Blythe E. Janowiak

Contribution of Glutathione Peroxidase to the Virulence of Streptococcus pyogenes

ABSTRACT 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.

GroEL as a molecular scaffold for structural analysis of the anthrax toxin pore

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.

Imaging the cell entry of the anthrax oedema and lethal toxins with fluorescent protein chimeras

To 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.

A Semisynthesis Platform for Investigating Structure−Function Relationships in the N-Terminal Domain of the Anthrax Lethal Factor

Many bacterial toxins act by covalently altering molecular targets within the cytosol of mammalian cells and therefore must transport their catalytic moieties across a membrane. The Protective-Antigen (PA) moiety of anthrax toxin forms multimeric pores that transport the two enzymatic moieties, the Lethal Factor (LF) and the Edema Factor, across the endosomal membrane to the cytosol. The homologous PA-binding domains of these enzymes contain N-terminal segments of highly charged amino acids that are believed to enter the pore and initiate N- to C-terminal translocation. Here we describe a semisynthesis platform that allows chemical control of this segment in LF(N), the PA-binding domain of LF. Semisynthetic LF(N) was prepared in milligram quantities by native chemical ligation of synthetic LF(N)(14-28)alphathioester with recombinant N29C-LF(N)(29-263) and compared with two variants containing alterations in residues 14-28 of the N-terminal region. The properties of the variants in blocking ion conductance through the PA pore and translocating across planar phospholipid bilayers in response to a pH gradient were consistent with current concepts of the mechanism of polypeptide translocation through the pore. The semisynthesis platform thus makes new analytical approaches available to investigate the interaction of the pore with its substrates.

Effects of Introducing a Single Charged Residue into the Phenylalanine Clamp of Multimeric Anthrax Protective Antigen

Multimeric pores formed in the endosomal membrane by the Protective Antigen moiety of anthrax toxin translocate the enzymatic moieties of the toxin to the cytosolic compartment of mammalian cells. There is evidence that the side chains of the Phe427 residues come into close proximity with one another in the lumen of the pore and form a structure, termed the Phe clamp, that catalyzes the translocation process. In this report we describe the effects of replacing Phe427 in a single subunit of the predominantly heptameric pore with a basic or an acidic amino acid. Incorporating any charged residue at this position inhibited cytotoxicity ≥1,000-fold in our standard assay and caused strong inhibition of translocation in a planar phospholipid bilayer system. His and Glu were the most strongly inhibitory residues, ablating both cytotoxicity and translocation. Basic residues at position 427 prevented the Phe clamp from interacting with a translocation substrate to form a seal against the passage of ions and accelerated dissociation of the substrate from the pore. Acidic residues, in contrast, allowed the seal to form and the substrate to remain firmly bound, but blocked its passage, perhaps via electrostatic interactions with the positively charged N-terminal segment. Our findings are discussed in relation to the role of the Phe clamp in a Brownian ratchet model of translocation.

Evidence That Histidine Protonation of Receptor-Bound Anthrax Protective Antigen Is a Trigger for Pore Formation

The protective antigen (PA) component of the anthrax toxin forms pores within the low pH environment of host endosomes through mechanisms that are poorly understood. It has been proposed that pore formation is dependent on histidine protonation. In previous work, we biosynthetically incorporated 2-fluorohistidine (2-FHis), an isosteric analogue of histidine with a significantly reduced pK(a) ( approximately 1), into PA and showed that the pH-dependent conversion from the soluble prepore to a pore was unchanged. However, we also observed that 2-FHisPA was nonfunctional in the ability to mediate cytotoxicity of CHO-K1 cells by LF(N)-DTA and was defective in translocation through planar lipid bilayers. Here, we show that the defect in cytotoxicity is due to both a defect in translocation and, when bound to the host cellular receptor, an inability to undergo low pH-induced pore formation. Combining X-ray crystallography with hydrogen-deuterium (H-D) exchange mass spectrometry, our studies lead to a model in which hydrogen bonds to the histidine ring are strengthened by receptor binding. The combination of both fluorination and receptor binding is sufficient to block low pH-induced pore formation.

An approach to characterizing single‐subunit mutations in multimeric prepores and pores of anthrax protective antigen

Heptameric pores formed by the protective antigen (PA) moiety of anthrax toxin translocate the intracellular effector moieties of the toxin across the endosomal membrane to the cytosol of mammalian cells. We devised a protocol to characterize the effects of individual mutations in a single subunit of heptameric PA prepores (pore precursors) or pores. We prepared monomeric PA containing a test mutation plus an innocuous Cys‐replacement mutation at a second residue (Lys563, located on the external surface of the prepore). The introduced Cys was biotinylated, and the protein was allowed to cooligomerize with a 20‐fold excess of wild‐type PA. Finally, biotinylated prepores were freed from wild‐type prepores by avidin affinity chromatography. For the proof of principle, we examined single‐subunit mutations of Asp425 and Phe427, two residues where Ala replacements have been shown to cause strong inhibitory effects. The single‐subunit D425A mutation inhibited pore formation by >104 and abrogated activity of PA almost completely in our standard cytotoxicity assay. The single‐subunit F427A mutation caused ∼100‐fold inhibition in the cytotoxicity assay, and this effect was shown to result from a combination of strong inhibition of translocation and smaller effects on pore formation and ligand affinity. Our results show definitively that replacing a single residue in one subunit of the heptameric PA prepore can inhibit the transport activity of the oligomer almost completely—and by different mechanisms, depending on the specific residue mutated.

Cys−Cys Cross-Linking Shows Contact between the N-Terminus of Lethal Factor and Phe427 of the Anthrax Toxin Pore

Electrophysiological studies of wild-type and mutated forms of anthrax protective antigen (PA) suggest that the Phe clamp, a structure formed by the Phe427 residues within the lumen of the oligomeric PA pore, binds the unstructured N-terminus of the lethal factor and the edema factor during initiation of translocation. We now show by electrophysiological measurements and gel shift assays that a single Cys introduced into the Phe clamp can form a disulfide bond with a Cys placed at the N-terminus of the isolated N-terminal domain of LF. These results demonstrate direct contact of these Cys residues, supporting a model in which the interaction of the unstructured N-terminus of the translocated moieties with the Phe clamp initiates N- to C-terminal threading of these moieties through the pore.

pH effects on binding between the anthrax protective antigen and the host cellular receptor CMG2

The anthrax protective antigen (PA) binds to the host cellular receptor capillary morphogenesis protein 2 (CMG2) with high affinity. To gain a better understanding of how pH may affect binding to the receptor, we have investigated the kinetics of binding as a function of pH to the full‐length monomeric PA and to two variants: a 2‐fluorohistidine‐labeled PA (2‐FHisPA), which is ∼1 pH unit more stable to variations in pH than WT, and an ∼1 pH unit less stable variant in which Trp346 in the domain 2β3‐2β4 loop is substituted with a Phe (W346F). We show using stopped‐flow fluorescence that the binding rate increases as the pH is lowered for all proteins, with little influence on the rate of dissociation. In addition, we have crystallized PA and the two variants and examine the influence of pH on structure. In contrast to previous X‐ray studies, the domain 2β3‐2β4 loop undergoes little change in structure from pH ∼8 to 5.5 for the WT protein, but for the 2‐FHis labeled and W346F mutant there are changes in structure consistent with previous X‐ray studies. In accord with pH stability studies, we find that the average B‐factor values increase by ∼20–30% for all three proteins at low pH. Our results suggest that for the full‐length PA, low pH increases the binding affinity, likely through a change in structure that favors a more “bound‐like” conformation.

Crystallization and preliminary crystallographic analysis of bifunctional γ-glutamylcysteine synthetase-glutatione synthetase from Streptococcus agalactiae

gamma-Glutamylcysteine synthetase-glutathione synthetase (gammaGCS-GS) is a bifunctional enzyme that catalyzes two consecutive steps of ATP-dependent peptide formation in glutathione biosynthesis. Streptococcus agalactiae gammaGCS-GS is a target for the development of potential therapeutic agents. gammaGCS-GS was crystallized using the sitting-drop vapour-diffusion method. The crystals grew to dimensions of 0.3 x 0.2 x 0.2 mm under reducing conditions with 5 mM TCEP. X-ray data were collected to 2.8 A resolution from a tetragonal crystal that belonged to space group I4(1).