The roles of the NADPH phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) in host resistance to virulent Salmonella typhimurium were investigated in gp91phox −/−, iNOS −/−, and congenic wild-type mice. Although both gp91phox −/− and iNOS −/− mice demonstrated increased susceptibility to infection with S. typhimurium compared with wild-type mice, the kinetics of bacterial replication were dramatically different in the gp91phox −/− and iNOS −/− mouse strains. Greater bacterial numbers were present in the spleens and livers of gp91phox −/− mice compared with C57BL/6 controls as early as day 1 of infection, and all of the gp91phox −/− mice succumbed to infection within 5 d. In contrast, an increased bacterial burden was detected within reticuloendothelial organs of iNOS −/− mice only beyond the first week of infection. Influx of inflammatory CD11b+ cells, granuloma formation, and serum interferon γ levels were unimpaired in iNOS −/− mice, but the iNOS-deficient granulomas were unable to limit bacterial replication. The NADPH phagocye oxidase and iNOS are both required for host resistance to wild-type Salmonella, but appear to operate principally at different stages of infection.

We recently described the use of selective transposon mutagenesis to generate a series of avirulent mutants of a pathogenic strain of Salmonella typhimurium. Cloning and sequencing of the insertion sites from two of these mutants reveals that both have identical locations within an open reading frame that is highly homologous to a gene, htrA, encoding a heat-shock protein in Escherichia coli. DNA sequence analysis of S. typhimurium htrA reveals the presence of a gene capable of encoding a protein with a calculated Mr of 49316 that has 88.7% protein:protein homology with its E. coli counterpart. In E. coli, lesions in this gene, also known as degP, reduce proteolytic degradation of aberrant periplasmic proteins. Characteristics of the S. typhimurium htrA mutants, 046 and 014, in vivo and in vitro suggested that they are avirulent because of impaired ability to survive and/or replicate in host tissues. In vitro, the S. typhimurium htrA mutants 046 and 014 are not temperature-sensitive but were found to be more susceptible to oxidative stress than the parent, suggesting that they may be less able to withstand oxidative killing within macrophages.

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In the present study we evaluated the role of B cells in acquired immunity to Salmonella infection by using gene-targeted B-cell-deficient innately susceptible mice on a C57BL/6 background (Igh-6 −/−).Igh-6 −/− mice immunized with a live, attenuated aroA Salmonella enterica serovar Typhimurium vaccine strain showed impaired long-term acquired resistance against the virulent serovar Typhimurium strain C5.Igh-6 −/− mice were able to control a primary infection and to clear the inoculum from the reticuloendothelial system. However, Igh-6 −/− mice, unlikeIgh-6 +/+ C57BL/6 controls, did not survive an oral challenge with strain C5 at 4 months after vaccination. Transfer of immune serum did not restore resistance inIgh-6 −/− mice. Total splenocytes and purified CD4+ T cells obtained fromIgh-6 −/− mice 4 months after vaccination showed reduced ability to release Th1-type cytokines (interleukin 2 and gamma interferon) upon in vitro restimulation with serovar Typhimurium soluble cell extracts compared to cells obtained fromIgh-6 +/+ C57BL/6 control mice. Therefore, the impaired resistance to oral challenge with virulent serovar Typhimurium observed in B-cell-deficient mice, which cannot be restored by passive transfer of Salmonella-immune serum, may be in part due to a reduced serovar Typhimurium-specific T-cell response following primary immunization.