SummaryCopper/zinc-cofactored superoxide dismutase ([Cu,Zn]-SOD) has been found in the periplasm of many bacterial species but its biological function is unknown. Here we report the cloning and characterization of sodC, encoding [Cu,Zn]-SOD, from Salmonella typhimurium. The predicted protein sequence shows only 58% identity to Escherichia coli SodC, and from this its chromosomal location and its immediate proximity to a phage gene, sodC, in Salmonella is speculated to have been acquired by bacteriophagemediated horizontal transfer from an unknown donor. A sodC mutant of S. typhimurium was unimpaired on aerobic growth in rich medium but showed enhanced sensitivity in vitro to the microbicidal action of superoxide. S. typhimurium, S. choleraesuis and S. dublin sodC mutants showed reduced lethality in a mouse model of oral infection and persisted in significantly lower numbers in livers and spleens after intraperitoneal infection, suggesting that [Cu,Zn]-SOD plays a role in pathogenicity, protecting Salmonella against oxygen radical-mediated host defences. There was, however, no observable difference compared with wild type in the interaction of sodC mutants with porcine pleural, mouse peritoneal or J774 macrophages in vitro, perhaps reflecting the hierarchical capacity of different macrophage lines to kill Salmonella, the most efficient overwhelming the proposed protective effect of periplasmic SOD.

Meningococcal sodC encodes periplasmic copper-and zinc-cofactored superoxide dismutase (Cu,Zn SOD) which catalyzes the conversion of the superoxide radical anion to hydrogen peroxide, preventing a sequence of reactions leading to production of toxic hydroxyl free radicals. From its periplasmic location, Cu,Zn SOD was inferred to acquire its substrate from outside the bacterial cell and was speculated to play a role in preserving meningococci from the action of microbicidal oxygen free radicals produced in the context of host defense. A sodC mutant was constructed by allelic exchange and was used to investigate the role of Cu,Zn SOD in pathogenicity. Wild-type and mutant meningococci grew at comparable rates and survived equally long in aerobic liquid culture. The mutant showed no increased sensitivity to paraquat, which generates superoxide within the cytosol, but was approximately 1,000-fold more sensitive to the toxicity of superoxide generated in solution by the xanthine/xanthine oxidase system. These data support a role for meningococcal Cu,Zn SOD in protection against exogenous superoxide. In experiments to translate this into a role in pathogenicity, wild-type and mutant organisms were used in an intraperitoneal mouse infection model. The sodC mutant was significantly less virulent. We conclude that periplasmic Cu,Zn SOD contributes to the virulence of Neisseria meningitidis, most likely by reducing the effectiveness of toxic oxygen host defenses.

Superoxide dismutase cofactored by copper and zinc ([Cu,Zn]-SOD) contributes to the protection of opsonized serogroup B Neisseria meningitidis against phagocytosis by human monocytes/macrophages, with sodC mutant organisms being endocytosed in significantly higher numbers than are wild-type organisms. The influence of [Cu,Zn]-SOD was found to be exerted at the stage of phagocytosis, rather than at earlier (modulating surface association) or later (intracellular killing) stages.Superoxide dismutases (SODs) are metalloenzymes that catalyze the conversion of superoxide radical anions to hydrogen peroxide and molecular oxygen by removing key substrates for the formation of highly reactive, toxic small molecules such as peroxynitrite and hydroxyl radical (13,19). There are three classes of SODs in bacteria. Manganese-and iron-cofactored SODs are found in the cytosol, while members of the third class of bacterial SODs-those cofactored by copper and zinc ([Cu,Zn]-SODs; encoded by sodC)-are located in the periplasm or are lipid anchored in the outer envelope (4,5,10,11,21,28). In such a location, [Cu,Zn]-SOD can dismutate superoxide produced outside the bacterial cell by the action of phagocytic cells, for example, and so contribute to bacterial virulence (6,7,16,20,27).Neisseria meningitidis, a common colonist of the human upper respiratory tract, occasionally invades the epithelium by transcytosis (15) and proliferates in the bloodstream to cause life-threatening septicemia and meningitis in previously healthy children. [Cu,Zn]-SOD protects meningococci from the toxicity of exogenous superoxide generated by the oxidation of xanthine in vitro (8), and sodC mutants are significantly attenuated in a systemic mouse model of meningococcal infection (27). [Cu,Zn]-SOD has been shown to contribute to the survival of bacteria within nonprofessional phagocytes (2), so we have hypothesized that sodC contributes to the pathogenic potential of N. meningitidis by conferring resistance to bactericidal mechanisms based on the production of reactive oxygen intermediates that operate against bacteria in the circulation. Here we report the results of our investigation into the interaction of isogenic wild-type bacteria and sodC mutants with fresh human monocytes/macrophages, showing that meningococcal [Cu,Zn]-SOD enhances the capacity of bacteria to avoid internalization by these important contributors to host defense.The serogroup B meningococcal strain MC58 and its nonencapsulated mutant MC58¢2, created by a defined deletion in the capsulation locus, were kindly provided by Mumtaz Virji, University of Bristol, Bristol, United Kingdom (26). The sodC mutant of MC58 has been described previously (27). A sodC mutant of MC58¢2 was constructed by using plasmid pJSK207, which contains the sodC gene interrupted by a kanamycin cassette, as described by Wilks et al. (27). All experiments were performed with bacteria that had been subcultured once only. The comparative amounts of the surface components B capsule, nonsialylated lipopolysaccharide...

SummaryBrazilian purpuric fever (BPF) is a fulminant septicaemic infection of young children, caused by a clonal group of strains of Haemophilus influenzae biogroup aegyptius ( Hae ), an organism previously solely associated with conjunctivitis. Their special capacity to invade from the initial site of conjunctival infection is unexplained. A polymerase chain reaction (PCR)-amplified subtractive hybridization technique was used to identify genes specific to the BPF clonal group. A copy of bacteriophage HP1 and 46 further chromosomal loci were identified in the BPF but not in the conjunctivitis strain of Hae . Sixteen were characterized further, and one -encoding an analogue of the Legionella pneumophila epithelial cell entry-enhancing protein EnhC -was investigated in depth. Two genes, bpf001 and bpf002 , unique to the BPF clonal group were identified between homologues of HI1276 and HI1277 in a complex locus close to H. influenzae genetic island 1, recently identified in pathogenic H. influenzae type b. Bpf001 encodes a protein homologous to EnhC and to the previously uncharacterized product of the meningococcal gene NMB0419 . Functional studies of bpf001 proving intractable, NMB0419 was chosen as a surrogate for investigation and shown to modulate bacterial interaction with monolayers of human respiratory epithelial cells, promoting invasion, the first stage (for Hae ) in the pathogenesis of BPF.

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