Differential Modulation of TNF-α–Induced Apoptosis by Neisseria meningitidis

Infections by Neisseria meningitidis show duality between frequent asymptomatic carriage and occasional life-threatening disease. Bacterial and host factors involved in this balance are not fully understood. Cytopathic effects and cell damage may prelude to pathogenesis of isolates belonging to hyper-invasive lineages. We aimed to analyze cell–bacteria interactions using both pathogenic and carriage meningococcal isolates. Several pathogenic isolates of the ST-11 clonal complex and carriage isolates were used to infect human epithelial cells. Cytopathic effect was determined and apoptosis was scored using several methods (FITC-Annexin V staining followed by FACS analysis, caspase assays and DNA fragmentation). Only pathogenic isolates were able to induce apoptosis in human epithelial cells, mainly by lipooligosaccharide (endotoxin). Bioactive TNF-α is only detected when cells were infected by pathogenic isolates. At the opposite, carriage isolates seem to provoke shedding of the TNF-α receptor I (TNF-RI) from the surface that protect cells from apoptosis by chelating TNF-α. Ability to induce apoptosis and inflammation may represent major traits in the pathogenesis of N. meningitidis. However, our data strongly suggest that carriage isolates of meningococci reduce inflammatory response and apoptosis induction, resulting in the protection of their ecological niche at the human nasopharynx

Genome Wide Expression Profiling Reveals Suppression of Host Defence Responses during Colonisation by Neisseria meningitides but not N. lactamica

Both Neisseria meningitidis and the closely related bacterium Neisseria lactamica colonise human nasopharyngeal mucosal surface, but only N. meningitidis invades the bloodstream to cause potentially life-threatening meningitis and septicaemia. We have hypothesised that the two neisserial species differentially modulate host respiratory epithelial cell gene expression reflecting their disease potential. Confluent monolayers of 16HBE14 human bronchial epithelial cells were exposed to live and/or dead N. meningitidis (including capsule and pili mutants) and N. lactamica, and their transcriptomes were compared using whole genome microarrays. Changes in expression of selected genes were subsequently validated using Q-RT-PCR and ELISAs. Live N. meningitidis and N. lactamica induced genes involved in host energy production processes suggesting that both bacterial species utilise host resources. N. meningitidis infection was associated with down-regulation of host defence genes. N. lactamica, relative to N. meningitidis, initiates up-regulation of proinflammatory genes. Bacterial secreted proteins alone induced some of the changes observed. The results suggest N. meningitidis and N. lactamica differentially regulate host respiratory epithelial cell gene expression through colonisation and/or protein secretion, and that this may contribute to subsequent clinical outcomes associated with these bacteria

Antimicrobial activity of innate immune molecules against <it>Streptococcus pneumoniae, Moraxella catarrhalis </it>and nontypeable <it>Haemophilus influenzae</it>

<p>Abstract</p> <p>Background</p> <p>Despite its direct connection to the nasopharynx which harbors otitis media pathogens as part of its normal flora, the middle ear cavity is kept free of these bacteria by as yet unknown mechanisms. Respiratory mucosal epithelia, including those of the middle ear and eustachian tube, secrete antimicrobial effectors including lysozyme, lactoferrin and β defensins-1 and -2. To elucidate the role of these innate immune molecules in the normal defense and maintenance of sterility of respiratory mucosa such as that of the middle ear, we assessed their effect on the respiratory pathogens nontypeable <it>Haemophilus influenzae </it>(NTHi) 12, <it>Moraxella catarrhalis </it>035E, and <it>Streptococcus pneumoniae </it>3, and 6B.</p> <p>Methods</p> <p>Two assay methods, the radial assay and the liquid broth assay, were employed for testing the antimicrobial activity of the molecules. This was done in order to minimize the possibility that the observed effects were artifacts of any single assay system employed. Also, transmission electron microscopy (TEM) was employed to evaluate the effect of antimicrobial innate immune molecules on OM pathogens. For the statistical analysis of the data, Student's <it>t</it>-test was performed.</p> <p>Results</p> <p>Results of the radial diffusion assay showed that β defensin-2 was active against all four OM pathogens tested, while treatment with β defensin-1 appeared to only affect <it>M. catarrhalis</it>. The radial assay results also showed that lysozyme was quite effective against <it>S. pneumoniae </it>3 and 6B and was partially bacteriostatic/bactericidal against <it>M. catarrhalis</it>. Lysozyme however, appeared not to affect the growth of NTHi. Thus, lysozyme seems to have a more pronounced impact on the growth of the Gram-positive <it>S. pneumoniae </it>as compared to that of Gram-negative pathogens. Lactoferrin on the other hand, enhanced the growth of the bacteria tested. The results of the radial assays were confirmed using liquid broth assays for antimicrobial activity, and showed that lysozyme and β defensin-2 could act synergistically against <it>S. pneumoniae </it>6B. Moreover, in the liquid broth assay, β defensin-1 showed a modest inhibitory effect on the growth of <it>S. pneumoniae </it>6B. As assessed by ultrastructural analysis, lysozyme and β defensin-2, and to a much lesser extent, β defensin-1, appeared to be able to cause damage to the bacterial membranes.</p> <p>Conclusions</p> <p>Here we report that lysozyme and the β defensins can inhibit the growth of clinical isolates of otitis media pathogens – namely NTHi strain 12, <it>S. pneumoniae </it>strains 3 and 6B and <it>M. catarrhalis </it>strain 035E – and cause ultrastructural damage to these pathogens. Moreover, we demonstrate that lysozyme and β defensin-2 can act synergistically against <it>S. pneumoniae</it>. These findings are consistent with the concept that secreted antimicrobial peptides and other components of innate immunity constitute the first line of defense protecting host mucosal surfaces, including the tubotympanal (eustachian tube and middle ear cavity) mucosa, against pathogens.</p