Virulent Salmonella typhimurium has two periplasmic Cu, Zn-superoxide dismutases

Abstract

Periplasmic Cu, Zn-cofactored superoxide dismutase (SodC) protects Gram-negative bacteria from exogenous oxidative damage. The virulent Salmonella typhimurium strain ATCC 14028s has been found to contain two discrete periplasmic Cu, Zn-SOD enzymes that are only 57% identical at the amino acid level. SodCI is carried by a cryptic bacteriophage, and SodCII is closely related to the Cu, Zn-superoxide dismutase of Escherichia coli. All Salmonella serotypes appear to carry the sodCII locus, but the phage-associated sodCI gene is found only in certain strains belonging to the most highly pathogenic serotypes. Expression of either sodC locus appears to be enhanced during stationary phase, but only sodCII is regulated by the alternative sigma factor sigmas (RpoS). Mutants lacking both sodC genes are less lethal for mice than mutants possessing either sodC locus alone, indicating that both Cu, Zn-SOD enzymes contribute to Salmonella pathogenicity. The evolutionary acquisition of an additional sodC gene has contributed to the enhanced virulence of selected Salmonella strains.

Figure 1

Two-dimensional SDS/PAGE analysis of SodCII (ASP71) in wild-type Salmonella and a sodCII mutant. Wild-type UK1 (A and B) and sodCII mutant JF3912 (C and D) were grown to logarithmic phase at pH 7.7 in minimal EG medium. Cells were either processed directly for two-dimensional SDS/PAGE (A and C) or acid shocked for 1 hr at pH 4.4 before processing (B and D). The arrow indicates the position of SodCII. The panels show equivalent small areas of larger gels.

Figure 2

Sequence Comparison of E. coli and S. typhimurium SodC proteins. The two S. typhimurium SodC proteins share 57% identity. S. typhimurium SodCII is 82% identical to E. coli SodC, but SodCI is only 54% identical to the E. coli protein. Each SodC possesses a typical hydrophobic NH₂ terminus signal sequence (underlined), consistent with secretion of Cu, Zn-SOD into the periplasmic space.

Figure 3

Presence of sodCI and spvR in Salmonella Reference Collection B Strains. Strains and evolutionary tree are modified from ref. , with permission. Strains carrying sodCI are indicated in bold type. Strains carrying spvR are indicated by ∗. Abbreviations of Salmonella serotypes: Ag, S. agona; An, S. anatum; Ba, S. brandenburg; Cs, S. cholerasuis; De, S. derby; Di, S. duisburg; Dt, S. decatur; Du, S. dublin; Em, S. emek; En, S. enteritidis; Ga, S. gallinarum; Ha, S. haifa; He, S. heidelberg; Id, S. indiana; In, S. infantis; Mi, S. miami; Mo, S. montevideo; Mu, S. muenchen; Np, S. newport; Pa, S. paratyphi A; Pb, S. paratyphi B; Pc, S. paratyphi C; Pn, S. panama; Pu, S. pullorum; Re, S. reading; Ru, S. rubislaw; Se, S. sendai; Sf, S. senftenberg; Sp, S. saintpaul; St, S. stanley; Sv, S. stanleyville; Sw, S. schwarzengrund; Th, S. thompson; Tm, S. typhimurium; Tp, S. typhi; Ts, S. typhisuis; Wi, S. wien. [Reproduced with permission from ref. (Copyright 1993, Society for General Microbiology)].

Figure 4

Effects of growth phase and σ^S on sodCI and sodCII expression. Expression of both P_sodC1-lacZ and P_sodCII-lacZ fusions increases during the transition from exponential growth to stationary phase. However, expression of P_sodC1-lacZ is σ^S-independent and subsequently declines, whereas σ^S -dependent expression of P_sodCII-lacZ is sustained. β-Galactosidase activity in Miller units (32) from S. typhimurium 14028s (wild-type) or SF1005 (rpoS) carrying P_sodC1-lacZ or P_sodCII-lacZ fusions is shown in A. The OD₆₀₀ of S. typhimurium 14028s carrying P_sodC1-lacZ is shown in B; growth curves were highly similar for all strains tested.

Figure 5

Mortality of C57BL/6 or C3H/HeN mice infected with sodC mutant S. typhimurium. Intraperitoneal inocula of wild-type, sodCI mutant, sodCII mutant, or sodCI/sodCII double mutant S. typhimurium were administered to C57BL/6 (Ity^s) mice (A) or C3H/HeN (Ity^r) mice (B). Mortality was recorded daily. The virulence of S. typhimurium carrying mutations in single sodC loci was minimally attenuated in susceptible C57BL/6 mice, but the loss of both sodC loci had a marked effect on virulence. In contrast, mutations in either or both sodC loci significantly reduced virulence in resistant C3H/HeN mice.