Global population structure and phase variation of serotype 12F Streptococcus pneumoniae following the introduction of pneumococcal conjugate vaccine

Abstract Background After the global deployment of pneumococcal conjugate vaccines (PCVs), serotype 12F has become the predominant serotype responsible for invasive pneumococcal disease (IPD) worldwide. As PCVs that include serotype 12F are gradually introduced, we aim to characterise the global population structure and genetic diversity of the 12F capsule locus using whole-genome sequencing. Capsule variants with vaccine evasion potential were further investigated by functional experiments. Methods A global collection of pneumococcal serotype 12F genomes (n=806) from 37 countries across six continents were included in this study. To characterise the serotype 12F population, Global Pneumococcal Sequence Cluster (GPSC), in silico serotype, and antimicrobial resistance profile were inferred from whole-genome data for each isolate. The capsule biosynthesis (cps) locus was analysed for gene content variations that could alter polysaccharide capsule production or structure, thereby influencing recognition by vaccine-induced antibodies. These isolates were further investigated by assessing their capsule production using immunofluorescence assays and its susceptibility to vaccine-elicited antibody killing by opsonophagocytosis assays. Findings The global increase in serotype 12F was driven by both distinct pneumococcal lineages across different continents, and a globally-disseminated and multidrug-resistant lineage GPSC26. We identified six capsule variants in nine isolates that had disruptive mutations in cps genes including wze, wcil, wciJ and fnlA. Most (6/9) of the disruptive mutations were a result of strand-slippage mutations. A convergent strand-slippage mutation disrupting the glycosyltransferase gene wciJ was identified in four isolates from distinct lineages and countries. Despite the truncation, three of four isolates with available Quellung typing results still identified them as 12F, indicating the production of the capsule. We then created a genetically engineered lab strain with wciJ knockout and complemented with wciJ containing the strand-slipppage mutation. The knockout strain did not produce any capsule. In contrast, the lab strain with wciJ containing the strand-slippage mutation produced a mixed population of encapsulated and non-encapsulated pneumococci, even within the same chain of pneumococcal cells. This observation indicated encapsulated subpopulation possesses a functional WciJ and rapidly reversible strand-slippage mutation during replication. Opsonophagocytosis assays indicated that the clinical 12F strain with strand-slippage mutation in wciJ exhibited reduced susceptibility to vaccine-elicited serum killing, compared to a genetically closely related 12F clinical strain with an intact wciJ. However, substantial inter-individual antisera variation limits definitive interpretation. Interpretation Our work revealed the global rise of serotype 12F pneumococci has been driven by both regional-specific lineages, and a globally-disseminated and multidrug-resistant lineage GPSC26. We demonstrated that strand-slippage mutation is one of the major drivers of serotype 12F capsule variants and represents a novel mechanism enabling reversible on-off switching of capsule production. The ability to switch off capsule expression in a subpopulation may enable evasion of antibody-mediated killing but increase susceptibility to innate immune clearance. Funding Bill & Melinda Gates Foundation, Wellcome Sanger Institute, and the US Centers for Disease Control and Prevention.