Selection for phage resistance reduces virulence of Shigella flexneri

<p>There is increasing interest in phage therapy as an alternative to chemical antibiotics for treating bacterial infections, especially using phages that select for evolutionary trade-offs between increased phage resistance and decreased fitness traits such as virulence in target bacteria. A vast repertoire of virulence factors allows the opportunistic bacterial pathogen, <i>Shigella flexneri</i>, to invade human gut epithelial cells, to replicate intracellularly, and to evade host immunity through intercellular spread; the latter is an infection stage that causes tissue damage, contributing to host mortality. It is previously shown that outer membrane porin OmpA of <i>S. flexneri</i> is necessary for polar localization of virulence factor IcsA, which polymerizes host actin and results in bacterial motility within infected cells. We hypothesized that a phage which uses OmpA as a receptor to recognize, bind and infect <i>S. flexneri</i>, should select for phage-resistant mutants with attenuated intercellular spread.  Here we show that a naturally-isolated <i>Myoviridae</i> virus, phage A1-1, requires OmpA as a receptor, and selects for reduced virulence in <i>S. flexneri</i>. We characterized five phage-resistant mutants by measuring their phenotypic changes relative to wildtype bacteria in various traits: cell-membrane permeability, total lipopolysaccharide (LPS), sensitivity to four antibiotics, and susceptibility to infection by phages T7 and 60B. Results separated the mutants into two groups: R1 and R2 phenotypically resembled <i>ompA</i> knockouts, whereas R3, R4 and R5 were similar to LPS-deficient strains. Whole genome sequencing confirmed that R1 and R2 had mutations in <i>ompA</i>, while R3, R4 and R5 showed mutations in LPS inner-core biosynthesis genes <i>gmhA</i> and <i>gmhC</i>. Bacterial plaque assays in Vero cells confirmed that all five phage-resistant mutants were incapable of intercellular spread. We concluded that selection for <i>S. flexneri</i> resistance to phage A1-1 generally reduced the virulence trait intercellular spread, but this trade-off could be mediated either by mutations in <i>ompA</i> or those in LPS-core genes that likely altered OmpA conformation.</p>