Complement resistance is a virulence factor of Branhamella (Moraxella) catarrhalis

Abstract The purpose of this study was to investigate complement resistance in Branhamella (Moraxella) catarrhalis isolated from healthy schoolchildren or sputum-producing adult patients. Two techniques were used: a serum bactericidal assay as the gold standard and an easier ‘culture and spot’ test. Children (age 4–13; n = 303) and patients ( n = 1047) showed high colonization/infection rates with B. catarrhalis (31% and 19%, respectively). Complement resistance or intermediate sensitivity occurred frequently in patient isolates (62% and 27%, respectively) and less often in children (33% and 8.5%, respectively; P ⪡ 0.0001). In young children (age 4–5 years), the proportion of complement-resistant strains was around 50%. Complement resistance in B. catarrhalis is associated with illness and may hence be considered a virulence factor.     

Purification and characterization of outer membrane protein P6, a vaccine antigen of non-typeable Haemophilus influenzae

Outer membrane protein P6 is a promising vaccine antigen with potential to prevent infections caused by non-typeable Haemophilus influenzae. A convenient and reliable method for the purification of P6 and an assessment of the purity, yield, protein structure, antigenicity and immunogenicity of the purified protein are described. The method begins with intact H. influenzae and utilizes a series of incubations and centrifugations using a single buffer to remove all cell components with the exception of the peptidoglycan to which the P6 is associated. P6 is dissociated from the complex with heat and the insoluble peptidoglycan is removed by centrifugation. The procedure yields highly purified P6. Contamination with lipooligosaccharide is less than 0.025 endotoxin U per microgr P6. The yield of P6 is approximately 2 mg of P6 per l H. influenzae culture. The purified P6 retains both the secondary and tertiary structure as measured by circular dichroism and analysis with monoclonal antibodies. The purified P6 is immunogenic in animals. A convenient method for purifying P6 which retains antigenicity and immunogenicity will be an important tool for future studies of the vaccine potential of P6.     

Exploration of Moraxella catarrhalis outer membrane proteins, CD and UspA, as new carriers for lipooligosaccharide-based conjugates

Moraxella catarrhalis outer membrane proteins, CD and ubiquitous surface protein A (UspA), were used as carriers for M. catarrhalis detoxified lipooligosaccharide (dLOS)-based conjugates. Our study was designed to investigate the feasibility of CD and UspA as protein carriers for dLOS-based conjugates and their possible synergic effects on protection from both anti-LOS and anti-CD or anti-UspA antibody responses. Female Balb/c mice were immunized subcutaneously three times with dLOS-CD or dLOS-UspA conjugate in Ribi adjuvant. Antisera elicited by the conjugates showed high titers of specific anti-LOS antibodies with complement-dependent bactericidal activity towards M. catarrhalis strain 25238. In a mouse aerosol challenge model, mice immunized with both conjugates showed a significant enhancement of the clearance of strain 25238 from lungs as compared with the control mice. Although both conjugates elicited reduced (relative to unconjugated CD or UspA) but significant levels of anti-CD or UspA antibodies, they did not show synergetic effects with anti-LOS antibodies on the bactericidal activity or the pulmonary bacterial clearance. Nevertheless, CD and UspA are safe and effective new carriers for dLOS-based or other potential carbohydrate-based conjugate vaccines to help thymus-independent carbohydrate antigens for production of anti-carbohydrate antibodies against target pathogens.     

The major heat-modifiable outer membrane protein CD is highly conserved among strains of Branhamella catarrhalis

The outer membrane of Branhamella catarrhalis contains a major, heat-modifiable outer membrane protein called CD which has epitopes on the surface of the intact bacterium. The gene encoding CD was cloned and expressed in Escherichia coli. The protein migrates in gels as a doublet, indicating that CD is encoded by single gene whose gene product has two stable conformations. The nucleotide sequence of the gene encoding CD was determined and shows homology with the OprF outer membrane protein of Pseudomonas species. The CD protein contains a proline-rich region, which appears to account for its aberrant migration in gels. Restriction fragment-length analysis of 30 isolates of B. catarrhalis with oligonucleotide probes corresponding to sequences in the CD gene produced identical patterns in Southern blot assays. The major heat-modifiable outer membrane protein CD shares homology with the OprF protein and is highly conserved among strains of B. catarrhalis.     

Moraxella (Branhamella) catarrhalis Adherence to Human Bronchial and Oropharyngeal Cells: The Role of Adherence in Lower Respiratory Tract Infections

To study the role of Moraxella (subgenus Branhamella) catarrhalis (B. catarrhalis) adherence to airway cells in lower respiratory tract infections, the in vitro attachments of B. catarrhalis to upper airway (oropharyngeal) and lower airway (bronchial) epithelial cells were compared. The adherence of 4 strains (1 nonfimbriated and 3 fimbriated) of B. catarrhalis to respiratory tract epithelial cells collected from 11 patients with chronic pulmonary disease (CPD) and 11 healthy individuals was evaluated. Both the fimbriated and nonfimbriated strains showed increased attachment to oropharyngeal cells in the CPD patients (mean ± SEM; 25.0 ± 3.2/cell; P < 0.01) when compared to the control subjects (12.1 ± 1.1/cell). On the average, the attachment to bronchial cells was 6.1 to 13.6 times greater per surface area (bacteria/μ²) than the attachment to oropharyngeal cells. The fimbriated strains tended to adhere in higher numbers to bronchial cells (19.0 ± 1.8/cell) than the nonfimbriated strain (8.7 ± 1.2/cell), although there was no difference between the CPD and control groups. In conclusion, the attachment of B. catarrhalis to oropharyngeal cells may be an enhancing factor for colonization in the upper respiratory tract in patients with CPD, and elevated adherence of the bacteria to bronchial cells may suggest pathogenic importance when mucociliary function is impaired.     

Human serum and mucosal antibody responses to outer membrane protein G1b of Moraxella catarrhalis in chronic obstructive pulmonary disease

Moraxella catarrhalis is an important human pathogen that causes otitis media, sinusitis, and lower respiratory tract infections in adults with chronic obstructive pulmonary disease. Outer membrane protein G1b is a approximately 29-kDa protein that has a high degree of homology among strains, contains surface-exposed epitopes, and is a potential vaccine candidate. The ompG1b gene was cloned, expressed in Escherichia coli, and purified. To assess the expression of outer membrane protein G1b during human infection, paired serum and sputum supernatants from patients with chronic obstructive pulmonary disease followed prospectively were studied by enzyme-linked immunosorbent assays with recombinant outer membrane protein G1b to detect antibodies made specifically during carriage of M. catarrhalis. Overall, 39% of patients developed either a serum IgG (28.6%) or a sputum supernatant IgA (19.2%) response to outer membrane protein G1b following 100 episodes of acquisition and clearance of M. catarrhalis. A sputum supernatant IgA response was more likely following exacerbations compared with asymptomatic colonizations, whereas a serum IgG response occurred at similar rates. Serum IgG antibodies following natural infection were directed toward surface-exposed epitopes of outer membrane protein G1b. Overall, these studies show that outer membrane protein G1b is expressed during infection of the human respiratory tract and that human antibodies bind to outer membrane protein G1b epitopes on the bacterial surface. These observations indicate that outer membrane protein G1b should be evaluated further as a vaccine antigen.     

Omp52 is a growth-phase-regulated outer membrane protein of Leptospira santarosai serovar Shermani

We report the expression and characterization of the omp52 gene of Leptospira santarosai serovar Shermani strain CCF that is isolated in Taiwan. omp52 was identified among pathogenic leptospires but not among non-pathogenic leptospires by using suppression subtractive hybridization in our previous study. With an open reading frame of 1371 bp that encodes 456 amino acids and a predicted molecular mass of 52.6 kDa, Omp52 was shown to be an outer membrane protein containing a C-terminal OmpA consensus domain and exposed on the cell surface. Furthermore, Omp52 increases dramatically during the stationary phase, indicating that the expression of Omp52 is environmentally regulated. By using immunoblotting analysis, we proved that Omp52 was expressed in human patients infected with leptospires. These observations suggest that Omp52 may play roles in the interaction of host cells and pathogens during infection.     

A major outer membrane protein of Serratia marcescens which was easily solubilized and had a capacity to bind to calcium

Easily solubilized major outer membrane protein was found in Serratia marcescens. The protein was originally obtained as a membrane-associated, insoluble protein in the outer membrane when the cells were slightly disrupted. However, the amount of this protein in the outer membrane gradually decreased with the time of sonication. The decrease was not due to decomposition of the protein but to solubilization into a soluble fraction after a long period of disruption. The molecular weight of this protein was 47 kDa and it bound calcium. Another 40 kDa calcium binding protein was also found in the outer membrane of S. marcescens.     

Identification and characterization of a novel outer membrane protein (OMP J) of Moraxella catarrhalis that exists in two major forms

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.     

An outer membrane protein A (ompA) homologue from the photosynthetic purple sulfur bacterium Allochromatium vinosum

A 991 bp DNA fragment, consisting of a 225 amino acid reading frame homologous to outer membrane protein coding ompA gene, was cloned from a purple sulfur bacterium Allochromatium vinosum. The homology analysis revealed up to 51% similarity to other bacterial species. The absence of branching within diazotrophs or other taxonomically related groups shows the structural importance of the protein regardless of the metabolism and evolution of the species.     

Identification and characterisation of a novel conserved outer membrane protein from Neisseria meningitidis

We have identified a homologue of the adhesin AIDA-I of Escherichia coli in Neisseria meningitidis. This gene was designated nhhA (Neisseria hia homologue), as analysis of the complete coding sequence revealed that it is more closely related to the adhesins Hia and Hsf of Haemophilus influenzae. The sequence of nhhA was determined from 10 strains, and found to be highly conserved. Studies of the localisation by Western immunoblot analysis of total cell proteins and outer membrane complex preparations and by immunogold electron microscopy revealed that NhhA is located in the outer membrane. A strain survey showed that nhhA is present in 85/85 strains of N. meningitidis representative of all the major disease-associated serogroups, based on Southern blot analysis. It is expressed in the majority of strains tested by Western immunoblot.     

A 26-kDa outer membrane protein, OmpK, common to Vibrio species is the receptor for a broad-host-range vibriophage, KVP40

Abstract KVP40 is a broad-host-range vibriophage forming plaques on strains of at least eight Vibrio and one Photobacterium species. A spontaneous KVP40-resistant mutant, R4000, derived from Vibrio parahaemolyticus 1010 lacked a 26-kDa outer membrane protein designated OmpK. KVP40 was inactivated by outer membrane and OmpK prepared from 1010, but not by outer membrane from R4000. These results strongly suggest that OmpK is the receptor for KVP40. Immunoblotting analyses using an anti-OmpK rabbit serum revealed that OmpK or its homologs of molecular masses 25–29 kDa were distributed widely among Vibrio and Photobacterium strains including those naturally resistant to KVP40.     

The outer membrane protein of enteropathogenic Escherichia coli, described as the ''localised adherence factor'', is OmpF and probably not involved in adhesion to HEp-2 cells

Strains of enteropathogenic Escherichia coli (EPEC) were examined for a factor, described as an outer membrane protein (OMP) of 32 kilodaltons (kDa) and reported to be involved in the adhesion of EPEC to HeLa cells. A comparable OMP of 35 kDa was detected in strains of EPEC, although expression of this protein was not related to the ability of strains to adhere to HEp-2 cells. The 35 kDa OMP was found to be heat-modifiable and peptidoglycan associated, and considered to be the porin protein OmpF.     

Vaccination with major outer membrane protein proteosomes elicits protection in mice against a Chlamydia respiratory challenge

Vaccines formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) have so far been shown to elicit the most robust protection against this pathogen. nMOMP is a membrane protein and therefore, detergents are used to keep it in solution. Detergents however, have toxic effects. To address this limitation, we tested a nMOMP proteosome vaccine and compared its ability to elicit protection against nMOMP solubilized in the detergent Z3-14. The two preparations were formulated with or without CpG + Montanide (C/M). As a control antigen we used ovalbumin. Mice vaccinated with nMOMP developed strong humoral and cell mediated Chlamydia-specific immune responses. Based on the IgG2a/IgG1 levels in serum and amounts of IFN-γ in splenocytes supernatants the immune responses were predominantly Th1-biased. The animals were subsequently challenged intranasally with 2 × 10³ Chlamydia inclusion forming units (IFU) and the course of the infection was followed for 10 days when the mice were euthanized. Based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, mice immunized with nMOMP-Ps and nMOMP + Z3-14 adjuvanted with C/M showed the most robust protection. In summary, nMOMP-Ps should be considered as Chlamydia vaccine candidates.     

Cloning and sequence analysis of Vibrio parahaemolyticus ompK gene encoding a 26-kDa outer membrane protein, OmpK, that serves as receptor for a broad-host-range vibriophage, KVP40

Abstract The ompK gene of Vibrio parahaemolyticus 1010 (RIMD 2210001) encoding an outer membrane protein (OMP), OmpK, which serves as the receptor for a broad-host-range vibriophage, KVP40, was cloned and sequenced. The gene consisted of 789 nucleotides encoding 263 amino acids. Since the first 20 amino acids most likely constitute the signal peptide, mature OmpK would consist of 243 amino acids with a calculated molecular mass of 27458 Da. Sequence comparisons indicate that OmpK is unique among Vibrio OMPs so far sequenced, but may be distantly related to Tsx of enteric bacteria and is homologous to an Aeromonas hydrophila OMP, protein IV.     

The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.