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Glucose 6-phosphate dehydrogenase (G6PD) encoded by the zwf gene catalyzes the first enzymatic step in the pentose phosphate cycle. This pathway provides ribose for nucleoside synthesis and reducing equivalents in the form of NADPH for reductive biosynthetic reactions and maintenance of the cellular redox state. Notably, NADPH is the electron source for several reductases that repair oxidative damage and regenerate antioxidant species, including glutathione reductase, thioredoxin reductase, and methionine sulfoxide reductase (10, 25).

In Escherichia coli, zwf expression is subject to at least three forms of regulation: growth rate-dependent regulation (22, 26), induction by the MarA (multiple antibiotic resistance) regulon (16), and induction by the SoxRS (superoxide radical response) regulon (11, 17). SoxRS can augment zwf expression during specific conditions of oxidative stress but is not required for basal levels of expression (11). A chromosomal deletion encompassing the zwf and edd genes has been correlated with increased susceptibility of E. coli to redox-cycling agents, nitric oxide gas, and killing by murine macrophages (15, 19), suggesting that G6PD is both induced by and involved in resistance to the antimicrobial activity of phagocyte-derived reactive oxygen and nitrogen species.

The present study examines the function of G6PD in Salmonella typhimurium, a pathogenic bacterium specifically adapted to survival within phagocytic cells (12, 13, 21). Phagocyte-derived oxygen and nitrogen intermediates have been strongly implicated in host defense against salmonellosis (2, 5, 6, 18), although essential antioxidant and antinitrosative defenses of Salmonella have been incompletely defined. The transcriptional regulator SoxS was recently found to be nonessential for survival of Salmonella within phagocytic cells (9), in contrast to observations for E. coli (19). We constructed and phenotypically characterized a zwf mutant S. typhimurium strain to examine the specific role of G6PD in Salmonella virulence.

S. typhimurium mutants with interruptions of the zwf gene were constructed by two approaches. First, oligonucleotide primers corresponding to nucleotides 525 to 551 and 964 to 988 of the published E. coli zwf sequence (23) were used to amplify an internal fragment of the zwf gene from S. typhimurium ATCC 14028s (12) genomic DNA. The sequenced fragment, which is 87% identical to the corresponding region of the E. coli gene, was ligated into the suicide vector pRR10[trfA 250V] (8). Conjugation of this plasmid from E. coli S17-1 (24) into S. typhimurium ATCC 14028s produced S. typhimurium BL850 carrying a zwf mutation. Interruption of zwf was confirmed by Southern blotting and a biochemical assay of G6PD activity (14). The zwf mutant lacked detectable G6PD, which was restored by introduction of the cloned E. coli zwf gene on plasmid pDR17 (23) in trans (data not shown). Moreover, bacteriophage P22-mediated transduction of a pgi::Tn5 mutation from S. typhimurium CH1021 into the zwf mutant resulted in a strain unable to grow on minimal medium with glucose. An additional zwf mutant derivative of S. typhimurium 14028s was obtained by transduction of zwf::Tn10 from S. typhimurium LT2-derivative DM653 (7), producing strain BL851.

Susceptibility to hydrogen peroxide (H₂O₂) or S-nitrosoglutathione (GSNO) was determined by a disk diffusion method (4). Fifteen microliters of 3% H₂O₂ or 500 mM GSNO was added to a 0.25-in. paper disk placed over a lawn of 10⁶ bacteria on M9 minimal agar with 0.2% glucose. The zone of inhibition after overnight incubation is a measure of susceptibility. We were able to confirm increased susceptibility of E. coli HB351 ([edd-zwf]22) (1) to reactive oxygen or nitrogen intermediates in comparison to wild-type parental strain E. coli W3110 (Fig. 1). However, although the zwf gene on plasmid pDR17 (23) restored wild-type levels of resistance to H₂O₂, pDR17 failed to restore HB351 resistance to GSNO. This suggests that phenotypic analyses of this E. coli strain should be interpreted with caution; it is likely that loci in addition to zwf contribute to the increased susceptibility of HB351 to reactive nitrogen intermediates. Plating of E. coli DR612 (pgi zwf) (23) carrying pDR17 on gluconate-bromthymol blue medium (27) confirmed expression of zwf from plasmid pDR17 (not shown). The S. typhimurium zwf mutant strains BL850 and BL851 were also found to be hypersusceptible to H₂O₂ and GSNO, but introduction of the cloned zwf gene was able to restore wild-type resistance levels to both compounds (Fig. 1), in contrast to the E. coli mutant.

View larger version (44K): [in this window] [in a new window]   FIG. 1.   Susceptibility of E. coli and S. typhimurium strains to hydrogen peroxide (A) and GSNO (B). Susceptibility was determined by a disk diffusion method (4); the zone of inhibition is a measure of susceptibility. pDR17 carries the E. coli zwf gene. Complementation with the cloned zwf gene restores hydrogen peroxide (H2O2) resistance to all strains and GSNO resistance to the S. typhimurium zwf mutant strains but fails to restore GSNO resistance to E. coli HB351 (23).

The virulence of zwf mutant S. typhimurium was determined by intraperitoneal inoculation of 1 × 10³ to 2 × 10³ organisms into 6-week-old female C57BL/6 (Ity^S) mice. By this route of infection, zwf mutant S. typhimurium BL850 was found to be avirulent (Fig. 2). Genetic abrogation of the NADPH phagocyte oxidase in congenic C57BL/6-derived gp91 phox knockout (KO) mice (20) restored 100% mortality following intraperitoneal challenge (mean time to death, 4.4 days). Administration of 2.5% (wt/vol) aminoguanidine (3, 6), an inhibitor of inducible nitric oxide synthase, also restored virulence to zwf mutant S. typhimurium BL850, but mortality occurred significantly later (mean time to death, 17 days). No deaths occurred in C57BL/6 or congenic gp91 phox KO mice receiving intraperitoneal injections of phosphate-buffered saline alone.

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Virulence of wild-type or zwf mutant S. typhimurium in mice. Mortality was determined following intraperitoneal inoculation of 1 × 103 to 2 × 103 wild-type (WT) or zwf mutant (zwf) organisms in C57BL/6 mice (BL6), congenic phox KO mice (phox), or C57BL/6 mice with 2.5% aminoguanidine added to their drinking water (AG). The results of a single experiment are shown (n = 5 mice per group). The virulence study was performed twice, with essentially identical results.

This represents the first demonstration of an essential role of G6PD in microbial pathogenesis. Both reactive oxygen and nitrogen intermediates produced by phagocytic cells appear to contribute to host defense against Salmonella (2, 5, 6, 18). By providing reducing equivalents in the form of NADPH, G6PD encoded by the zwf gene plays an important role in antioxidant and antinitrosative defenses by maintaining the cellular redox state, regenerating reduced thiols, and repairing oxidative or nitrosative damage (10, 25). In this work, construction of G6PD-deficient S. typhimurium mutants, determination of their susceptibility to reactive oxygen and nitrogen species, and measurement of virulence for wild-type and immunodeficient mice demonstrate an essential role of G6PD in resisting antimicrobial effects of the phagocyte respiratory burst and nitric oxide synthase. Oxidative antimicrobial mechanisms are required for an effective early primary immune response to zwf mutant S. typhimurium, but NO-dependent host defenses also appear to play an important role later in the course of infection.