Genetic characterisation of Streptococcus pneumoniae serotype 1 isolates in relation to invasiveness.

Abstract

Streptococcus pneumoniae (the pneumococcus) is one of the most significant causes of human mortality and morbidity, and is a leading cause of diseases such as pneumonia, invasive disease (including bacteraemia and meningitis [IPD]) and otitis media. However, the pneumococcus is more commonly carried asymptomatically within the nasopharynx. The likelihood of the pneumococcus progressing from asymptomatic carriage to IPD varies between strains, and is associated with certain serotypes and clones. In particular, serotype 1 strains have a high-attack rate as they readily progress from a state of transient carriage to IPD. Recently, a closely-related group of hypervirulent serotype 1 clones have been responsible for epidemics of IPD with unusually high mortality rates. In contrast, epidemic asymptomatic carriage of serotype 1 clones has been found in a number of remote indigenous communities in the Northern Territory of Australia. Such isolates of serotype 1 from asymptomatic carriage are unusual and provided a rare opportunity to perform genomic comparisons with invasive serotype 1 isolates in order to identify serotype-independent factors that contribute to differences in the invasive potential of the pneumococcus. Preliminary work using the non-invasive serotype 1 isolates from the Northern Territory and a collection of invasive human isolates of both indigenous and non-indigenous origin identified three virulence profiles that were non-invasive, intermediately virulent, or highly virulent in mice. Subsequently, phenomic analyses did not identify differences in the amount of capsule or differences in the apparent molecular weight or relative expression of a selection of well-characterised protein virulence factors that correlated with a virulence phenotype. However, in preliminary genomic comparisons the chromosomal toxin-antitoxin (TA) system of the PPI-1 variable region (PezAT) was identified in only highly virulent serotype 1 isolates, but absent from intermediately virulent and non-invasive serotype 1 isolates. Therefore, the broad objectives of this study where to determine the clonal relatedness of isolates representing all three virulence phenotypes, characterise the potential role of the PPI-1 variable region in IPD and identify additional variable regions of the pneumococcal genome that were associated with heightened virulence. Interestingly, it was shown that the highly virulent strain 1861 was a one-locus variant of the sequence type 217 clone of lineage B, responsible for severe IPD in parts of Africa. Therefore, the highly virulent nature of strain 1861 (and strain 4496) in mice is likely to also be reflected in humans. In contrast, the non-invasive and intermediately virulent strains were of lineage A, which includes the most frequently detected clones in Europe and the United States. In addition, different organisations of the PPI-1 variable region correlated with certain lineages of serotype 1. For example, the lineage A isolates lacked pezAT and instead contained a transcriptionally active immunity system against the bacteriocin, mersacidin. Interestingly, following a survey of a variety of S. pneumoniae strains representing a broad array of serotypes, the mersacidin immunity system was identified as the most common feature of the PPI-1 variable region, and is also present in the pandemic carriage Spanish ² ³F ST81 clone. In contrast, the highly virulent isolates of lineages B and C encoded pezAT and a number of genes predicted to encode enzymes that catalyse the rate-limiting steps of pathways involved in the degradation and biosynthesis of some amino acids and the biosynthesis and conversion of UDP-sugars. Interestingly, key components of this region exhibited preferential expression in the lungs and blood when compared to the nasopharynx of infected mice. Subsequently, it was shown using replacement mutants of the PPI-1 variable region in a D39 background that the region from the highly virulent strains promotes greater competitive fitness within the blood, lungs and nasopharyngeal tissue, compared to the equivalent region from the intermediately virulent and non-invasive strains in co-infected mice. Whilst the mechanism by which the PPI-1 variable region contributes to survival in vivo is not clear, a possibility is that centralised regulation of a number of metabolic pathways may enhance the survival of the pneumococcus in the lungs and blood. Whilst the PPI-1 variable region was important for the competitive fitness of D39 during disease, it was not clear whether this region was solely responsible for the differences observed in invasive potential between the highly virulent, intermediately virulent and non-invasive serotype 1 isolates. Therefore, comparative genomic hybridisation (CGH) and next generation genome sequencing were used to identify additional regions of the genome that are associated with the highly virulent isolates. It was found that genes homologous to the platelet-binding protein B (PblB) and a Streptococcus mitis lysogenic phage endolysin were present in the genome of only the highly virulent strains, and not in either the intermediately virulent and non-invasive strains. In addition, regions encoding a putative ABC transporter and enzymes predicted to be involved in the degradation of sialic acid, ZmpD, and a 64-kb Tn5253-like conjugative transposon that included a TA system that is highly homologous to pezAT, were found in only the highly virulent strains and not in the intermediately virulent or non-invasive isolates. Subsequent in vivo gene expression comparisons revealed that the phage-associated endolysin exhibited significantly greater expression in the lungs and blood of infected mice than the nasopharynx, which highlighted a potential mechanism for increased surface display of PblB in the lungs and blood. Whilst yet to be proven experimentally, it is thought that greater surface display of PblB could contribute to the rapid invasion of the blood that is characteristic of the highly virulent serotype 1 strains. In addition to PblB, greater expression of the sialic acid-associated ABC transporter was observed in the blood when compared to the lungs and nasopharynx of infected mice. Therefore, whilst the role of the region remains to be determined, it might be possible that the region enables the utilisation of host-derived sialic acids as an energy source in the blood, thus promoting survival and growth. However, a significant roadblock encountered in this study was the inability to genetically manipulate the highly virulent serotype 1 isolates. In order to confirm the importance of genes such as that in the PPI-1 variable region and pblB in virulence, mutagenesis of these regions was attempted. However, despite numerous attempts to optimise the transformation protocol, it is possible that some defect in the competence system that is linked to the over-expression of comW might be responsible for the inability to transform strains 1861 and 4496. In this study a number of genomic regions were identified that via putative roles in metabolism, sugar acquisition and degradation and adherence to human platelets and their patterns of expression in vivo promote the invasion and survival of the pneumococcus in the blood and lungs. Such findings broaden the understanding of the progression to IPD from asymptomatic carriage and highlight strain-specific differences that could make some strains more virulent than others.