Lysozyme M deficiency leads to an increased susceptibility to Streptococcus pneumoniae-induced otitis media

<p>Abstract</p> <p>Background</p> <p>Lysozyme is an antimicrobial innate immune molecule degrading peptidoglycan of the bacterial cell wall. Lysozyme shows the ubiquitous expression in wide varieties of species and tissues including the tubotympanum of mammals. We aim to investigate the effects of lysozyme depletion on pneumococcal clearance from the middle ear cavity.</p> <p>Methods</p> <p>Immunohistochemistry was performed to localize lysozyme in the Eustachian tube. Lysozyme expression was compared between the wild type and the lysozyme M^-/-mice using real time quantitative RT-PCR and western blotting. Muramidase activity and bactericidal activity of lysozyme was measured using a lysoplate radial diffusion assay and a liquid broth assay, respectively. To determine if depletion of lysozyme M increases a susceptibility to pneumococal otitis media, 50 CFU of <it>S. pneumoniae </it>6B were transtympanically inoculated to the middle ear and viable bacteria were counted at day 3 and 7 with clinical grading of middle ear inflammation.</p> <p>Results</p> <p>Immunolabeling revealed that localization of lysozyme M and lysozyme P is specific to some/particular cell types of the Eustachian tube. Lysozyme P of lysozyme M^-/-mice was mainly expressed in the submucosal gland but not in the tubal epithelium. Although lysozyme M^-/-mice showed compensatory up-regulation of lysozyme P, lysozyme M depletion resulted in a decrease in both muramidase and antimicrobial activities. Deficiency in lysozyme M led to an increased susceptibility to middle ear infection with <it>S. pneumoniae </it>6B and resulted in severe middle ear inflammation, compared to wild type mice.</p> <p>Conclusion</p> <p>The results suggest that lysozyme M plays an important role in protecting the middle ear from invading pathogens, particularly in the early phase. We suggest a possibility of the exogenous lysozyme as an adjuvant therapeutic agent for otitis media, but further studies are necessary.</p

The Vein Patterning 1 (VEP1) Gene Family Laterally Spread through an Ecological Network

Lateral gene transfer (LGT) is a major evolutionary mechanism in prokaryotes. Knowledge about LGT— particularly, multicellular— eukaryotes has only recently started to accumulate. A widespread assumption sees the gene as the unit of LGT, largely because little is yet known about how LGT chances are affected by structural/functional features at the subgenic level. Here we trace the evolutionary trajectory of VEin Patterning 1, a novel gene family known to be essential for plant development and defense. At the subgenic level VEP1 encodes a dinucleotide-binding Rossmann-fold domain, in common with members of the short-chain dehydrogenase/reductase (SDR) protein family. We found: i) VEP1 likely originated in an aerobic, mesophilic and chemoorganotrophic α-proteobacterium, and was laterally propagated through nets of ecological interactions, including multiple LGTs between phylogenetically distant green plant/fungi-associated bacteria, and five independent LGTs to eukaryotes. Of these latest five transfers, three are ancient LGTs, implicating an ancestral fungus, the last common ancestor of land plants and an ancestral trebouxiophyte green alga, and two are recent LGTs to modern embryophytes. ii) VEP1's rampant LGT behavior was enabled by the robustness and broad utility of the dinucleotide-binding Rossmann-fold, which provided a platform for the evolution of two unprecedented departures from the canonical SDR catalytic triad. iii) The fate of VEP1 in eukaryotes has been different in different lineages, being ubiquitous and highly conserved in land plants, whereas fungi underwent multiple losses. And iv) VEP1-harboring bacteria include non-phytopathogenic and phytopathogenic symbionts which are non-randomly distributed with respect to the type of harbored VEP1 gene. Our findings suggest that VEP1 may have been instrumental for the evolutionary transition of green plants to land, and point to a LGT-mediated ‘Trojan Horse’ mechanism for the evolution of bacterial pathogenesis against plants. VEP1 may serve as tool for revealing microbial interactions in plant/fungi-associated environments

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