Plasticity and maintenance of the virulence plasmid in Shigella flexneri and Shigella sonnei

Shigella is the major cause of human bacillary dysentery worldwide. Shigella flexneri and Shigella sonnei account for >90% of shigellosis globally, and evolved from commensal Escherichia coli following acquisition of a large plasmid, pINV. This plasmid is essential for virulence as it harbours genes encoding a Type three secretion system (T3SS) on a 32 kb pathogenicity island (T3SS PAI). pINV is present in the bacterium in low copy number, and possesses multiple partitioning systems and toxin-antitoxin (TA) systems whose role in plasmid maintenance is unclear. Furthermore, during growth in the laboratory both S. flexneri and S. sonnei spontaneously lose T3SS expression, becoming avirulent. In this work I evaluated how insertion sequences (IS), TA systems and partitioning systems influence the maintenance of virulence in S. flexneri and S. sonnei. I demonstrate that in S. flexneri avirulence mainly results from discrete deletions involving the T3SS PAI mediated by insertion sequences (IS). Repositioning of vapBC, which encodes a TA system essential for pINV maintenance, near the T3SS PAI prevents loss of the PAI, indicating that VapBC maintains the entire plasmid as well as local sequences. IS also mediate reversible integration of pINV into the chromosome, which results in down-regulation of T3SS expression, and reversible avirulence. In contrast, S. sonnei becomes avirulent by mainly losing pINV, which I found is caused by the absence of the two TA systems present in S. flexneri, CcdAB and GmvAT, as well as amino acid substitutions in VapBC. The contribution of the partitioning system ParAB on pINV maintenance is influenced by bacterial growth conditions and the presence of TA systems. However, the function of the second partitioning system StbAB is unknown. The absence of this system in S. sonnei pINV suggests that the two partitioning systems may be redundant. Finally, I have designed and evaluated CRISPR-Cas to induce the loss of virulence or reduced viability in Shigella by targeting T3SS genes and vapBC, thus, validating a novel approach to treat shigellosis.