Ecology of virulence genes in the human pathogen Streptococcus pneumoniae

Streptococcus pneumoniae, also known as the pneumococcus, is an important human pathogen, with high burdens of disease and mortality worldwide. There are over 90 serotypes of this pathogen, demonstrating the vast amounts of diversity present. Currently, there are two pneumococcal vaccines, both targeting the polysaccharide capsule. However, one vaccine is ineffective in the paediatric population, whilst the other only targets a minority of disease-causing serotypes, and has increased disease caused by serotypes not present in the vaccine. One solution is a new pneumococcal vaccine targeting a protein virulence factor possessed by all pneumococci, which would afford cross-serotype protection. As a result, it is important to assess the diversity of pneumococcal virulence factors in order to determine their potential as vaccine candidates, as excess diversity present may prevent full serotype-independent protection of a vaccine. Furthermore, diversity studies offer important insight on pneumococcal biology, epidemiology and pathogenesis. The diversity in the toxin pneumolysin (Ply) was greater than previously thought, with 14 protein alleles discovered. However, diversity remained significantly lower than surface-exposed virulence factors, indicating this toxin may be more suitable as a vaccine candidate. Furthermore, all 14 alleles were recognised by polyclonal antibodies, showing the potential cross-serotype protection of a vaccine targeting this toxin. A novel non-haemolytic Ply allele was associated with clones recently expanding in the pneumococcal population, as well as serotypes associated with outbreaks of pneumococcal disease. The non-haemolytic toxin may therefore play a role in driving clonal expansion in certain genetic backgrounds, or be involved in establishing outbreaks of pneumococcal disease. The diversity in the neuraminidase A (NanA) enzyme was significantly higher than in Ply, with many point mutations, mosaic blocks and insertions regions present in 18 divergent alleles. This level of diversity should not be prohibitive to the use of this protein as a vaccine candidate, as polyclonal antibodies recognised 4 NanA alleles of significant diversity, indicating the possibility of cross-serotype protection. The role of NanA in pathogenesis of pneumococcal haemolytic uraemic syndrome (p-HUS) was investigated, although there was no correlation between p-HUS and NanA allele or activity. The novel discovery that pneumococcal NanA was inhibited by viral neuraminidase inhibitors of influenza allowed insight into the synergistic relationship between these two deadly pathogens, and showed for the first time that treatment with these drugs acts on both the primary and secondary pathogen. One of these inhibitors, Oseltamivir, was found to have potential in treating secondary pneumococcal pneumonia, which may help decrease the significant burden of this disease, as well as reducing the over-reliance on antibiotics for treating pneumococcal diseases. Homologues of Ply and NanA were identified and characterised in the related species Streptococcus mitis and Streptococcus pseudopneumoniae, giving insight into the evolutionary relationships between these species. Furthermore, the presence of these homologues in related species gives rise to the possibility of pneumococci acquiring altered genes through horizontal gene transfer. The selective pressure of a vaccine targeting these proteins may give evolutionary advantage to these pneumococci, resulting in evasion of a vaccine. Microarray studies have been used to assess pneumococcal diversity at a genome-wide level. Gene expression studies identified candidate genes which may play a role in p-HUS pathogenesis. Further studies into this area will improve the diagnosis and treatment of this disease. Furthermore, a large number of established pneumococcal virulence factors, many of which are vaccine candidates, were found to have homologues in closely related commensal species. These results must be taken into consideration for future protein-based pneumococcal vaccine design.