AttenuatedSalmonella typhimurium htrAmutants cause fatal infections in mice deficient in NADPH oxidase and destroy NADPH oxidase-deficient macrophage monolayers

Salmonellalive vaccine strains harbouring mutations inhtrA, a stress protein gene, display increased susceptibility to oxidative stress in vitro. This is believed to be connected to their reduced virulence, perhaps due to impaired survival inside phagocytes, although this has never been formally proven. We report that the in vitro phenotype of increased susceptibility to oxidative stress ofSalmonella typhimurium htrAmutants newly prepared by transduction is rapidly lost on subculture, with the mutants becoming as resistant as the parent for reasons that remain unclear. However, despite this change,htrAmutants are still attenuated in normal mice. In contrast, they were found to be lethal for gene targeted gp91phox-/-mice deficient in NADPH oxidase, as was aS. typhimuriumSPI-2 mutant known to be virulent in gp9lphox-/-mice. Infection withhtrAmutants caused little damage to primary bone marrow macrophage cultures from normal mice; conversely, they caused extensive damage to macrophages from gp9lphox-/-mice, with more than 60% reduction in cell numbers 2.5h after being infected. The parental wild type strain similarly caused extensive damage to macrophages from both normal and gp9lphox-/-mice, whereas anaroAlive vaccine strain had no effect on either normal or gp9lphox-/-macrophages. Taken collectively, the present results suggest thathtrAis somehow involved in resistance to oxidative stress in vivo, with the avirulence ofhtrAmutants in mice being due to mechanisms which involve NADPH oxidase and suppression of bacterial growth within macrophages.