Catarrhalis Cells Research Articles

Moraxella catarrhalis NucM is an entry nuclease involved in extracellular DNA and RNA degradation, cell competence and biofilm scaffolding

Moraxella catarrhalis is a host-adapted bacterial pathogen that causes otitis media and exacerbations of chronic obstructive pulmonary disease. This study characterises the conserved M. catarrhalis extracellular nuclease, a member of the ββα metal finger family of nucleases, that we have named NucM. NucM shares conserved sequence motifs from the ββα nuclease family, including the DRGH catalytic core and Mg2+ co-ordination site, but otherwise shares little primary sequence identity with other family members, such as the Serratia Nuc and pneumococcal EndA nucleases. NucM is secreted from the cell and digests linear and circular nucleic acid. However, it appears that a proportion of NucM is also associated with the cell membrane and acts as an entry nuclease, facilitating transformation of M. catarrhalis cells. This is the first example of a ββα nuclease in a Gram negative bacteria that acts as an entry nuclease. In addition to its role in competence, NucM affects cell aggregation and biofilm formation by M. catarrhalis, with ΔnucM mutants having increased biofilm biomass. NucM is likely to increase the ability of cells to survive and persist in vivo, increasing the virulence of M. catarrhalis and potentially affecting the behaviour of other pathogens that co-colonise the otorhinolaryngological niche.

Moraxella catarrhalis uses a twin-arginine translocation system to secrete the β-lactamase BRO-2

BackgroundMoraxella catarrhalis is a human-specific gram-negative bacterium readily isolated from the respiratory tract of healthy individuals. The organism also causes significant health problems, including 15-20% of otitis media cases in children and ~10% of respiratory infections in adults with chronic obstructive pulmonary disease. The lack of an efficacious vaccine, the rapid emergence of antibiotic resistance in clinical isolates, and high carriage rates reported in children are cause for concern. Virtually all Moraxella catarrhalis isolates are resistant to β-lactam antibiotics, which are generally the first antibiotics prescribed to treat otitis media in children. The enzymes responsible for this resistance, BRO-1 and BRO-2, are lipoproteins and the mechanism by which they are secreted to the periplasm of M. catarrhalis cells has not been described.ResultsComparative genomic analyses identified M. catarrhalis gene products resembling the TatA, TatB, and TatC proteins of the well-characterized Twin Arginine Translocation (TAT) secretory apparatus. Mutations in the M. catarrhalis tatA, tatB and tatC genes revealed that the proteins are necessary for optimal growth and resistance to β-lactams. Site-directed mutagenesis was used to replace highly-conserved twin arginine residues in the predicted signal sequence of M. catarrhalis strain O35E BRO-2, which abolished resistance to the β-lactam antibiotic carbanecillin.ConclusionsMoraxella catarrhalis possesses a TAT secretory apparatus, which plays a key role in growth of the organism and is necessary for secretion of BRO-2 into the periplasm where the enzyme can protect the peptidoglycan cell wall from the antimicrobial activity of β-lactam antibiotics.

Functionality of cross-reactive antibodies against Moraxella catarrhalis

Event Abstract Back to Event Functionality of cross-reactive antibodies against Moraxella catarrhalis Daria Augustyniak1*, Monika Piekut1 and Grażyna Majkowska-Skrobek1 1 University of Wroclaw, Department of Pathogen Biology and Immunology, Institute of Genetics and Microbiology, Poland Introduction: Antibody-dependent immune response against human respiratory tract pathogen Moraxella catarrhalis is critical for its effective elimination [1-5]. Outer membrane proteins (OMPs) and lipooligosaccharide are the major surface-exposed immune targets in this bacterium [1-3,6].The goal of the study was to characterize the broad-spectrum effectiveness of cross-reacting antibodies produced in response to whole cells (Mc) or purified outer membrane proteins (OMPs) of this pathogen. Methods: Antisera were obtained from mice immunized with whole bacteria (anti-Mc sera) or Zwittergent-isolated OMPs (anti-OMPMc sera) [3]. The titers of cross-reactive antibodies were measured by whole-cell ELISA whereas their avidities by ELISA elution assay with sodium thiocyanate. The following functional methods were used: (1) the antibody-dependent bactericidal assay, (2) the opsonophagocytic assay with human THP-1 cell line, and (3) the blocking adhesion assay with human A549 epithelial cell line. Results: There were high titers of cross-reactive antibodies both in anti-Mc and anti-OMPMc sera. The titers of anti-OMPMc sera were higher than that of anti-Mc sera. The cross-reactive IgG produced exclusively to OMPs had stronger avidity comparing to those elicited against whole M. catarrhalis cells. Additionally, these antibodies played a pivotal role in complement-mediated killing of heterologous M. catarrhalis isolates. The positive relationship between total titer of complement-fixing subclasses IgG2a and IgG2b and bactericidal titer was found for both anti-Mc and anti-OMPMc sera. The opsonophagocytic potency as well as reduction of bacterial attachment to human epithelium was much stronger for cross-reactive anti-OMPMc than cross-reactive anti-Mc antibodies. Conclusion: The presence of cross-reactive antibodies with bactericidal, opsonophagocytic and bacterial adhesion blocking potency may be additional source to control host-Moraxella catarrhalis interaction especially when the host immune status is prone to a new infection.

Laboratory Maintenance of Moraxella catarrhalis

AbstractMoraxella catarrhalis is a Gram‐negative bacterium that has recently emerged as the third leading cause of bacterial ear infections in children. This organism is also responsible for a variety of upper respiratory tract illnesses in adults, including ∼10% of all cases of respiratory exacerbations in patients with chronic obstructive pulmonary disease (COPD). There is interest in studying M. catarrhalis for vaccine development, and this unit provides guidelines for the laboratory maintenance of the organism. The three Basic Protocols presented in this unit describe how to culture and prepare M. catarrhalis cells for use in experiments pertaining to various biological aspects of this important respiratory pathogen. Curr. Protoc. Microbiol. 11:6B.1.1‐6B.1.11. © 2008 by John Wiley & Sons, Inc.

Diffusion of macrolide antibiotics through the outer membrane of Moraxella catarrhalis

We reported previously that the high susceptibility of Moraxella catarrhalis to macrolide antibiotics and other hydrophobic antimicrobial agents was related to the hydrophobicity of the cell surface. Electrophoretic analysis of lipopolysaccharide (LPS) extracted from M. catarrhalis revealed a deep rough-type profile similar to that of an LPS Re type mutant of Salmonella typhimurium, which also exhibits high susceptibility to macrolides. Moreover, treatment of 32P-labeled cells of M. catarrhalis by phospholipase C induced the release of radioactive materials. These results suggested that hydrophobic agents such as macrolides readily access the cell surface exposed by the deep roughtype LPS and phospholipids, and permeate into the cell interior through the lipid bilayer. In fact, M. catarrhalis cells rapidly accumulated large amounts of the macrolide antibiotics, erythromycin and rokitamycin, whereas no accumulation of the macrolides was observed in cells having smooth-type or Rc type LPS under the same conditions.

Experimental Otitis Media withMoraxella (Branhamella) Catarrhalis

Two hundred fifty gerbils and 7 chinchillas were utilized in 11 experiments to determine the effect of inoculating viable and heat-killed suspensions of Moraxella (Branhamella) catarrhalis into the middle ear cavity. Development of otitis media was observed by otoscopy and histopathology. Gerbils were found to be susceptible to 2 x 10(4) viable M catarrhalis cells. Depending on the number of cells inoculated, the resulting untreated inflammation was a very mild to moderately severe, self-limiting disease with no permanent sequelae except in animals inoculated with high [10(6) to 10(7)] numbers of bacteria. Viable bacteria could not be isolated from the middle ears 24 hours after inflammation was induced. Heat-killed cells produced less severe acute inflammation with no permanent sequelae. We conclude 1) gerbils and chinchillas are susceptible to a self-limited inflammation caused by M catarrhalis, 2) no infection occurs, since viable bacteria cannot be recovered from middle ear aspirates, and 3) viable cells produce more severe inflammation than heat-killed cells.

Cell wall lipopolysaccharides from Neisseria catarrhalis

Lipopolysaccharides (LPS) prepared from N. catarrhalis cells were separated into a chloroform-soluble fraction (26%) and a chloroform-insoluble fraction (74%). Both LPS fractions contained D-glucose, D-galactose, D-glucosamine, galactosamine, lipid A, ethanolamine, fatty acids, acetyl, phosphate, and protein in approximately equal proportions. The lipid A moieties prepared from the two LPS fractions were also similar in composition to each other. The fatty acids and galactosamine of the LPS fractions were recovered quantitatively in their lipid A fractions. The major fatty acid component was β-hydroxylauric acid in contrast with β-hydroxymyristic acid, which is the major fatty acid component of the lipid A of N. perflava and other Gram-negative bacteria. The lipid A of N. catarrhalis also contained a considerable amount of D-glucose and D-galactose, which are not normal constituents of lipid A fractions. The presence of amino acids (ca. 2%) in all fractions suggested that proteins were an integral part of the LPS molecules. The absence of heptose and 3-deoxyoctulosonic acid (KDO) from the N. catarrhalis LPS shows that it lacks a lipopolysaccharide "core" structure similar to that present in the LPS of N. perflava; the polysaccharide part of the LPS molecule is also compositionally different from that of N. perflava. These differences may provide additional evidence to that already accumulated from other sources that N. catarrhalis is taxonomically a "false neisseria".

Transforming activities and base contents of deoxyribonucleate preparations from various Neisseriae.

SUMMARY: Genetic transformation was investigated among Neisseria spp. whose normal habitat is the nasopharynx of humans. Seven species, as characterized in Bergey's Manual (1957), were represented. Deoxyribonucleate (DNA) preparations from streptomycin-resistant mutants of N. meningitidis, N. perflava, N. flava, N. subflava, N. sicca, and N. flavescens conferred resistance upon streptomycin-susceptible parent strains of the corresponding species (intraspecific transformation) and of each other species (interspecific transformation). Ratios of interspecific to intraspecific transformation were 0.01 or higher for all possible combinations of DNA and recipient cells of the six species. On the other hand, N. catarrhalis cells, which exhibited high frequencies of intraspecific transformation, were not transformed at detectable frequencies by DNA from any of the six Neisseria species listed above. In turn, DNA from N. catarrhalis had little or no transforming activity for these other neisseriae. Possible evidence of structural differences between these DNA's was sought by analysing the base contents of transforming preparations. The bases adenine, thymine, guanine and cytosine were present in about equal proportions in the DNA's of the six Neisseria: meningitidis, perflava, flava, subflava, sicca and flavescens. In DNA preparations from two strains of N. catarrhalis, however, adenine and thymine predominated. The ratio (adenine + thymine/guanine + cytosine) was higher than 1.4 compared to 1.0 for the others.