Periplasmic superoxide dismutase in meningococcal pathogenicity

Abstract

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.

FIG. 1

Southern blot of seven meningococcal strains and one gonococcal strain. Genomic DNA (lane 1, MS11 [gonococcal]; lane 2, MC19 [serogroup B]; lane 3, MC54 [serogroup B]; lane 4, MC12 [serogroup A]; lane 5, MC14 [serogroup A]; lane 6, MC58 [serogroup B]; lane 7, C311 [serogroup B]; lane 8, MC50 [serogroup C]) was digested with ClaI and hybridized with a sodC-containing, DIG-labelled HindIII/EcoRI fragment from pJSK204.

FIG. 2

SOD activity of WCE, uninhibited (A) or inhibited with DEDC (B) or H₂O₂ (C), visualized in a 10% nondenaturing gel. Lanes 1, MC58; lanes 2, E. coli DH5α (expressing SodA and SodB); lanes 3, QC779 containing pJSK205. The arrow highlights the position of the cloned meningococcal Cu,Zn SOD in QC779, which has the same electrophoretic mobility as the wild-type enzyme.

FIG. 3

Nondenaturing gel stained to visualize SOD activity. Lane 1, QC779 containing pJSK205; lane 2, MC58 WCE; lane 3, MC58 sodC mutant WCE. The arrow indicates the position of meningococcal Cu,Zn SOD activity.

FIG. 4

Survival of wild-type and sodC mutant N. meningitidis MC58 exposed to 0.7 mU of XO/ml in the presence of 100 μM X. Circles, wild type; squares, sodC mutant; open symbols, control cultures; solid symbols, XO present; dashed line, sodC mutant in the presence of XO plus added exogenous SOD (0.01 U/ml).

FIG. 5

Cumulative deaths of i.p.-infected mice. Fifty mice were infected with 5 × 10⁵ wild-type (continuous line) or sodC mutant (dashed line) meningococci and monitored for 4 days.