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Selection for phage resistance reduces virulence of Shigella flexneri


Kortright, Kaitlyn et al. (2021), Selection for phage resistance reduces virulence of Shigella flexneri, Dryad, Dataset,


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, Shigella flexneri, 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 S. flexneri 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 S. flexneri, should select for phage-resistant mutants with attenuated intercellular spread.  Here we show that a naturally-isolated Myoviridae virus, phage A1-1, requires OmpA as a receptor, and selects for reduced virulence in S. flexneri. 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 ompA knockouts, whereas R3, R4 and R5 were similar to LPS-deficient strains. Whole genome sequencing confirmed that R1 and R2 had mutations in ompA, while R3, R4 and R5 showed mutations in LPS inner-core biosynthesis genes gmhA and gmhC. Bacterial plaque assays in Vero cells confirmed that all five phage-resistant mutants were incapable of intercellular spread. We concluded that selection for S. flexneri resistance to phage A1-1 generally reduced the virulence trait intercellular spread, but this trade-off could be mediated either by mutations in ompA or those in LPS-core genes that likely altered OmpA conformation.