Diverse regulatory pathways modulate bet hedging of competence induction in epigenetically-differentiated phase variants of Streptococcus pneumoniae.

Despite enabling Streptococcus pneumoniae to acquire antibiotic resistance and evade vaccine-induced immunity, transformation occurs at variable rates across pneumococci. Phase variants of isolate RMV7, distinguished by altered methylation patterns driven by the translocating variable restriction-modification (tvr) locus, differed significantly in their transformation efficiencies and biofilm thicknesses. These differences were replicated when the corresponding tvr alleles were introduced into an RMV7 derivative lacking the locus. RNA-seq identified differential expression of the type 1 pilus, causing the variation in biofilm formation, and inhibition of competence induction in the less transformable variant, RMV7domi. This was partly attributable to RMV7domi's lower expression of ManLMN, which promoted competence induction through importing N-acetylglucosamine. This effect was potentiated by analogues of some proteobacterial competence regulatory machinery. Additionally, one of RMV7domi's phage-related chromosomal island was relatively active, which inhibited transformation by increasing expression of the stress response proteins ClpP and HrcA. However, HrcA increased competence induction in the other variant, with its effects depending on Ca2+ supplementation and heat shock. Hence the heterogeneity in transformation efficiency likely reflects the diverse signalling pathways by which it is affected. This regulatory complexity will modulate population-wide responses to synchronising quorum sensing signals to produce co-ordinated yet stochastic bet hedging behaviour.

Post-vaccine epidemiology of serotype 3 pneumococci identifies transformation inhibition through prophage-driven alteration of a non-coding RNA.

BACKGROUND: The respiratory pathogen Streptococcus pneumoniae (the pneumococcus) is a genetically diverse bacterium associated with over 101 immunologically distinct polysaccharide capsules (serotypes). Polysaccharide conjugate vaccines (PCVs) have successfully eliminated multiple targeted serotypes, yet the mucoid serotype 3 has persisted despite its inclusion in PCV13. This capsule type is predominantly associated with a single globally disseminated strain, GPSC12 (clonal complex 180). METHODS: A genomic epidemiology study combined previous surveillance datasets of serotype 3 pneumococci to analyse the population structure, dynamics, and differences in rates of diversification within GPSC12 during the period of PCV introductions. Transcriptomic analyses, whole genome sequencing, mutagenesis, and electron microscopy were used to characterise the phenotypic impact of loci hypothesised to affect this strain's evolution. RESULTS: GPSC12 was split into clades by a genomic analysis. Clade I, the most common, rarely underwent transformation, but was typically infected with the prophage ϕOXC141. Prior to the introduction of PCV13, this clade's composition shifted towards a ϕOXC141-negative subpopulation in a systematically sampled UK collection. In the post-PCV13 era, more rapidly recombining non-Clade I isolates, also ϕOXC141-negative, have risen in prevalence. The low in vitro transformation efficiency of a Clade I isolate could not be fully explained by the ~100-fold reduction attributable to the serotype 3 capsule. Accordingly, prophage ϕOXC141 was found to modify csRNA3, a non-coding RNA that inhibits the induction of transformation. This alteration was identified in ~30% of all pneumococci and was particularly common in the unusually clonal serotype 1 GPSC2 strain. RNA-seq and quantitative reverse transcriptase PCR experiments using a genetically tractable pneumococcus demonstrated the altered csRNA3 was more effective at inhibiting production of the competence-stimulating peptide pheromone. This resulted in a reduction in the induction of competence for transformation. CONCLUSION: This interference with the quorum sensing needed to induce competence reduces the risk of the prophage being deleted by homologous recombination. Hence the selfish prophage-driven alteration of a regulatory RNA limits cell-cell communication and horizontal gene transfer, complicating the interpretation of post-vaccine population dynamics.