Physiologic Cold Shock Increases Adherence of Moraxella catarrhalis to and Secretion of Interleukin 8 in Human Upper Respiratory Tract Epithelial Cells

Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid and prolonged downshifts of environmental temperature when humans breathe cold air. In the present study, we show that a 26°C cold shock up-regulates the expression of UspA1, a major adhesin and putative virulence factor of M. catarrhalis, by prolonging messenger RNA half-life. Cold shock promotes M. catarrhalis adherence to upper respiratory tract cells via enhanced binding to fibronectin, an extracellular matrix component that mediates bacterial attachment. Exposure of M. catarrhalis to 26°C increases the outer membrane protein-mediated release of the proinflammatory cytokine interleukin 8 in pharyngeal epithelial cells. Furthermore, cold shock at 26°C enhances the binding of salivary immunoglobulin A on the surface of M. catarrhalis. These data indicate that cold shock at a physiologically relevant temperature of 26°C affects the nasopharyngeal host-pathogen interaction and may contribute to M. catarrhalis virulenc

Down-regulation of porin M35 in Moraxella catarrhalis by aminopenicillins and environmental factors and its potential contribution to the mechanism of resistance to aminopenicillins

Objectives The outer membrane protein M35 of Moraxella catarrhalis is an antigenically conserved porin. Knocking out M35 significantly increases the MICs of aminopenicillins. The aim of this study was to determine the biological mechanism of this potentially new antimicrobial resistance mechanism of M. catarrhalis and the behaviour of M35 in general stress situations. Methods PCR using m35-specific primers was used to detect the m35 gene in clinical isolates. The m35 mRNA expression of strains 300, O35E and 415 after exposure to amoxicillin and different stress conditions was measured by real-time PCR and normalized in relation to their 16S rRNA expression. The expression of M35 protein was analysed by SDS-PAGE and western blotting. Results Screening of 52 middle ear isolates resulted in positive PCR products for all tested strains. The analysis of m35 mRNA expression after amoxicillin treatment showed 24%-85% down-regulation compared with the respective amoxicillin-free controls in all three strains tested. Also, analysis of protein concentrations revealed lower M35 expression after growth with amoxicillin. Investigation of M35 during general stress responses showed down-regulation of the porin with growth at 26°C and 42°C, under hyperosmolar stress and under iron restriction. Conclusions The reduced expression of M35 after aminopenicillin exposure indicates a novel resistance mechanism against aminopenicillins in M. catarrhalis, which may be relevant in vivo. The differences in expression after different stress treatments demonstrate that M35 is involved in general stress response

Physiologic cold shock of Moraxella catarrhalis affects the expression of genes involved in the iron acquisition, serum resistance and immune evasion

<p>Abstract</p> <p>Background</p> <p><it>Moraxella catarrhalis</it>, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. It was previously shown that the prevalence of pharyngeal colonization and respiratory tract infections caused by <it>M. catarrhalis </it>are greatest in winter. The aim of this study was to investigate how <it>M. catarrhalis </it>uses the physiologic exposure to cold air to upregulate pivotal survival systems in the pharynx that may contribute to <it>M. catarrhalis </it>virulence.</p> <p>Results</p> <p>A 26°C cold shock induces the expression of genes involved in transferrin and lactoferrin acquisition, and enhances binding of these proteins on the surface of <it>M. catarrhalis</it>. Exposure of <it>M. catarrhalis </it>to 26°C upregulates the expression of UspA2, a major outer membrane protein involved in serum resistance, leading to improved binding of vitronectin which neutralizes the lethal effect of human complement. In contrast, cold shock decreases the expression of Hemagglutinin, a major adhesin, which mediates B cell response, and reduces immunoglobulin D-binding on the surface of <it>M. catarrhalis</it>.</p> <p>Conclusion</p> <p>Cold shock of <it>M. catarrhalis </it>induces the expression of genes involved in iron acquisition, serum resistance and immune evasion. Thus, cold shock at a physiologically relevant temperature of 26°C induces in <it>M. catarrhalis </it>a complex of adaptive mechanisms that enables the bacterium to target their host cellular receptors or soluble effectors and may contribute to enhanced growth, colonization and virulence.</p

Topical curcumin can inhibit deleterious effects of upper respiratory tract bacteria on human oropharyngeal cells in vitro: potential role for patients with cancer therapy induced mucositis?

Purpose: Curcumin exerts its anti-inflammatory activity via inhibition of nuclear factor κB. Oropharyngeal epithelia and residing bacteria closely interact in inflammation and infection. This in vitro model investigated the effects of curcumin on bacterial survival, adherence to, and invasion of upper respiratory tract epithelia, and studied its anti-inflammatory effect. We aimed to establish a model, which could offer insights into the host-pathogen interaction in cancer therapy induced mucositis. Methods: Moraxella catarrhalis (Mcat) and the oropharyngeal epithelial cell line Detroit 562 were used. Time-kill curves assessed the inhibition of bacterial growth and adherence assays and gentamicin protection assays determined the effect of curcumin-preincubated cells on bacterial adherence and invasion. Curcumin-mediated inhibition of pro-inflammatory activation by Mcat was determined via interleukin-8 concentrations in the supernatants. The synergistic role of secretory IgA (sIgA) on adherence was investigated. Results: Curcumin was bactericidal at concentrations >50µM. Preincubation of Detroit cells for 60min demonstrated that concentrations >100µM inhibited bacterial adherence. Together with sIgA, curcumin inhibited adherence at concentrations ≥50µM. Both 100 and 200µM curcumin significantly inhibited Mcat cell invasion. Finally, curcumin inhibited Mcat-induced pro-inflammatory activation by strongly suppressing IL-8 release. At a concentration of 200µM, 10min of curcumin exposure inhibited IL-8 release significantly, and complete suppression required a pre-exposure time of ≥45min. Conclusion: Curcumin, in clinically relevant concentrations for topical use, displayed strong antibacterial effect against a facultative upper respiratory tract pathogen by inhibiting bacterial growth, adherence, invasion, and pro-inflammatory activation of upper respiratory tract epithelial cells in vitr

A Reservoir of Moraxella catarrhalis in Human Pharyngeal Lymphoid Tissue

Background. Early exposure of infants and long-term immunity suggest that colonization with Moraxella catarrhalis is more frequent than is determined by routine culture. We characterized a reservoir of M. catarrhalis in pharyngeal lymphoid tissue. Methods. Tissue from 40 patients (median age, 7.1 years) undergoing elective tonsillectomy and/or adenoidectomy was analyzed for the presence of M. catarrhalis by culture, real-time DNA and RNA polymerase chain reaction (PCR), immunohistochemical analysis (IHC), and fluorescent in situ hybridization (FISH). Histologic sections were double stained for M. catarrhalis and immune cell markers, to characterize the tissue distribution of the organism. Intracellular bacteria were identified using confocal laser scanning microscopy (CLSM). Results. Twenty-nine (91%) of 32 adenoids and 17 (85%) of 20 tonsils were colonized with M. catarrhalis. Detection rates for culture, DNA PCR, RNA PCR, IHC, and FISH were 7 (13%) of 52, 10 (19%) of 52, 21 (41%) of 51, 30 (61%) of 49, and 42 (88%) of 48, respectively (P < .001). Histologic analysis identified M. catarrhalis in crypts, intraepithelially, subepithelially, and (using CLSM) intracellularly. M. catarrhalis colocalized with macrophages and B cells in lymphoid follicles. Conclusions. Colonization by M. catarrhalis is more frequent than is determined by surface culture, because the organism resides both within and beneath the epithelium and invades host cell

Outer membrane porin M35 of Moraxella catarrhalis mediates susceptibility to aminopenicillins

<p>Abstract</p> <p>Background</p> <p>The outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen <it>Moraxella catarrhalis</it>. 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 <it>M. catarrhalis</it>.</p> <p>Results</p> <p>Isogenic <it>m35 </it>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 <it>m35 </it>mutants. Immunoblot analysis demonstrated that human saliva contains anti-M35 IgA. Wild-type strains and their respective <it>m35 </it>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 <it>m35 </it>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.</p> <p>Conclusion</p> <p>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 <it>in vivo </it>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.</p

Unveiling electrotransformation of Moraxella catarrhalis as a process of natural transformation

The human respiratory tract pathogen Moraxella catarrhalis is a naturally competent microorganism. However, electrotransformation has long been used to introduce foreign DNA into this organism. This study demonstrated that electrotransformants obtained with linear or circular nonreplicating plasmid DNA originated exclusively from natural transformation processes taking place during the recovery phase after the application of current. Only replicating plasmid DNA could be introduced into M. catarrhalis by electrotransformation, in a type IV pilus-independent manner. Electrotransformation with homologous genomic DNA indicated that restriction of double-stranded DNA was independent of type III restriction-methylation systems. Nontransformability of M. catarrhalis by electrotransformation was observed using double- as well as single-stranded DNA. In addition, the study showed that natural competence is a very constant feature of M. catarrhali

A reservoir of Moraxella catarrhalis in human pharyngeal lymphoid tissue

BACKGROUND: Early exposure of infants and long-term immunity suggest that colonization with Moraxella catarrhalis is more frequent than is determined by routine culture. We characterized a reservoir of M. catarrhalis in pharyngeal lymphoid tissue. METHODS: Tissue from 40 patients (median age, 7.1 years) undergoing elective tonsillectomy and/or adenoidectomy was analyzed for the presence of M. catarrhalis by culture, real-time DNA and RNA polymerase chain reaction (PCR), immunohistochemical analysis (IHC), and fluorescent in situ hybridization (FISH). Histologic sections were double stained for M. catarrhalis and immune cell markers, to characterize the tissue distribution of the organism. Intracellular bacteria were identified using confocal laser scanning microscopy (CLSM). RESULTS: Twenty-nine (91%) of 32 adenoids and 17 (85%) of 20 tonsils were colonized with M. catarrhalis. Detection rates for culture, DNA PCR, RNA PCR, IHC, and FISH were 7 (13%) of 52, 10 (19%) of 52, 21 (41%) of 51, 30 (61%) of 49, and 42 (88%) of 48, respectively (P<.001). Histologic analysis identified M. catarrhalis in crypts, intraepithelially, subepithelially, and (using CLSM) intracellularly. M. catarrhalis colocalized with macrophages and B cells in lymphoid follicles. CONCLUSIONS: Colonization by M. catarrhalis is more frequent than is determined by surface culture, because the organism resides both within and beneath the epithelium and invades host cells

Antibacterial and antiinflammatory kinetics of curcumin as a potential antimucositis agent in cancer patients

The antiinflammatory agent curcumin (diferuloylmethane) has a potential to mitigate cancer therapy-induced mucositis. We assessed the in vitro extent of its bactericidal activity and determined the kinetics of its antiinflammatory effect on pharyngeal cells. Bactericidal activity was assessed using the LIVE/DEAD® Kit after 4 h of exposure to curcumin (50-200 μM) in 18 oropharyngeal species commonly associated with bacteremia in febrile neutropenia. Moraxella catarrhalis or its outer membrane vesicles were used to determine the inhibitory effect of curcumin on bacteria-induced proinflammatory activity as determined by cytokine release into the supernatant of Detroit 562 pharyngeal cells using the Luminex® xMAP® technology. Curcumin exerted a concentration-dependent bactericidal effect on all 18 species tested. After 4 h at 200 μM, 12 species tested were completely killed. Preincubation of Detroit cells with 200 μM curcumin for 5 to 60 min resulted in complete suppression of the release of tumor necrosis factor-α, interleukin (IL)-6, IL-8, monocyte chemoattractant protein 1, granulocyte macrophage-colony stimulating factor, and vascular endothelial growth factor. Fibroblast growth factor-2 and interferon-γ were not affected. Repetitive exposure to curcumin resulted in repetitive suppression of cytokine/chemokine expression lasting from 4 to 6 h. Through reduction of oral microbial density as well as suppression of inflammation cascades curcumin may prevent cancer therapy-induced oral mucositis, e.g., when applied as multiple daily mouth washes