Plasmid pWW115, a cloning vector for use with Moraxella catarrhalis.
ABSTRACT The plasmid shuttle vector pWW102B is able to replicate in only a modest number of Moraxella catarrhalis strains. Plasmid pWW115, a spontaneous deletion mutant of pWW102B, was shown to lack both the pACYC184-derived origin of replication and the associated chloramphenicol-resistance gene but was able to replicate in every M. catarrhalis strain tested in this study, including one strain that had been previously refractory to all types of genetic manipulations. To test the utility of this plasmid, a M. catarrhalis gene encoding the UspA2 serum-resistance factor was cloned into pWW115 and the resultant recombinant plasmid was shown to confer serum-resistance on a serum-sensitive M. catarrhalis uspA2 mutant.
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ABSTRACT: Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. We have identified and characterized a zinc uptake ABC transporter that is present in all strains of M. catarrhalis tested. A mutant in which the znu gene cluster is knocked out shows markedly impaired growth compared to wild type in media that contains trace zinc; growth is restored to wild type levels by supplementing media with zinc but not with other divalent cations. Thermal shift assays showed that purified recombinant substrate binding protein ZnuA binds zinc but does not bind other divalent cations. Adherence and invasion assays with human respiratory epithelial cells demonstrated that the zinc ABC transporter of M. catarrhalis is critical for invasion of respiratory epithelial cells, an observation that is especially relevant because an intracellular reservoir of M. catarrhalis is present in the human respiratory tract and this reservoir is important for persistence. The znu knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to wild type in a mouse pulmonary clearance model. We conclude that the zinc uptake ABC transporter mediates uptake of zinc in environments of very low zinc concentration and is critical for full virulence of M. catarrhalis in the respiratory tract by facilitating intracellular invasion of epithelial cells and persistence in the respiratory tract.Infection and immunity 07/2013; DOI:10.1128/IAI.00589-13 · 4.16 Impact Factor
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ABSTRACT: Moraxella catarrhalis is a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality. M. catarrhalis has a growth requirement for arginine, thus acquiring arginine is important for fitness and survival. M. catarrhalis has a putative oligopeptide permease ABC transport operon (opp) consisting of 5 genes (oppB, C, D, F, and A), encoding 2 permeases, 2 ATPases, and a substrate binding protein. Thermal-shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which the oppBCDFA gene cluster is knocked out shows impaired growth in minimal media where the only source of arginine came from a peptide 5-10 aa residues in length. Whether methylated arginine supports growth of M. catarrhalis is important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild type M. catarrhalis was observed in minimal media in which arginine was present only in methylated form, indicating that the bacterium requires L-arginine. An oppA knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract.Infection and Immunity 08/2014; 82(11). DOI:10.1128/IAI.02185-14 · 4.16 Impact Factor
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ABSTRACT: Colonization of the human nasopharynx by Moraxella catarrhalis is presumed to involve attachment of this bacterium to the mucosa. DNA microarray analysis was used to determine whether attachment of M. catarrhalis to human bronchial epithelial (HBE) cells in vitro affected gene expression in this bacterium. Attachment affected expression of at least 454 different genes, with 163 being up-regulated and 291 being down-regulated. Among the up-regulated genes was one (ORF 113) previously annotated as encoding a protein with some similarity to outer membrane protein A (OmpA). The protein encoded by ORF 113 was predicted to have a signal peptidase II cleavage site, and globomycin inhibition experiments confirmed that this protein was indeed a lipoprotein. The ORF 113 protein also contained a predicted peptidoglycan-binding domain in its C-terminal half. The use of mutant and recombinant M. catarrhalis strains confirmed that the ORF 113 protein was present in outer membrane preparations, and this protein was also shown to be at least partially exposed on the bacterial cell surface. A mutant unable to produce the ORF 113 protein showed little or no change in its growth rate in vitro, in its ability to attach to HBE cells in vitro, or in its autoagglutination characteristics, but did exhibit a reduced ability to survive in the chinchilla nasopharynx. This is the first report of a lipoprotein essential to the ability of M. catarrhalis to persist in an animal model.Infection and immunity 03/2014; 82(6). DOI:10.1128/IAI.01745-14 · 4.16 Impact Factor