Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase

Abstract

Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to lambda phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria. Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo.

Figure 1

(A) Map of S. typhimurium sodC region. An ORF encoding a predicted protein with homology to the Ail protein of Yersinia enterocolitica and ORFs highly homologous to genes that encode bacteriophage λ tail proteins are designated. (B) Sequence alignment of S. typhimurium and E. coli SodC proteins. The full-length SodC proteins from S. typhimurium and E. coli (22) are 54% identical at the amino acid level. The S. typhimurium sodC gene encodes a protein of 18.3 kDa predicted size. The protein possesses a typical hydrophobic NH₂ terminus signal sequence (underlined); cleavage would produce a mature protein of 16.3 kDa predicted size.

Figure 2

Confirmation of SodC enzymatic activity. Sonicated cell extracts from E. coli overexpressing the S. typhimurium sodC gene (first and third lanes) or carrying the plasmid vector alone (second and fourth lanes) were subjected to SOD activity gel analysis (13). The left arrow designates SodC, and right arrows designate Fe- and Mn-SOD proteins, respectively. Inhibition of SodC activity by 1 mM sodium cyanide is characteristic of Cu,Zn-SODs (14).

Figure 3

In vitro susceptibility of sodC mutant S. typhimurium to extracellular superoxide and NO. Wild-type or sodC mutant (MF1005) bacteria in PBS with 250 μM hypoxanthine were treated with 0.1 unit/ml xanthine oxidase (XO), 1 mM SPER/NO (NO), or the combination of xanthine oxidase and SPER/NO (XONO) as described in Materials and Methods.

Figure 4

Susceptibility of sodC mutant S. typhimurium to killing by murine peritoneal macrophages. IFN-γ-stimulated periodate-elicited peritoneal exudate cells from C3H/HeN mice were used to kill wild-type (WT) or sodC mutant (MF1005) S. typhimurium. N^G-d-monomethyl arginine (250 μM) was added to control wells, 250 μM N^G-l-monomethyl arginine (MMA) was added to inhibit NO synthase, and 250 μM acetovanillone (AV) was added to inhibit the phagocyte NADPH-oxidase.

Figure 5

(A) Virulence of sodC mutant S. typhimurium in wild-type and respiratory burst-deficient mice. C57BL/6 (Ity^s) and congenic phox-91 ko mice (19) were intraperitoneally challenged with wild-type or sodC mutant (MF1005) S. typhimurium. The time to death for wild-type and sodC mutant organisms was significantly different in C57BL/6 mice (P < 0.01) but not in phox ko (knock-out) mice (P > 0.05). (B) Virulence of sodC and katEG mutant S. typhimurium and the effect of NO synthase inhibition. C3H/HeN (Ity^r) mice were intraperitoneally challenged with wild-type, sodC mutant (MF1005), or katEG mutant (XF1001) S. typhimurium. Separate groups of mice received drinking water treated with 2.5% (wt/vol) aminoguanidine (25) (AG), an inhibitor of inducible NO synthase. The survival of untreated mice challenged with sodC mutant organisms was significantly different from the other experimental groups (P < 0.01) after adjustment for multiple comparisons.

Figure 6

Proposed mechanism of SodC-mediated protection against products of NO synthase and the respiratory burst NADPH-oxidase. NO·, nitric oxide; O₂, superoxide; H₂O₂, hydrogen peroxide; ·OH, hydroxyl radical; SodC, periplasmic superoxide dismutase; KatG, periplasmic catalase.