In Vivo-Selected Mutations in Methyl-Directed Mismatch Repair Suppress the Virulence Attenuation ofSalmonella damMutant Strains following Intraperitoneal, but Not Oral, Infection of Naïve Mice

Salmonella enterica serovar Typhimurium that lacks the DNA adenine methylase (Dam) ectopically expresses multiple genes that are preferentially expressed during infection, is attenuated for virulence, and confers heightened immunity in vaccinated hosts. The safety ofdam mutantSalmonella vaccines was evaluated by screening within infected mice for isolates that have an increased capacity to cause disease relative to the attenuated parental strain. Sincedam mutant strains are sensitive to the DNA base analog 2-aminopurine (2-AP), we screened for 2-AP-resistant (2-APr ) isolates in systemic tissues of mice infected withdam mutantSalmonella . Such 2-APr derivatives were isolated following intraperitoneal but not oral administration and were shown to be competent for infectivity via intraperitoneal but not oral infection of naïve mice. These 2-APr derivatives were deficient in methyl-directed mismatch repair and were resistant to nitric oxide, yet they retained the bile-sensitive phenotype of the parentaldam mutant strain. Additionally, introduction of amutH null mutation intodam mutant cells suppressed the inherent defects in intraperitoneal infectivity and nitric oxide resistance, as well as overexpression of SpvB, an actin cytotoxin required forSalmonella systemic survival. These data suggest that restoration of intraperitoneal virulence ofdam mutant strains is associated with deficiencies in methyl-directed mismatch repair that correlate with the production of systemically related virulence functions.