We analyzed the 440-kDa transmembrane pore formed by the protective antigen (PA) moiety of anthrax toxin in the presence of GroEL by negative-stain electron microscopy. GroEL binds both the heptameric PA prepore and the PA pore. The latter interaction retards aggregation of the pore, prolonging its insertion-competent state. Two populations of unaggregated pores were visible: GroEL-bound pores and unbound pores. This allowed two virtually identical structures to be reconstructed, at 25-Å and 28-Å resolution, respectively. The structures were mushroom-shaped objects with a 125-Å-diameter cap and a 100-Å-long stem, consistent with earlier biochemical data. Thus, GroEL provides a platform for obtaining initial glimpses of a membrane protein structure in the absence of lipids or detergents and can function as a scaffold for higher-resolution structural analysis of the PA pore.

Glutathione peroxidases are widespread among eukaryotic organisms and function as a major defense against hydrogen peroxide and organic peroxides. However, glutathione peroxidases are not well studied among prokaryotic organisms and have not previously been shown to promote bacterial virulence. Recently, a gene with homology to glutathione peroxidase was shown to contribute to the antioxidant defenses of Streptococcus pyogenes (group A streptococcus). Since this bacterium causes numerous suppurative diseases that require it to thrive in highly inflamed tissue, it was of interest to determine if glutathione peroxidase is important for virulence. In this study, we report that GpoA glutathione peroxidase is the major glutathione peroxidase in S. pyogenes and is essential for S. pyogenes pathogenesis in several murine models that mimic different aspects of streptococcal suppurative disease. In contrast, glutathione peroxidase is not essential for virulence in a zebrafish model of streptococcal myositis, a disease characterized by the absence of an inflammatory cell infiltrate. Taken together, these data suggest that S. pyogenes requires glutathione peroxidase to adapt to oxidative stress that accompanies an inflammatory response, and the data provide the first demonstration of a role for glutathione peroxidase in bacterial virulence. The fact that genes encoding putative glutathione peroxidases are found in the genomes of many pathogenic bacterial species suggests that glutathione peroxidase may have a general role in bacterial pathogenesis.

␥-Glutamylcysteine synthetase (␥-GCS) and glutathione synthetase (GS), distinct enzymes that together account for glutathione (GSH) synthesis, have been isolated and characterized from several Gram-negative prokaryotes and from numerous eukaryotes including mammals, amphibians, plants, yeast, and protozoa. Glutathione synthesis is relatively uncommon among the Gram-positive bacteria, and, to date, neither the genes nor the proteins involved have been identified. In the present report, we show that crude extracts of Streptococcus agalactiae catalyze the ␥-GCS and GS reactions and can synthesize GSH from its constituent amino acids. The putative gene for S. agalactiae ␥-GCS was identified and cloned, and the corresponding protein was expressed and purified. Surprisingly, it was found that the isolated enzyme catalyzes both the ATP-dependent synthesis of L-␥-glutamyl-L-cysteine from L-glutamate and L-cysteine and the ATP-dependent synthesis of GSH from L-␥-glutamyl-L-cysteine and glycine. This novel bifunctional enzyme, referred to as ␥-GCS-GS, has been characterized in terms of catalytic activity, substrate specificity, and inhibition by GSH, cystamine, and transition state analog sulfoximines. The N-terminal 518 amino acids of ␥-GCS-GS (total M r 85,000) show 32% identity and 43% similarity with E. coli ␥-GCS (M r 58,000), but the C-terminal putative GS domain (remaining 202 amino acids) of ␥-GCS-GS shows no significant homology with known GS sequences. The C terminus (360 amino acids) is, however, homologous to D-Ala, D-Ala ligase (24% identity; 38% similarity), an enzyme having the same protein fold as known GS proteins. These results are discussed in terms of the evolution of GSH synthesis and the possible occurrence of a similar bifunctional GSH synthesis enzyme in other bacterial species.

The action of anthrax toxin relies in part upon the ability of the protective antigen (PA) moiety to form a heptameric pore in the endosomal membrane, providing a portal for entry of the enzymic moieties of the toxin into the cytosol. Pore formation is dependent on a conformational change in the heptameric prepore that occurs in the neutral to mildly acidic pH range, and it has been hypothesized that protonation of one or more histidine residues triggers this transition. To test this hypothesis, we used biosynthetic methods to incorporate the unnatural amino acid analogue 2-fluorohistidine (2-FHis) into PA. 2-FHis is isosteric with histidine but resists protonation at physiological pH values due to a dramatically reduced side-chain pKa ( approximately 1). We found that 2-FHis-labeled PA was biologically inactive, as judged by its inability to deliver a model intracellular effector, LFN-DTA, to the cytosol of CHO-K1 cells. However, whereas 2-FHis blocked a conformational transition in the full-length PA83 protein in the pH 5-6 range, the pH dependence of prepore-to-pore conversion of (PA63)7 was unchanged from the wild-type protein, implying that this conversion is not dependent on His protonation. Consistent with this result, the labeled, trypsin-activated PA was able to permeabilize liposomes to K+ and retained pore-forming activity in planar phospholipid bilayers. The pores in planar bilayers were incapable, however, of translocating a model ligand in response to a transmembrane pH gradient or elevated voltage. The results indicate that protonation of residues other than His, presumably Glu and/or Asp side chains, triggers pore formation in vitro, but His residues are nonetheless important for PA functioning in vivo.

SummaryTo investigate the cell entry and intracellular trafficking of anthrax oedema factor (EF) and lethal factor (LF), they were C-terminally fused to the enhanced green fluorescent protein (EGFP) and monomeric Cherry (mCherry) fluorescent proteins. Both chimeras bound to the surface of BHK cells treated with protective antigen (PA) in a patchy mode. Binding was followed by rapid internalization, and the two anthrax factors were found to traffic along the same endocytic route and with identical kinetics, indicating that their intracellular path is essentially dictated by PA. Colocalization studies indicated that anthrax toxins enter caveolin-1 containing compartments and then endosomes marked by phoshatidylinositol 3-phoshate and Rab5, but not by early endosome antigen 1 and transferrin. After 40 min, both EF and LF chimeras were observed to localize within late compartments. Eventually, LF and EF appeared in the cytosol with a time-course consistent with translocation from late endosomes. Only the EGFP derivatives reached the cytosol because they are translocated by the PA channel, while the mCherry derivatives are not. This difference is attributed to a higher resistance of mCherry to unfolding. After translocation, LF disperses in the cytosol, while EF localizes on the cytosolic face of late endosomes.