Attenuation of bacterial virulence by quorum sensing-regulated lysis

Genetically attenuated pathogenic bacteria have been extensively considered as vaccine candidates. However, insufficient attenuation has been a frequent limitation of this approach. Many pathogens use quorum sensing to escape host defense mechanism. Here, we hypothesized that quorum sensing can be manipulated to diminish pathogenesis. To test this hypothesis, we modified the quorum sensing circuitry of a live cholera vaccine strain to add a second layer of attenuation. Attenuation resulted from the expression of phage PhiX174 lysis gene E on a balanced lethal plasmid from the quorum sensing-regulated luxC promoter. For conditional expression of quorum sensing and positive selection in vivo, the host strain was deleted of its cqsA and thyA genes encoding cholera autoinducer 1 (CAI-1) synthase and thymidylate synthase, respectively. A recombinant cqsA gene expressed from the cholera toxin (CT) promoter and an active thyA gene was provided in trans. The resulting strain expressed CAI-1 in AKI cultures (CT permissive condition) but not in LB medium. Additionally, it expressed elevated biofilm in LB medium compared to AKI conditions where CAI-1 is synthesized to repress biofilm formation. Induction of lysis gene E by quorum sensing restricted growth to a lower cell density in AKI medium, the suckling mouse intestine or LB supplemented with exogenous CAI-1. Microscopic examination revealed the presence of Vibrio cholerae ghost cells at high cell density. Lysis was accompanied by the release of intracellular β-galactosidase to the culture medium. We conclude that it is possible to manipulate quorum sensing to attenuate a live vaccine vector and restrict its shedding to the environment and diminish its subsequent dissemination.