Anthrax Toxin Receptor 2–Dependent Lethal Toxin Killing In Vivo

Anthrax toxin receptors 1 and 2 (ANTXR1 and ANTXR2) have a related integrin-like inserted (I) domain which interacts with a metal cation that is coordinated by residue D683 of the protective antigen (PA) subunit of anthrax toxin. The receptor-bound metal ion and PA residue D683 are critical for ANTXR1-PA binding. Since PA can bind to ANTXR2 with reduced affinity in the absence of metal ions, we reasoned that D683 mutant forms of PA might specifically interact with ANTXR2. We show here that this is the case. The differential ability of ANTXR1 and ANTXR2 to bind D683 mutant PA proteins was mapped to nonconserved receptor residues at the binding interface with PA domain 2. Moreover, a D683K mutant form of PA that bound specifically to human and rat ANTXR2 mediated killing of rats by anthrax lethal toxin, providing strong evidence for the physiological importance of ANTXR2 in anthrax disease pathogenesis

PA^D683N and PA^D683K Bind and Support Intoxication via ANTXR2

<p>CHO-R1.1 cells stably expressing human ANTXR2-EGFP (A), human ANTXR1-EGFP (B), and control CHO-R1.1 cells expressing no receptors (C) were analyzed by flow cytometry after incubation with 100 nM purified PA or PA^D683K proteins, followed by an anti-PA serum, and an APC-conjugated secondary antibody. Triplicate samples of CHO-R1.1 cells stably expressing human ANTXR2-EGFP (D) or human ANTXR1-EGFP (E) were incubated with 10⁻¹⁰ M LF_N-DTA, and with increasing amounts of either purified PA, PA^D683N, or PA^D683K proteins. Cell viability was measured by CellTiter-Glo reagent and is represented as the percentage of signal seen with cells incubated with LF_N-DTA alone (100% viable).</p

ANTXR2 Binding to PA Domains 2 and 4

<p>Ribbon model of the PA-ANTXR2 complex generated with UCSF Chimera [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020111#ppat-0020111-b032" target="_blank">32</a>]. PA domains 2 (D2) and 4 (D4) are shown in dark cyan and aquamarine, respectively, and the remainder of PA is depicted in gray. The ANTXR2 I domain is depicted in pink with its chelated metal ion in green. The ANTXR2 G153 and L154 residues (orange), which contact PA domain 2, and PA D683 residue (blue), which binds the receptor cation, are shown in stick representation. The ANTXR2 152–157 region is shown in yellow.</p

ANTXR2 Contact with PA Domain 2 Confers Sensitivity to PA^D683N-Mediated Intoxication

<div><p>(A) Triplicate samples of CHO-R1.1 cells transiently expressing ANTXR2, or ANTXR2 proteins with alterations in PA contacts to nonconserved ANTXR1 residues were incubated with 10⁻¹⁰ M LF_N-DTA and 10⁻⁷ M PA^D683N, or LF_N-DTA only, and analyzed by flow cytometry. The average percentage of live EGFP-positive cells in the samples with both parts toxin is divided by that in the LF_N-DTA only samples to determine percent cell viability (black bars). Individual samples of the same cells were also incubated with an anti-ANTXR2 antibody, followed by an AlexaFluor-633–conjugated secondary antibody and analyzed by FACS to determine the receptor cell surface expression level. The geometric mean of AlexaFluor-633 fluorescence after staining cells with antibodies (gray bars) is depicted next to the cell viability data for comparison purposes.</p><p>(B) Receptor sequences responsible for contacting PA domain 2 were exchanged between ANTXR1 and ANTXR2 proteins in an analysis similar to (A). Cells expressing ANTXR1^154–159 bound anti-ANTXR2 antibodies, suggesting that an epitope for the polyclonal antibody mapped to this site. Saturable binding studies performed with transfected cells and with increasing amounts of an AlexaFluor-633–labeled monomeric PA protein confirmed that the mutant 154–159 ANTXR1 protein is expressed on the cell surface at levels similar to wild-type ANTXR1 and ANTXR2 (unpublished data).</p></div

PA^D683K Supports Lethal Toxin-Mediated Killing of Rats via rANTXR2

<div><p>(A) Triplicate samples of CHO-R1.1 cells transiently expressing the different receptor-EGFP proteins were incubated with 10⁻¹⁰ M LF_N-DTA and with either 10⁻⁸ M wild-type PA or 10⁻⁷ M PA^D683K or without PA. These amounts of wild-type and mutant PA proteins were used because they gave rise to maximal levels of killing when incubated with cells expressing human ANTXR2 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020111#ppat-0020111-g001" target="_blank">Figure 1</a>D). Cell viability was measured by counting the percentage of live EGFP-positive cells in the sample by flow cytometry and is expressed as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020111#ppat-0020111-g003" target="_blank">Figure 3</a>A legend.</p><p>(B) Triplicate samples of CHO-R1.1 cells transiently expressing ANTXR1^R72,S136-EGFP were incubated with toxin and analyzed as in (A) except that increasing amounts of either the wild-type or mutant PA protein were used.</p><p>(C) Groups of five rats each were injected by jugular vein cannula with 8 μg of LF and either 100 μg of PA^D683K or 10 to 40 μg of PA and time until death postinjection was recorded. One rat in the 40 μg PA group died immediately after injection from pulmonary embolism and was excluded from analysis. Each data point represents a single animal, and the vertical line represents the mean for each group. <i>P</i>-values were determined by Student's unpaired <i>t</i>-test. Control rats injected with PBS all survived (unpublished data).</p></div

CD44⁻ cells are resistant to iota and iota-like toxins versus CD44⁺ cells.

<p>(<b>A</b>) Dose-response of iota toxin on cells with controls consisting of cells in media only. The Y-axis represents the “% control” of F-actin content (Alexa-488 phalloidin stained after 5 h) in intoxicated cells versus controls in media only. (<b>B</b>) Like iota toxin, CD44⁺ RPM cells are also susceptible to <i>C. difficile</i> (CDT) and <i>C. spiroforme</i> (CST) binary toxins. Each assay was done in duplicate and values represent mean +/− standard deviation from three separate experiments.</p

Binding of iota-family B components to purified CD44 in solution.

<p>(<b>A and B</b>) The B component (10 µg) of each toxin was added to CD44-IgG (10 µg in 50 µl) at room temperature for 60 min. Protein A - agarose beads were then added for 5 min at room temperature, gently centrifuged, and washed with buffer. SDS-PAGE sample buffer containing reducing agent was added to the beads, the mixture heated, and protein separated from the beads by centrifugation. Supernatant proteins were then resolved on a 10% gel, transferred onto nitrocellulose, and clostridial B component detected with either 1∶1000 dilutions of rabbit anti-Ib (Panel A) or anti-C2II sera (Panel B). Protein A - peroxidase conjugate was used to detect bound antibody, and following washes, specific bands were visualized with chemiluminescent substrate. <b>(C)</b> Like the CD44-IgG construct, Ib also binds specifically to a CD44-GST construct. A GST version of <i>C. botulinum</i> C3 exoenzyme, used as a negative control, does not bind to Ib in pull-down experiments done similarly for panels A and B, with an exception being the use of glutathione-sepharose (instead of Protein A-agarose) beads.</p