Graeme J.M. Cowan

Presence of Nonhemolytic Pneumolysin in Serotypes of Streptococcus pneumoniae Associated with Disease Outbreaks

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.

The human-specific action of intermedilysin, a homolog of streptolysin O, is dictated by domain 4 of the protein.

Intermedilysin is a pore‐forming cytolysin belonging to the streptolysin O gene family known as the ‘Cholesterol‐binding/dependent cytolysins’ and is unique within the family in that it is highly human‐specific. This specificity suggests interaction with a component of human cells other than cholesterol, the proposed receptor for the other toxins of the gene family. Indeed, intermedilysin showed no significant degree of affinity to free or liposome‐embedded cholesterol. Characterization of intermedilysin undecapeptide mutants revealed that this lack of affinity to cholesterol was a result of the substitutions of intermedilysin in this region. Absorption assays with erythrocyte membranes from various animals, competitive inhibition with domain 4 of intermedilysin and liposome‐binding assays of streptolysin O and intermedilysin indicated that cell membrane binding is the human‐specific step of intermedilysin action, that the host cell membrane‐binding site is located within domain 4 in common with other members of the family and that the receptor for this toxin is not cholesterol. The species specificity of undecapeptide mutants of intermedilysin and streptolysin O and chimeric mutants between intermedilysin and streptolysin O, and intermedilysin and pneumolysin indicated that domain 4 of intermedilysin determines the human‐specific action step and the cell‐binding site of domain 4 lies within the 56 amino acids of the C‐terminal, excluding the undecapeptide region.

Novel mucosal vaccines generated by genetic conjugation of heterologous proteins to pneumolysin (PLY) from Streptococcus pneumoniae

Induction of immunity at mucosal surfaces is thought to be an essential feature in the protection of the host against the many pathogens that gain access through these surfaces. Here we describe how strong local and systemic immune responses can be generated when proteins are genetically conjugated to pneumolysin (PLY) from Streptococcus pneumoniae. Using green fluorescent protein (eGFP) and PsaA from S. pneumoniae, we have shown that genetic fusion (eGFPPLY and PsaAPLY) is essential to ensure high levels of antigen specific IgG and IgA in the serum and at mucosal surfaces. This form of vaccination is highly effective with antigen specific antibodies detected after a single dose of nanogram quantities of the conjugated proteins. In addition, generation of a non-toxic variant (eGFPDelta6PLY) indicated that while the toxic activity of PLY was not essential for adjuvanticity, it contributed to the magnitude of the response generated. Whilst vaccination with the PsaAPLY fusion proteins did not protect the animals from challenge, these studies confirm the utility of pneumolysin to act as a novel mucosal adjuvant to substantially increase the local and systemic humoral response to genetically fused protein antigens.

The mechanism of pneumolysin‐induced cochlear hair cell death in the rat

Streptoccocus pneumoniae infection can result in local and systemic diseases such as otitis media, pneumonia and meningitis. Sensorineural hearing loss associated with this infection is mediated by the release of an exotoxin, pneumolysin. The goal of the present study was to characterize the mechanisms of pneumolysin toxicity in cochlear hair cells in vitro. Pneumolysin induced severe damage in cochlear hair cells, ranging from stereocilia disorganization to total cell loss. Surprisingly, pneumolysin‐induced cell death preferentially targeted inner hair cells. Pneumolysin triggered in vitro cell death by an influx of calcium. Extracellular calcium appeared to enter the cell through a pore formed by the toxin. Buffering intracellular calcium with BAPTA improved hair cell survival. The mitochondrial apoptotic pathway involved in pneumolysin‐induced cell death was demonstrated by the use of bongkrekic acid. Binding of pneumolysin to the hair cell plasma membrane was required to induce cell death. Increasing external calcium reduced cell toxicity by preventing the binding of pneumolysin to hair cell membranes. These results showed the significant role of calcium both in triggering pneumolysin‐induced hair cell apoptosis and in preventing the toxin from binding to its cellular target.

Identification of the high affinity binding site in the Streptococcus intermedius toxin intermedilysin for its membrane receptor, the human complement regulator CD59

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.