Selection for phage resistance reduces virulence of Shigella flexneri

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.