Pneumolysin is an important virulence factor of the human pathogen Streptococcus pneumoniae. Sequence analysis of the ply gene from 121 clinical isolates of S. pneumoniae uncovered a number of alleles. Twenty-two strains were chosen for further analysis, and 14 protein alleles were discovered. Five of these had been reported previously, and the remaining 9 were novel. Cell lysates were used to determine the specific hemolytic activities of the pneumolysin proteins. Six strains showed no hemolytic activity, and the remaining 16 were hemolytic, to varying degrees. We report that the nonhemolytic allele reported previously in serotype 1, sequence type (ST) 306 isolates is also present in a number of pneumococcal isolates of serotype 8 that belong to the ST53 lineage. Serotype 1 and 8 pneumococci are known to be associated with outbreaks of invasive disease. The nonhemolytic pneumolysin allele is therefore associated with the dominant clones of outbreak-associated serotypes of S. pneumoniae.

We have demonstrated that longitudinal non-invasive duplexed optical fluorescence imaging provides a simple assessment of arthritic disease activity within the joints of mice following the induction of CIA and may represent a powerful tool to monitor the efficacy of drug treatments in preclinical studies.

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word count: 246 11Total word count (Abstract to Acknowledgments): 3493 12

The unique species specificity of the bacterial cytolysin intermedilysin is explained by its requirement for the human complement regulator CD59 as the primary receptor. Binding studies using individual domains of intermedilysin mapped the CD59-binding site to domain 4 and swap mutants between human and rabbit (non-intermedilysin-binding) CD59 implicated a short sequence (residues 42–59) in human CD59 in binding intermedilysin. We set out to map more closely the CD59 binding site in intermedilysin. We first looked for regions of homology between domain 4 in intermedilysin and the terminal complement components that bind CD59, C8 and C9. A nine amino acid sequence immediately adjacent the undecapeptide segment in intermedilysin domain 4 matched (5 of 9 identical, 3 of 9 conserved) a sequence in C9. A peptide containing this sequence caused dose-dependent inhibition of intermedilysin-mediated lysis of human erythrocytes and rendered erythrocytes more susceptible to complement lysis. Surface plasmon resonance analysis of intermedilysin binding to immobilized CD59 revealed saturable fast-on, fast-off binding and a calculated affinity of 4.9 nM. Substitution of three residues from the putative binding site caused a 5-fold reduction in lytic potency of intermedilysin and reduced affinity for immobilized CD59 by 2.5-fold. The demonstration that a peptide modeled on the CD59-binding site inhibits intermedilysin-mediated haemolysis leads us to suggest that such peptides might be useful in treating infections caused by intermedilysin-producing bacteria.