title: Attenuated Salmonella typhimurium htrA mutants cause fatal infections in mice deficient in NADPH oxidase and destroy NADPH oxidase-deficient macrophage monolayers.
abstract: Salmonella live vaccine strains harbouring mutations in htrA, 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 of Salmonella typhimurium htrA mutants 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, htrA mutants 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 a S. typhimurium SPI-2 mutant known to be virulent in gp9lphox-/- mice. Infection with htrA mutants 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 an aroA live vaccine strain had no effect on either normal or gp9lphox-/- macrophages. Taken collectively, the present results suggest that htrA is somehow involved in resistance to oxidative stress in vivo, with the avirulence of htrA mutants in mice being due to mechanisms which involve NADPH oxidase and suppression of bacterial growth within macrophages.
authors.affiliation: School of Cell and Molecular Biosciences, The Medical School, University of Newcastle, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
authors.forename: Mbithe
authors.initials: M
authors.lastname: Mutunga
authors.forename: Sarah
authors.initials: S
authors.lastname: Graham
authors.forename: Raquel Demarco
authors.initials: RD
authors.lastname: De Hormaeche
authors.forename: Julie A
authors.initials: JA
authors.lastname: Musson
authors.forename: John H
authors.initials: JH
authors.lastname: Robinson
authors.forename: Pietro
authors.initials: P
authors.lastname: Mastroeni
authors.forename: C M Anjam
authors.initials: CM
authors.lastname: Khan
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Hormaeche
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D005798:Genes, Bacterial; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000382:microbiology; D051379:Mice; D018345:Mice, Knockout; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D012481:Salmonella Infections, Animal / Q000382:microbiology*; D022562:Salmonella Vaccines; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D014161:Transduction, Genetic; D014613:Vaccines, Attenuated; D014774:Virulence / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D016877:Oxidants; D022562:Salmonella Vaccines; D014613:Vaccines, Attenuated; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases