-terminal leader peptide or signal sequence, (ii) the secreted mature protein (or passenger domain), and (iii) a C-terminal translocation domain responsible for the formation of a pore in the outer membrane to allow passage of the passenger domain to the cell surface (26).The passenger domains of previously described autotransporter systems have been shown to have widely different functions in gram-negative bacteria, including but not limited to proteolytic, adhesive, and cytotoxic activities (27). M. catarrhalis has been shown to synthesize at least three proteins (i.e., UspA1, UspA2, and Hag) that have been classified as trimeric autotransporters and one additional protein that is considered a conventional autotransporter (i.e., McaP) (for reviews, see references 11, 19, and 35). These four previously characterized M. catarrhalis autotransporters have been shown to be involved in adherence (1), serum resistance (6), binding of immunoglobulin D (18), autoaggregation (51), and lipolysis (68).Acid phosphatases catalyze the hydrolysis of phosphomonoesters at an acidic pH (9). Bacterial nonspecific acid phosphatases...

BackgroundBacteriocins are antimicrobial proteins and peptides ribosomally synthesized by some bacteria which can effect both intraspecies and interspecies killing.ResultsMoraxella catarrhalis strain E22 containing plasmid pLQ510 was shown to inhibit the growth of M. catarrhalis strain O35E. Two genes (mcbA and mcbB) in pLQ510 encoded proteins predicted to be involved in the secretion of a bacteriocin. Immediately downstream from these two genes, a very short ORF (mcbC) encoded a protein which had some homology to double-glycine bacteriocins produced by other bacteria. A second very short ORF (mcbI) immediately downstream from mcbC encoded a protein which had no significant similarity to other proteins in the databases. Cloning and expression of the mcbI gene in M. catarrhalis O35E indicated that this gene encoded the cognate immunity factor. Reverse transcriptase-PCR was used to show that the mcbA, mcbB, mcbC, and mcbI ORFs were transcriptionally linked. This four-gene cluster was subsequently shown to be present in the chromosome of several M. catarrhalis strains including O12E. Inactivation of the mcbA, mcbB, or mcbC ORFs in M. catarrhalis O12E eliminated the ability of this strain to inhibit the growth of M. catarrhalis O35E. In co-culture experiments involving a M. catarrhalis strain containing the mcbABCI locus and one which lacked this locus, the former strain became the predominant member of the culture after overnight growth in broth.ConclusionThis is the first description of a bacteriocin and its cognate immunity factor produced by M. catarrhalis. The killing activity of the McbC protein raises the possibility that it might serve to lyse other M. catarrhalis strains that lack the mcbABCI locus, thereby making their DNA available for lateral gene transfer.

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