Selection for phage resistance reduces virulence of Shigella flexneri

There has recently been an increasing interest in phage therapy as an alternative option for infection management, especially when phages have been shown to cause an evolutionary trade-off between phage resistance and bacterial fitness traits such as virulence. A vast repertoire of virulence factors allows Shigella flexneri to invade gut epithelial cells, replicate intracellularly, and evade the immune system through intercellular spread. In particular, the intercellular spread stage of S. flexneri infection causes tissue damage, contributing to mortality. It has previously been shown that the porin OmpA is necessary for the polar localization of virulence factor IcsA, which polymerizes host actin and allows the bacterium to be motile inside host cells. We hypothesize that a phage which uses OmpA as a receptor to recognize, bind and infect S. flexneri, will select for phage-resistant bacteria that are attenuated for intercellular spread. Here we show that a naturally isolated Myoviridae phage, A1-1, requires OmpA as a receptor. Using phenotypic traits observed from efficiency of plaquing assays, membrane integrity assays, total LPS measurements, MIC measurements as well as phage cross resistance profiles, five A1-1 resistant S. flexneri mutants can be placed into two phenotypic groups. R1 and R2 phenotypically resemble ompA knockouts and R3, R4 and R5 phenotypically resemble LPS-deficient strains. Whole genome sequencing confirms that R1 and R2 have mutations in ompA, while R3, R4 and R5 have a mutation in gmhA, a mutation in gmhC and a 30,000 base pair deletion spanning gmhA, respectively. Bacterial plaque assays confirm that all five phage-resistant mutants are unable to spread intercellularly. Thus, selection for phage A1-1 resistance results in a trade-off between phage resistance and bacterial virulence.