Outer membrane porin M35 of Moraxella catarrhalis mediates susceptibility to aminopenicillins
© Jetter et al; licensee BioMed Central Ltd. 2009
Received: 16 March 2009
Accepted: 4 September 2009
Published: 4 September 2009
The outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen Moraxella catarrhalis. It was previously shown that M35 is involved in the uptake of essential nutrients required for bacterial growth and for nasal colonization in mice. The aim of this study was (i) to characterize the potential roles of M35 in the host-pathogen interactions considering the known multifunctionality of porins and (ii) to characterize the degree of conservation in the phylogenetic older subpopulation (type 2) of M. catarrhalis.
Isogenic m35 mutants of the type 1 strains O35E, 300 and 415 were tested for their antimicrobial susceptibility against 15 different agents. Differences in the MIC (Minimum Inhibitory Concentration) between wild-type and mutant strains were found for eight antibiotics. For ampicillin and amoxicillin, we observed a statistically significant 2.5 to 2.9-fold MIC increase (p < 0.03) in the m35 mutants. Immunoblot analysis demonstrated that human saliva contains anti-M35 IgA. Wild-type strains and their respective m35 mutants were indistinguishable with respect to the phenotypes of autoagglutination, serum resistance, iron acquisition from human lactoferrin, adherence to and invasion of respiratory tract epithelial cells, and proinflammatory stimulation of human monocytes. DNA sequencing of m35 from the phylogenetic subpopulation type 2 strain 287 revealed 94.2% and 92.8% identity on the DNA and amino acid levels, respectively, in comparison with type 1 strains.
The increase in MIC for ampicillin and amoxicillin, respectively, in the M35-deficient mutants indicates that this porin affects the outer membrane permeability for aminopenicillins in a clinically relevant manner. The presence of IgA antibodies in healthy human donors indicates that M35 is expressed in vivo and recognized as a mucosal antigen by the human host. However, immunoblot analysis of human saliva suggests the possibility of antigenic variation of immunoreactive epitopes, which warrants further analysis before M35 can be considered a potential vaccine candidate.
Moraxella catarrhalis is an exclusively human, mucosal respiratory tract commensal and pathogen causing between 5%  and 20% of cases of acute otitis media in children  across all regions of the world. The recent introduction of routine infant immunization with pneumococcal conjugate vaccines has - in some studies  - led to a substantial increase in otitis media caused by M. catarrhalis . It is thus a major cause of the most common bacterial infection in children requiring medical attention. M. catarrhalis also triggers approximately 10% acute exacerbations of chronic obstructive pulmonary disease (COPD) in adults 
In our attempts to identify cold shock regulated outer membrane proteins (OMP) of M. catarrhalis  we investigated a recently described OMP called M35. We found no evidence of cold shock regulation, but the construction of an isogenic mutant lacking the expression of a currently incompletely described OMP of M. catarrhalis provided us with the opportunity to conduct a phenotypic analysis of the function of M35. Meanwhile, in an elegant series of experiments, Easton and co-workers  demonstrated that M35 is a typical Gram-negative OM porin, which also is essential for short-term nasal colonization of mice. Importantly, porins of Gram-negative bacteria not only assure bacterial homeostasis by acting as transport channels, but are also known to afford virulence mechanisms such as adhesion, invasion [7–11], and pro-inflammatory stimulation. [11–17]. In addition, porins are often involved in antimicrobial resistance [18–26]. Porins of M. catarrhalis have received little attention in the scientific literature. Gotho et al. described the permeability for beta-lactam antibiotics across the OM of M. catarrhalis suggesting that porins may be involved . Lafontaine et al investigated the porin-like OMP CD, which acts as an adhesin on lung cells . Thus, M35 is currently the only well characterized porin of M. catarrhalis [6, 28].
The aims of the present study were (i) to provide an overview of phenotypic differences between the strains O35E, 300 and 415 and their respective isogenic m35 mutants, (ii) to investigate whether M35 is a human mucosal antigen and thus a potential vaccine candidate, (iii) to evaluate the role of M35 in the susceptibility of M. catarrhalis to various classes of antimicrobial agents, and (iv) to provide the DNA sequence m35 of strain 287, which is a representative of the phylogenetically older major lineage (type 2) of M. catarrhalis .
Bacterial strains and culture conditions
Bacterial strains used in this study
Source or reference
M. catarrhalis O35E
middle ear isolate
M. catarrhalis O35E.m35
isogenic mutant strain, kanR
M. catarrhalis 300
nasopharyngeal isolate from child
M. catarrhalis 300.m35E
isogenic mutant strain, kanR
M. catarrhalis 415
nasopharyngeal isolate from child
M. catarrhalis 415.m35
M. catarrhalis 287
isogenic mutant strain, kanR
nasopharyngeal isolate from child
Escherichial coli DH5α
Host strain for plasmid constructs
Plasmids were isolated using the Wizard Plus SV Miniprep DNA purification system (Promega Corp., Madison, Wis.) DH5α was transformed as described previously . Restriction enzymes were purchased from New England Biolabs, Inc., Beverly, MA. Electrocompetent M. catarrhalis was prepared and DNA was electroporated as described . DNA sequencing was performed by using an ABI PRISM 310 genetic analyzer (PE Biosystems, Rotkreuz, Switzerland) with the Big Dye Terminator cycle sequencing ready reaction kit (PE Biosystems, Rotkreuz, Switzerland). Sequences were analyzed with the Lasergene software (DNASTAR Inc., Madison, WI). For sequencing of m35 of strain 287, DNA was amplified using the primers m35 B5 (5'-TCGATACCAGAACACTACCTAAGC-3'), m35 F2 (5' -GTCTGAGGGCAAGGTAGGCG-3'), m35 RMJ1 (5' -CGTAGCAGTTTTCATCTCACCAC 3'), m35 F3 (5'-CTTGCTCTAGCAACCGCAG-3'), m35 R3 (5'-GCAAGACCTAGGTAAGTATC-3') and m35 FMJ4 (5'-TGCGTGCATGGGTCGTGA-3').
Construction of the isogenic mutants O35E.m35, 300.m35 and 415.m35
Preparation of OMP
OMP were prepared by the EDTA buffer method as described . Bacteria were harvested from a stationary phase culture, resuspended in EDTA buffer (0.05 M Na2HPO4, 0.15 M NaCl, 0.01 M EDTA, ph 7.4), homogenized and incubated at 55°C at 300 rpm for 1 h. Cells and cell debris were eliminated by centrifugation at 10,000 × g for 15 min at 4°C. Finally, OMP were collected by ultracentrifugation at 100.00 × g for 2 h at 4°C.
2D-Gel electrophoresis and MALDI-TOF
Analysis of M35 and other OMP spots of strain O35E was performed a described previously , except for the precipitation of the OMP, which was omitted.
SDS-PAGE gel electrophoresis and immunoblot for detection of human anti-M35 IgA
Samples were resolved by SDS-PAGE using a 7.5% polyacrylamide gel. Band intensity was quantified using the AlphaEaseFC® program from Inotech, Inc. Antibody detection was performed by Western blot analysis. Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore Corp., Bedford, MA). IgA binding was detected using human saliva samples as primary antibody source and goat anti-human IgA, respectively, labeled with horseradish peroxidase (SIGMA) as secondary antibody. Super Signal West Pico Chemiluminescent Substrate (Pierce Chemical Co., Rockford, IL) was used for detection of antibody binding. Unstimulated human saliva was collected from healthy volunteers using Salivette sponges® (Sarstedt, Nümbrecht, Germany), centrifuged for 5 minutes at 2000 rpm and stored at -20°C. All volunteers were laboratory researchers and provided oral informed consent. Sampling of saliva from healthy volunteers was approved by the local ethics committee.
Antimicrobial resistance testing
The minimum inhibitory concentrations (MIC) of penicillin, ampicillin, amoxicillin, amoxicillin-clavulanate, cefuroxime, ceftriaxone, imipenem, meropenem, erythromycin, doxycycline, gentamicin, vancomycin, ciprofloxacin, levofloxacin, and moxifloxacin were determined by E-test® (AB Biodisk, Sweden) according to the manufacturer's instructions.
Autoagglutination and serum bactericidal assay
Overnight cultures were resuspended in PBS and adjusted to an OD600 of 2.0 in glass tubes. OD600 of the supernatants were determined after 15 and 60 minutes, respectively. Serum bactericidal assay were performed as previously reported .
Human cell lines and growth conditions
Chang conjunctival cells and A549 lung cells were maintained in Eagle's minimal essential medium (Invitrogen, Basel, Switzerland) supplemented with 10% of heat-inactivated fetal calf serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine at 37°C in 5% CO2. The THP-1 human monocytic cell line was maintained in RPMI 1640 (Lonza, Basel, Switzerland) supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal calf serum, 0.05 mM β-mercaptoethanol, 10 mM HEPES, 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO2.
Adherence and Invasion assay
The ability of M. catarrhalis to adhere to and invade human epithelial cells in vitro was measured as described previously . Adherence and invasion was assessed on both Chang conjunctival cells and A549 lung cells as described [36, 37]. Each strain was analyzed in triplicate in each experiment.
Proinflammatory activity of M35 on human monocytes
The pro-inflammatory potential of M. catarrhalis OMP was described previously . To investigate if M35 is an important mediator of proinflammatory cytokine release on the bacterial cell surface, THP-1 cells (1 × 106/ml) were stimulated with different concentrations (1 × 105/ml, 1 × 106/ml, or 1 × 107/ml) of heat inactivated strain O35E or the O35E.m35 mutant and incubated for 18 h at 37°C and 5% CO2. After incubation, cells were centrifuged for 2 min at 11,800 × g and supernatants were stored at -80°C. Cytokines were measured using the R&D Systems DY208 for human CXCL8/IL-8 and R&D Systems DY210 for human TNFα/TNFSF1A (R&D, Minneapolis, USA), respectively.
Comparison of several test series was evaluated by analysis of variance (ANOVA). The significance of differences between treatment and control groups was determined using the two-tailed t- test. P < 0.05 was considered as statistically significant. Each value represents the mean ± one standard deviation of at least three independent experiments performed in triplicate.
In vitro growth of m35 mutants
M35 knockout is not associated with upregulation of a 40 kDa OMP
Easton et al.  described the upregulation of a 40 kDa protein in one of their isogenic m35 mutants. In order to confirm this observation, we compared Coomassie-blue stained OMP profiles of our three strains with their respective m35 mutants, but failed to detect any discernible upregulation of other OMP as determined by measuring protein band intensities (figure 1A). Thus, removal of M35 does not appear to affect the OMP composition when bacteria are grown in BHI. Taken together, the data presented thus far indicate that M35 is not essential for growth in vitro and that its removal from the OM does not otherwise affect the OMP composition.
M35 is expressed in vivo
The human mucosal antibody response to OM components of M. catarrhalis has been described in detail [42–45], but M35 has never been paid specific attention. In order to search for human antibodies against M35, OMP of the strains O35E, 300, 415 and their isogenic mutants were resolved by SDS-PAGE, transferred to PVDF membranes, and incubated with human saliva for detection of IgA. Western Blot analysis for anti-M35 IgA with 7 different donors resulted in antibody signals to all three strains with the strains 300 and 415 displaying broader immunogenicity than O35E (figure 1B). Four of seven donor saliva contained anti-M35 IgA against strain 300 and 415, respectively. One donor displayed anti-M35 IgA for all three strains. OMP of the m35 mutants were used as negative controls (figure 1B). These data suggest that M. catarrhalis expresses M35 in vivo and that the mucosa-associated lymphoid tissue recognizes M35 as an antigen.
Aminopenicillin susceptibility is mediated by M35
Expression of M35 and putative virulence traits of M. catarrhalis
The capacity to autoagglutinate is mediated by hemagglutinin (also called Moraxella IgD-binding protein) , but some hemagglutinin knock-out mutants still autoagglutinate (unpublished data). Thus, we investigated whether the absence of M35 affected autoagglutination, but failed to identify any difference between strain O35E and O35E.m35 (data not shown). Similarly, resistance of M. catarrhalis to human complement, which is associated with disease-causing isolates [47–49] and which requires expression of several OMP [37, 50–52], was not impaired by the lack of M35 (data not shown). Growth of M. catarrhalis in vivo is dependent on the ability to acquire iron from the human host by retrieving Fe3+ from iron-containing host proteins by a number of specific binding and uptake systems [30, 53–59]. Because of its abundance on mucosal surfaces, we chose to investigate the ability of the m35 mutant to use iron bound to human lactoferrin using a standard disk feeding assay on iron depleted BHI agar plates . The experiment resulted in no differences in growth between the three m35 mutants and their respective wild-type parents (data not shown).
Adherence and invasion of the m35 mutant
Proinflammatory activity of M35 on human monocytes
Proinflammatory activity is typically induced by OMP, lipopolysaccharide or lipoteichoic acids. Porins have also been described to induce proinflammatory cascades by activating innate immune receptors mediating the expression of several chemokines and cytokines [11–17]. We investigated M35 with regard to its proinflammatory effect on human monocytes. THP-1 cells were stimulated with strain O35E or its O35E.m35 mutant overnight and cytokine release in the supernatant was measured by determining the concentrations of IL-8 and TNFα by ELISA. There was no difference in the release of IL-8 and TNFα, respectively, between wild-type and mutant (data not shown).
M35 sequence analysis of type 2 strain 287
Porins are essential components of the gram-negative outer membrane and contribute to nutrient transport, antimicrobial resistance, response to osmostress and other processes, which are essential for bacterial homeostasis. M35 is the first functionally characterized OM porin of M. catarrhalis  and as of today all isolates examined carry a highly conserved m35 gene on their chromosome. This may indicate that strains not expressing M35 are not viable in vivo, but the only evidence in support of this hypothesis is the observation that a m35 mutant was unable to colonize the nasal mucosa of mice , which are not a natural host species for M. catarrhalis.
Our observation that healthy humans have mucosal IgA directed against M35 indicates that this protein is expressed in vivo. However, the fact that some salivary samples did not recognize all three M35 proteins tested suggests that either (i) antigenic variation occurs at immunoreactive, surface exposed epitopes, or (ii) it is a weak antigen and some individuals lose or never acquire anti-M35 IgA, or (iii) some isolates lack expression of M35 in vivo. If the latter were the case, screening of large collections of clinical M. catarrhalis isolates should identify strains either lacking a m35 gene or isolates carrying silent genes. The answer to this question is of clinical relevance, because our data indicate that the absence of M35 is a previously unknown mechanism of aminopenicillin resistance in M. catarrhalis. This effect could occur in vivo by alterations in porin expression to prevent antibiotic influx, which is a well known mechanism of resistance in other pathogens [18, 22], and which often is associated with the expression of degradative enzymes to confer high level resistance [23, 24]. The specificity of M35 for aminopenicillins could be explained by an effect Bezrukov et al described for OmpF of E. coli. They found that the nature and position of specific charges on both the antibiotic molecule and the porin play a major role in these interactions [25, 26]. Strong binding to the pore correlates with high diffusion rates whereas weak binding is associated with reduced diffusion. As of today, the only known mechanism of aminopenicillin resistance in M. catarrhalis is the expression of one of three chromosomally encoded BRO betalactamases, which are detectable in greater than 90% of clinical isolates  and explain the fact that the MIC for amoxicillin-clavulanate in our experiments was at least 10-fold lower than the MIC for amoxicillin. This finding indicates that clavulanate penetrates the OM by ways other than M35. It also demonstrates that, in the presence of clavulanate, the reduced amount of aminopenicillins still penetrating the OM in m35 mutants suffices to inhibit growth. Thus, it appears that clavulanate inhibits essentially all betalactamase activity available and that naturally occurring isolates lacking M35 would not currently pose a substantial therapeutic problem in patients treated with betalactamase-resistant betalactams. However, standard dose or high dose amoxicillin still is the therapeutic standard for antimicrobial therapy of acute otitis media. Based on our data (figure 3), currently accepted pharmakinetic/pharmcodynamic (PK/PD) breakpoints for resistance against standard dose (≥ 1.0 μg/ml) or high dose amoxicillin (≥ 8 μg/ml), respectively,  thus predict that isolates lacking functional M35 may display clinically relevant aminopenicillin resistance. This is particularly relevant for the treatment of acute otitis media. Drug concentrations reached in the middle ear cavity are low in comparison with serum concentrations  and treatment failure is typically caused by insufficient drug concentrations in the middle ear fluid . The list of betalactam antibiotics tested in this study is not exhaustive and it is conceivable that other drugs may also be affected by m35 mutations. Thus, further studies are needed to explore the potential impact of m35 mutants on antimicrobial treatment failures.
Multiple phenotypic tests that we carried out with three wild-type/mutant pairs failed to uncover an additional functions attributable to M35. None of these results is particularly surprising. The strength of these "negative" data lies precisely in the fact that we did not study one, but three different isolates and their respective mutants, which, taken together, provide firm evidence that M35 is not involved these various phenotypes in vitro. The analysis of three different wild-type/mutant pairs also lead to the conclusion that knocking-out m35 does not necessarily upregulate expression of a 40 kDa OMP as stated by Easton et al (figure 1A). Because these authors used a different strain, it is conceivable that they observed a strain-specific phenomenon, which does not represent the entire species. Finally, we sequenced m35 of a strain belonging to the phylogenetically old, second subpopulation of M. catarrhalis , which differs from the younger subpopulation by a considerably larger genetic diversity . Indeed, we found a substantial number of sequence deviations, which, interestingly, were even greater at the amino acid level than at the DNA level. It is thus conceivable that type 2 strains exhibit functional and/or antigenic differences with respect to M35, which warrant further investigation.
The significant increases in MIC for ampicillin and amoxicillin of the m35 mutants indicate that the OM porin M35 is involved in the uptake of aminopenicillins. This is a previously unknown mechanism of resistance in M. catarrhalis. It remains to be elucidated whether naturally occurring, disease causing strains of M. catarrhalis devoid of functional M35 exist, and whether they may contribute to clinical treatment failure. The fact that normal human saliva contains anti-IgA indicates that M35 is expressed in vivo, but that antigenic variation may be greater than previously appreciated. Thus, further studies are needed before M35 can be considered a potential vaccine candidate against M. catarrhalis.
analysis of variance
chronic obstructive pulmonary disease
matrix-assisted laser desorption/ionisation-time of flight
outer membrane protein
polymerase chain reaction
Roswell Park Memorial Institute medium
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
transferring-binding protein B
tumor-necrosis factor α.
This work was supported by the Swiss National Science Foundation (SNF) grants 3100A0-102246 and 3100A0-116053 (to CA). Professor George Syrogiannopoulos, Larissa, Greece, provided the nasopharyngeal M. catarrhalis isolates 287, 300 and 415.
- Marchant CD: Spectrum of disease due to Branhamella catarrhalis in children with particular reference to acute otitis media. Am J Med. 1990, 88 (5A): 15S-19S. 10.1016/0002-9343(90)90255-C.PubMedView ArticleGoogle Scholar
- Palmu A, Herva E, Savolainen H, Karma P, Mäkela PH, Kilpi T: Association of clinical signs and symptoms with bacterial findings in acute otitis media. 2004, 38: 234-242.Google Scholar
- Eskola J, Kilpi T, Palmu A, Jokinen J, Haapakoski J, Herva E, Takala A, Kayhty H, Karma P, Kohberger R, et al: Efficacy of a pneumococcal conjugate vaccine against acute otitis media. N Engl J Med. 2001, 344 (6): 403-409. 10.1056/NEJM200102083440602.PubMedView ArticleGoogle Scholar
- Murphy TF, Brauer AL, Grant BJ, Sethi S: Moraxella catarrhalis in chronic obstructive pulmonary disease: burden of disease and immune response. Am J Respir Crit Care Med. 2005, 172 (2): 195-199. 10.1164/rccm.200412-1747OC.PubMed CentralPubMedView ArticleGoogle Scholar
- Heiniger N, Troller R, Meier PS, Aebi C: Cold shock response of the UspA1 outer membrane adhesin of Moraxella catarrhalis. Infect Immun. 2005, 73 (12): 8247-8255. 10.1128/IAI.73.12.8247-8255.2005.PubMed CentralPubMedView ArticleGoogle Scholar
- Easton DM, Maier E, Benz R, Foxwell AR, Cripps AW, Kyd JM: Moraxella catarrhalis M35 is a general porin that is important for growth under nutrient-limiting conditions and in the nasopharynges of mice. J Bacteriol. 2008, 190 (24): 7994-8002. 10.1128/JB.01039-08.PubMed CentralPubMedView ArticleGoogle Scholar
- Holm MM, Vanlerberg SL, Foley IM, Sledjeski DD, Lafontaine ER: The Moraxella catarrhalis Porin-Like Outer Membrane Protein CD Is an Adhesin for Human Lung Cells. Infection and Immunity. 2004, 72 (4): 1906-1913. 10.1128/IAI.72.4.1906-1913.2004.PubMed CentralPubMedView ArticleGoogle Scholar
- Rolhion N, Carvalho FA, Darfeuille-Michaud A: OmpC and the sigma(E) regulatory pathway are involved in adhesion and invasion of the Crohn's disease-associated Escherichia coli strain LF82. Mol Microbiol. 2007, 63 (6): 1684-1700. 10.1111/j.1365-2958.2007.05638.x.PubMedView ArticleGoogle Scholar
- Kim KS, Rao NN, Fraley CD, Kornberg A: Inorganic polyphosphate is essential for long-term survival and virulence factors in Shigella and Salmonella spp. Proceedings of the National Academy of Sciences. 2002, 112210499-Google Scholar
- Bernardini ML, Sanna MG, Fontaine A, Sansonetti PJ: OmpC is involved in invasion of epithelial cells by Shigella flexneri. Infect Immun. 1993, 61 (9): 3625-3635.PubMed CentralPubMedGoogle Scholar
- Achouak W, Heulin T, Pages JM: Multiple facets of bacterial porins. FEMS Microbiol Lett. 2001, 199 (1): 1-7. 10.1111/j.1574-6968.2001.tb10642.x.PubMedView ArticleGoogle Scholar
- Tancharoen S, Matsuyama T, Abeyama K, Matsushita K, Kawahara K, Sangalungkarn V, Tokuda M, Hashiguchi T, Maruyama I, Izumi Y: The role of water channel aquaporin 3 in the mechanism of TNF-alpha-mediated proinflammatory events: Implication in periodontal inflammation. J Cell Physiol. 2008, 217 (2): 338-349. 10.1002/jcp.21506.PubMedView ArticleGoogle Scholar
- Galdiero M, Vitiello M, Galdiero S: Eukaryotic cell signaling and transcriptional activation induced by bacterial porins. FEMS Microbiol Lett. 2003, 226 (1): 57-64. 10.1016/S0378-1097(03)00562-7.PubMedView ArticleGoogle Scholar
- Galdiero M, D'Isanto M, Vitiello M, Finamore E, Peluso L: Porins from Salmonella enterica serovar Typhimurium induce TNF-alpha, IL-6 and IL-8 release by CD14-independent and CD11a/CD18-dependent mechanisms. Microbiology. 2001, 147 (Pt 10): 2697-2704.PubMedView ArticleGoogle Scholar
- Galdiero M, Finamore E, Rossano F, Gambuzza M, Catania MR, Teti G, Midiri A, Mancuso G: Haemophilus influenzae porin induces Toll-like receptor 2-mediated cytokine production in human monocytes and mouse macrophages. Infect Immun. 2004, 72 (2): 1204-1209. 10.1128/IAI.72.2.1204-1209.2004.PubMed CentralPubMedView ArticleGoogle Scholar
- Massari P, Visintin A, Gunawardana J, Halmen KA, King CA, Golenbock DT, Wetzler LM: Meningococcal porin PorB binds to TLR2 and requires TLR1 for signaling. J Immunol. 2006, 176 (4): 2373-2380.PubMedView ArticleGoogle Scholar
- Vitiello M, D'Isanto M, Galdiero M, Raieta K, Tortora A, Rotondo P, Peluso L: Interleukin-8 production by THP-1 cells stimulated by Salmonella enterica serovar Typhimurium porins is mediated by AP-1, NF-kappaB and MAPK pathways. Cytokine. 2004, 27 (1): 15-24. 10.1016/j.cyto.2004.03.010.PubMedView ArticleGoogle Scholar
- Davin-Regli A, Bolla JM, James CE, Lavigne JP, Chevalier J, Garnotel E, Molitor A, Pages JM: Membrane permeability and regulation of drug "influx and efflux" in enterobacterial pathogens. Curr Drug Targets. 2008, 9 (9): 750-759. 10.2174/138945008785747824.PubMedView ArticleGoogle Scholar
- Delcour AH: Solute uptake through general porins. Front Biosci. 2003, 8: d1055-1071. 10.2741/1132.PubMedView ArticleGoogle Scholar
- Nikaido H: Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev. 2003, 67 (4): 593-656. 10.1128/MMBR.67.4.593-656.2003.PubMed CentralPubMedView ArticleGoogle Scholar
- Schulz GE: The structure of bacterial outer membrane proteins. Biochim Biophys Acta. 2002, 1565 (2): 308-317. 10.1016/S0005-2736(02)00577-1.PubMedView ArticleGoogle Scholar
- Delcour AH: Outer membrane permeability and antibiotic resistance. Biochim Biophys Acta. 2009, 1794 (5): 808-816.PubMed CentralPubMedView ArticleGoogle Scholar
- Pages JM: [Bacterial porin and antibiotic susceptibility]. Med Sci (Paris). 2004, 20 (3): 346-351.View ArticleGoogle Scholar
- Nikaido H: Outer membrane barrier as a mechanism of antimicrobial resistance. Antimicrob Agents Chemother. 1989, 33 (11): 1831-1836.PubMed CentralPubMedView ArticleGoogle Scholar
- Danelon C, Nestorovich EM, Winterhalter M, Ceccarelli M, Bezrukov SM: Interaction of zwitterionic penicillins with the OmpF channel facilitates their translocation. Biophys J. 2006, 90 (5): 1617-1627. 10.1529/biophysj.105.075192.PubMed CentralPubMedView ArticleGoogle Scholar
- Nestorovich EM, Danelon C, Winterhalter M, Bezrukov SM: Designed to penetrate: time-resolved interaction of single antibiotic molecules with bacterial pores. Proc Natl Acad Sci USA. 2002, 99 (15): 9789-9794. 10.1073/pnas.152206799.PubMed CentralPubMedView ArticleGoogle Scholar
- Gotoh N, Tanaka S, Nishino T: Permeability of the outer membrane of Moraxella catarrhalis for beta-lactam antibiotics. J Antimicrob Chemother. 1992, 29 (3): 279-285. 10.1093/jac/29.3.279.PubMedView ArticleGoogle Scholar
- Easton DMSA, Gallego SG, Foxwell AR, Cripps AW, Kyd JM: Characterization of a novel porin protein from Moraxella catarrhalis and identification of an immunodominant surface loop. 2005, 187: 6528-6535.Google Scholar
- Wirth T, Morelli G, Kusecek B, van Belkum A, Schee van der C, Meyer A, Achtman M: The rise and spread of a new pathogen: seroresistant Moraxella catarrhalis. Genome Res. 2007, 17 (11): 1647-1656. 10.1101/gr.6122607.PubMed CentralPubMedView ArticleGoogle Scholar
- Aebi C, Stone B, Beucher M, Cope LD, Maciver I, Thomas SE, McCracken GH, Sparling PF, Hansen EJ: Expression of the CopB outer membrane protein by Moraxella catarrhalis is regulated by iron and affects iron acquisition from transferrin and lactoferrin. Infection and Immunity. 1996, 64: 2024-2030.PubMed CentralPubMedGoogle Scholar
- Hanahan D: Studies on transformation of Escherichia coli with plasmids. 1983, 166: 557-580.Google Scholar
- Helminen ME, Maciver I, Latimer JL, Lumbley SL, Cope LD, McCracken GH: A mutation affecting expression of a major outer membrane protein of Moraxella catarrhalis alters serum resistance and survival of this organism in vivo. 1993, 168: 1194-1201.Google Scholar
- Murphy TF, Loeb MR: Isolation of the outer membrane of Branhamella catarrhalis. 1989, 6: 159-174.Google Scholar
- Schaller A, Troller R, Molina D, Gallati S, Aebi C, Stutzmann Meier P: Rapid typing of Moraxella catarrhalis subpopulations based on outer membrane proteins using mass spectrometry. Proteomics. 2006, 6 (1): 172-180. 10.1002/pmic.200500086.PubMedView ArticleGoogle Scholar
- Meier PS, Troller R, Heiniger N, Grivea IN, Syrogiannopoulos GA, Aebi C: Moraxella catarrhalis strains with reduced expression of the UspA outer membrane proteins belong to a distinct subpopulation. Vaccine. 2005, 23 (16): 2000-2008. 10.1016/j.vaccine.2004.09.036.PubMedView ArticleGoogle Scholar
- Spaniol V, Heiniger N, Troller R, Aebi C: Outer membrane protein UspA1 and lipooligosaccharide are involved in invasion of human epithelial cells by Moraxella catarrhalis. Microbes Infect. 2008, 10 (1): 3-11. 10.1016/j.micinf.2007.09.014.PubMedView ArticleGoogle Scholar
- Aebi C, Lafontaine ER, Cope LD, Latimer JL, Lumbley SL, McCracken GH, Hansen EJ: Phenotypic effect of isogenic uspA1 and uspA2 mutations on Moraxella catarrhalis O35E. Infection and Immunity. 1998, 66: 3113-3119.PubMed CentralPubMedGoogle Scholar
- Fink JML, Stewart GA, Graham PT, Steer JH, Joyce DA, McWilliam AS: Moraxella catarrhalis stimulates the release of proinflammatory cytokines and prostaglandin E from human respiratory epithelial cells and monocyte-derived macrophages. 2006, 46 (2): 198-208.Google Scholar
- Kaeriyama M, Machida K, Kitakaze A, Wang H, Lao Q, Fukamachi T, Saito H, Kobayashi H: OmpC and OmpF are required for growth under hyperosmotic stress above pH 8 in Escherichia coli. Lett Appl Microbiol. 2006, 42 (3): 195-201. 10.1111/j.1472-765X.2006.01845.x.PubMedView ArticleGoogle Scholar
- Massari P, Ram S, Macleod H, Wetzler LM: The role of porins in neisserial pathogenesis and immunity. Trends Microbiol. 2003, 11 (2): 87-93. 10.1016/S0966-842X(02)00037-9.PubMedView ArticleGoogle Scholar
- Thomas AD, Booth IR: The regulation of expression of the porin gene ompC by acid pH. J Gen Microbiol. 1992, 138 (9): 1829-1835.PubMedView ArticleGoogle Scholar
- Stutzmann Meier P, Entenza JM, Vaudaux P, Francioli P, Glauser MP, Moreillon P: Study of Staphylococcus aureus Pathogenic Genes by Transfer and Expression in the Less Virulent Organism Streptococcus gordonii. Infection and Immunity. 2001, 69 (2): 657-10.1128/IAI.69.2.657-664.2001.PubMed CentralPubMedView ArticleGoogle Scholar
- Stutzmann Meier P, Heiniger N, Troller R, Aebi C: Salivary antibodies directed against outer membrane proteins of Moraxella catarrhalis in healthy adults. Infect Immun. 2003, 71 (12): 6793-6798. 10.1128/IAI.71.12.6793-6798.2003.PubMed CentralPubMedView ArticleGoogle Scholar
- Murphy TF, Brauer AL, Aebi C, Sethi S: Identification of surface antigens of Moraxella catarrhalis as targets of human serum antibody responses in chronic obstructive pulmonary disease. Infect Immun. 2005, 73 (6): 3471-3478. 10.1128/IAI.73.6.3471-3478.2005.PubMed CentralPubMedView ArticleGoogle Scholar
- Adlowitz DG, Kirkham C, Sethi S, Murphy TF: Human serum and mucosal antibody responses to outer membrane protein G1b of Moraxella catarrhalis in chronic obstructive pulmonary disease. FEMS Immunol Med Microbiol. 2006, 46 (1): 139-146. 10.1111/j.1574-695X.2005.00020.x.PubMedView ArticleGoogle Scholar
- Pearson MM, Lafontaine ER, Wagner NJ, Geme JW, Hansen EJ: A hag Mutant of Moraxella catarrhalis Strain O35E Is Deficient in Hemagglutination, Autoagglutination, and Immunoglobulin D-Binding Activities. Infection and Immunity. 2002, 70 (8): 4523-4533. 10.1128/IAI.70.8.4523-4533.2002.PubMed CentralPubMedView ArticleGoogle Scholar
- Hol C, Verduin CM, van Dijke E, Verhoef J, Fleer A, van Dijk H: Complement resistance is a virulence factor of Branhamella (Moraxella) catarrhalis. 1995, 11: 207-212.Google Scholar
- Brandtzaeg P, Halstensen TS: Immunology and immunopathology of tonsils. 1992, 47: 64-75.Google Scholar
- Murphy S, Fitzgerald M, Mulcahy R, Keane C, Coakley D, Scott T: Studies on heamagglutination and serum resistance status of strains of Moraxella catarrhalis isolated from the elderly. Gerontology. 1997, 43: 277-282. 10.1159/000213863.PubMedView ArticleGoogle Scholar
- Attia AS, Lafontaine ER, Latimer JL, Aebi C, Syrogiannopoulos GA, Hansen EJ: The UspA2 protein of Moraxella catarrhalis is directly involved in the expression of serum resistance. Infect Immun. 2005, 73 (4): 2400-2410. 10.1128/IAI.73.4.2400-2410.2005.PubMed CentralPubMedView ArticleGoogle Scholar
- Nordstrom T, Blom AM, Forsgren A, Riesbeck K: The emerging pathogen Moraxella catarrhalis interacts with complement inhibitor C4b binding protein through ubiquitous surface proteins A1 and A2. Journal of Immunology. 2004, 173 (7): 4598-4606.View ArticleGoogle Scholar
- Nordstrom T, Blom AM, Forsgren A, Riesbeck K: A novel interaction of C4b-binding protein (C4BP) with ubiquitous surface proteins A1 and A2 (UspA1 and A2) of Moraxella catarrhalis. Molecular Immunology. 2004, 41 (2-3): 286-Google Scholar
- Campagnari AA, Ducey TF, Rebmann CA: Outer membrane protein B1, an iron-repressible protein conserved in the outer membrane of Moraxella (Branhamella) catarrhalis, binds human transferrin. Infection and Immunity. 1996, 64 (9): 3920-3924.PubMed CentralPubMedGoogle Scholar
- Campagnari AA, Shanks KL, Dyer DW: Growth of Moraxella catarrhalis with human transferrin and lactoferrin: expression of iron-repressible proteins without siderophore production. Infect Immun. 1994, 62 (11): 4909-4914.PubMed CentralPubMedGoogle Scholar
- Furano K, Campagnari AA: Inactivation of the Moraxella catarrhalis 7169 Ferric Uptake Regulator Increases Susceptibility to the Bactericidal Activity of Normal Human Sera. Infection and Immunity. 2003, 71 (4): 1843-1848. 10.1128/IAI.71.4.1843-1848.2003.PubMed CentralPubMedView ArticleGoogle Scholar
- Luke NR, Campagnari AA: Construction and characterization of Moraxella catarrhalis mutants defective in expression of transferrin receptors. Infection and Immunity. 1999, 67: 5815-5819.PubMed CentralPubMedGoogle Scholar
- Schryvers AB, Bonnah R, Yu RH, Wong H, Retzer M: Bacterial lactoferrin receptors. Adv Exp Med Biol. 1998, 443: 123-133.PubMedView ArticleGoogle Scholar
- Stojiljkovic I, Larson J, Hwa V, Anic S, So M: HmbR outer membrane receptors of pathogenic Neisseria spp.: iron-regulated, hemoglobin-binding proteins with a high level of primary structure conservation. J Bacteriol. 1996, 178 (15): 4670-4678.PubMed CentralPubMedGoogle Scholar
- Furano K, NR L, AJ H, AA C: Identification of a conserved Moraxella catarrhalis haemoglobin-utilization protein, MhuA. Microbiology. 2005, 151 (Pt4): 1151-1158. 10.1099/mic.0.27820-0.PubMedView ArticleGoogle Scholar
- Slevogt H, Seybold J, Tiwari KN, Hocke AC, Jonatat C, Dietel S, Hippenstiel S, Singer BB, Bachmann S, Suttorp N, et al: Moraxella catarrhalis is internalized in respiratory epithelial cells by a trigger-like mechanism and initiates a TLR2- and partly NOD1-dependent inflammatory immune response. Cell Microbiol. 2007, 9 (3): 694-707. 10.1111/j.1462-5822.2006.00821.x.PubMedView ArticleGoogle Scholar
- Lafontaine ER, Cope LD, Aebi C, Latimer JL, McCracken GH, Hansen EJ: The UspA1 protein and a second type of UspA2 protein mediate adherence of Moraxella catarrhalis to human epithelial cells in vitro. J Bacteriol. 2000, 182 (5): 1364-1373. 10.1128/JB.182.5.1364-1373.2000.PubMed CentralPubMedView ArticleGoogle Scholar
- Verduin CM, Hol C, Fleer A, van Dijk H, van Belkum A: Moraxella catarrhalis: from emerging to established pathogen. Clin Microbiol Rev. 2002, 15 (1): 125-144. 10.1128/CMR.15.1.125-144.2002.PubMed CentralPubMedView ArticleGoogle Scholar
- Harrison CJ, Woods C, Stout G, Martin B, Selvarangan R: Susceptibilities of Haemophilus influenzae, Streptococcus pneumoniae, including serotype 19A, and Moraxella catarrhalis paediatric isolates from 2005 to 2007 to commonly used antibiotics. J Antimicrob Chemother. 2009, 63 (3): 511-519. 10.1093/jac/dkn538.PubMedView ArticleGoogle Scholar
- Dagan R, Leibovitz E: Bacterial eradication in the treatment of otitis media. Lancet Infect Dis. 2002, 2 (10): 593-604. 10.1016/S1473-3099(02)00394-8.PubMedView ArticleGoogle Scholar
- Helminen ME, Maciver I, Latimer JL, Cope LD, McCracken GH, Hansen EJ: A major outer membrane protein of Moraxella catarrhalis is a target for antibodies that enhance pulmonary clearance of the pathogen in an animal model. Infection and Immunity. 1993, 61: 2003-2010.PubMed CentralPubMedGoogle Scholar
- Sambrook J, Fritsch E, Maniatis T: Molecular Cloning A Laboratory Manual. 1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, SecondGoogle Scholar