Pathogenesis of Streptococcus pneumoniae - a molecular characterisation

Abstract

The bacterium Streptococcus pneumoniae is a leading cause of human morbidity and mortality. With ever increasing antibiotic resistance rates and poor coverage of current vaccines, novel therapeutics and vaccines are urgently required. However, the mechanisms underlying the differences between capacity of individual strains to cause localised vs invasive pneumococcal diseases are poorly understood. This is largely due to vast genetic diversity of strains, which can be subdivided into 100 serotypes based on the capsular polysaccharide they produce, superimposed on >12000 clonal sequence types (ST) distinguished by multi-locus sequence typing. This poor understanding of underlying disease mechanisms is impeding novel treatment design. Previous studies have shown that even closely related strains within the same serotype and clonal type can display variations in virulence, corresponding to their isolation site in humans. In murine intranasal challenge models, serotype 14 ST15 and serotype 3 ST 180 clinical isolates from the blood tended to cause invasive disease, while corresponding ear isolates instead caused more localised disease. Focusing on the relatively few differences between these closely related strains offers an opportunity to bypass the large genetic diversity of S. pneumoniae. To determine the mechanisms that dictate their progression to specific sites of the body, the blood and ear isolates pairs had their genomes and transcriptomes sequenced and compared. No significant findings came out of the transcriptomic studies, but the genomic analyses found single nucleotide polymorphisms (SNPs) in rafR and rafK, respectively, that drastically alter pneumococcal disease progression. These genes encode proteins involved in the uptake and/or metabolism of the sugar raffinose. Growth assays with raffinose as the sole carbon source showed that blood isolates grew better than ear isolates, whereas there were no differences in growth in the presence of glucose. Finally, the swapping of rafR alleles by allelic-exchange mutagenesis between serotype 14 ST15 blood and ear isolates led to a concomitant swap in virulence phenotype in mice. These results suggest the ability to utilise raffinose plays a significant role in dictating pneumococcal tissue tropism and disease progression. Using in vivo dual RNA sequencing on infected murine lungs in the early stages of infection, it was established that the rafR SNP extensively impacts both bacterial and host transcriptomes. A crucial role for IL-17 induced neutrophil recruitment was predicted, with IL-17 pathway genes upregulated in the strains cleared from the lungs. Indeed, single cell flow cytometry analysis showed the rafR SNP leads to increased recruitment of neutrophils in the lungs of mice infected with the strains that were subsequently cleared. Importantly, in the murine intranasal challenge models, mice depleted of neutrophils or IL-17A had significantly higher bacterial loads in the lungs 24h post-challenge. Strikingly, the strains originally cleared from the lungs were now able to persist in this niche after neutrophil depletion, at similar levels at the non-neutrophil depleted invasive strains. The findings provide novel insights into the mechanisms underlying differences in virulence phenotype among S. pneumoniae clinical isolates and demonstrate the wide impact of a single bacterial SNP on both bacteria metabolism and host innate immune responses.