Vaccine 22 (2004) 4124–4131 Attenuated Salmonella typhimurium htrA mutants cause fatal infections in mice deficient in NADPH oxidase and destroy NADPH oxidase-deficient macrophage monolayers Mbithe Mutunga a,1 , Sarah Graham a , Raquel Demarco de Hormaeche a,b , Julie A. Musson a , John H. Robinson c , Pietro Mastroeni b , C.M. Anjam Khan a , Carlos E. Hormaeche a,b,∗ a School of Cell and Molecular Biosciences, The Medical School, University of Newcastle, Framlington Place, Newcastle upon Tyne NE2 4HH, UK b Department of Clinical Veterinary Medicine, Madingley Road, Cambridge CB3 0ES, UK c School of Clinical Medical Sciences (Rheumatology), The Medical School, University of Newcastle, Framlington Place, Newcastle upon Tyne NE2 4HH, UK Received 2 July 2003; accepted 20 October 2003 Available online 10 May 2004 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 2.5 h 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. © 2004 Published by Elsevier Ltd. Keywords: Salmonella typhimurium; Macrophage; Mutant 1. Introduction Production of reactive oxygen species (ROS) is consid- ered an important feature of intracellular bactericidal mech- anisms in phagocytes, and the phagocyte NADPH oxidase has been shown to be a key component in the mechanisms by which phagocytes kill virulent salmonellae [1,5,21,22]. To prevent oxidative damage, bacteria have evolved adaptive responses that result in the expression of numerous genes in response to the stresses exerted by the ROS. These adapta- ∗ Corresponding author. Tel.: +44-1223-764086; fax: +44-1223-337610; mobile: 07867-801115. E-mail address: ceh12@cam.ac.uk (C.E. Hormaeche). 1 Present address: Department of National Blood Service, Histocom- patibility and Immunogenetics, Holland Drive, Barrack Road, Newcastle upon Tyne NE2 4N0, UK tions include production of stress proteins such as HtrA, a periplasmic serine protease [17]. We have reported that Salmonella typhimurium htrA mu- tants show increased sensitivity to oxidative stress in vitro in a manner similar to that described for Escherichia coli htrA mutants, and further, that they are attenuated in mice [3,11,13,18]. S. typhimurium htrA mutants are effective as live vaccines and can be used for delivery of recombinant antigens [2]. Salmonella typhi strains with several attenuat- ing lesions, including htrA, are currently under evaluation as candidate live vaccines for human typhoid fever [12,19,20]. However, despite their attenuation in other species, htrA mutants were not attenuated when administered orally to calves [23]. In our original description of Salmonella htrA mutants [11], we suggested that the in vitro phenotype of increased 0264-410X/$ – see front matter © 2004 Published by Elsevier Ltd. doi:10.1016/j.vaccine.2003.10.053