Virtual Poster 21st Lancefield International Symposium for Streptococci and Streptococcal Diseases 2022

Streptococcus suis is an emerging zoonotic pathogen. A unique zoonotic serotype 2 clonal complex (CC) 20 clade has diverged from a non-zoonotic clade. The zoonotic CC20 clade acquired a phase-variable type I restriction-modification system (SsuCC20p). In Streptococcus pneumoniae a phase-variable type I restriction-modification system regulates virulence. Here we characterize SsuCC20p and assess its potential role in virulence.

Phase-variable hsdS alleles were quantified by fragment length analysis of endonuclease digested fluorescently labelled PCR products amplified from genomic DNA of strain 861160 (CC20). Mutants containing single hsdS alleles (‘locked’) were generated. Long read sequencing of isolated genomic DNA was used to quantify hsdS allele distribution in the wildtype and to analyze genome methylation patterns in wildtype and locked mutant strains. Virulence of locked hsdS mutants and wildtype was assessed in the zebrafish larvae infection model.

Within 861160, three unique and functional hsdS alleles were identified and expression of hsdS alleles was confirmed by RT-qPCR. Deletion of the recombinase xerD that is present within the SsuCC20p locus halted phase variation. Methylome analysis of the WT and hsdS knockout genomes showed that SsuCC20p actively methylates the genome and identified two different methylation patterns (GCAN⁵GTC/GACN⁵TGC and GCAN⁵CTC/GAGN⁵TGC). Experimental data in zebrafish larvae infection show differences in zebrafish mortality between locked hsdS mutants infections.

SsuCC20p phase variability is xerD dependent and three different hsdS alleles can be detected within a single isolate. SsuCC20p is expressed and actively methylates the S. suis genome. Phase variation of SsuCC20p results in differential genome methylation and potential changes in virulence.