SUMMARYStreptococcus pneumoniae is a leading cause of otitis media, sinusitis, pneumonia, bacteraemia and meningitis worldwide. The drawbacks associated with the limited number of various capsular polysaccharides that can be included in the polysaccharide-based vaccines focuses much attention on pneumococcal proteins as vaccine candidates. We extracted an enriched cell wall fraction from S. pneumoniae WU2. Approximately 150 soluble proteins could be identified by 2D gel electrophoresis. The proteins were screened by 2D-Western blotting using sera that were obtained longitudinally from children attending day-care centres at 18, 30 and 42 months of age and sera from healthy adult volunteers. The proteins were further identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Seventeen proteins were antigenic in children and adults, of which 13 showed an increasing antibody response with age in all eight children analysed. Two immunogenic proteins, fructose-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and a control protein with known low immunogenicity, heat shock protein 70 (DnaK), were expressed in Escherichia coli , purified and used to immunize mice. Mouse antibodies elicited to the recombinant (r) FBA and rGAPDH were cross-reactive with several genetically unrelated strains of different serotypes and conferred protection to respiratory challenge with virulent pneumococci. In addition, the FBA used in this study (NP_345117) does not have a human ortholog and warrants further investigation as a candidate for a pneumococcal vaccine. In conclusion, the immunoproteomics based approach utilized in the present study appears to be a suitable tool for identification of novel S. pneumoniae vaccine candidates.
The role of IL-1 in susceptibility to Streptococcus pneumoniae infection was studied in mice deficient in genes of the IL-1 family [i.e. IL-1alpha-/-, IL-1beta-/-, IL-1alpha/beta-/- and IL-1R antagonist (IL-1Ra)-/- mice] following intra-nasal inoculation. Intra-nasal inoculation of S. pneumoniae of IL-1beta-/- and IL-1alpha/beta-/- mice displayed significantly lower survival rates and higher nasopharyngeal and lung bacterial load as compared with control, IL-1alpha-/- and IL-1Ra-/- mice. Treatment of IL-1beta-/- mice with rIL-1beta significantly improved their survival. A significant increase in blood neutrophils was found in control, IL-1alpha-/- and IL-1Ra-/- but not in IL-1beta-/- and IL-1alpha/beta-/- mice. Local infiltrates of neutrophils and relatively preserved organ architecture were observed in the lungs of IL-1alpha-/- and control mice. However, S. pneumoniae-infected IL-1beta-/-, IL-1alpha/beta-/- and IL-1Ra-/- mice demonstrated diffuse pneumonia and tissue damage. Altogether, all three isoforms contribute to protection against S. pneumoniae; our results point to differential role of IL-1alpha and IL-1beta in the pathogenesis and control of S. pneumoniae infection and suggest that IL-1beta has a major role in resistance to primary pneumococcal infection while the role of IL-1alpha is less important.
Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligandhigh lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-liganddull macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC.
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