The Poly-γ- d -Glutamic Acid Capsule of Bacillus anthracis Enhances Lethal Toxin Activity

Abstract

The poly-␥-D-glutamic acid (PGA) capsule is one of the major virulence factors of Bacillus anthracis, which causes a highly lethal infectious disease. The PGA capsule disguises B. anthracis from immune surveillance and allows its unimpeded growth in the host. The PGA capsule recently was reported to be associated with lethal toxin (LT) in the blood of experimentally infected animals (M. H. Cho, et al., Infect. Immun. 78:387-392, 2010). The effect of PGA, either alone or in combination with LT, on macrophages, which play an important role in the progression of anthrax disease, has not been thoroughly investigated. In this study, we investigated the effect of PGA on LT cytotoxicity using the mouse macrophage cell line J774A.1. PGA produced a concentrationdependent enhancement of the cytotoxicity of LT on J774A.1 cells through an enhancement in the binding and accumulation of protective antigen to its receptors. The increase of LT activity was confirmed using Western blot analysis, which showed that the combination of PGA and LT produced a greater degree of degradation of mitogen-activated protein kinase kinases and an increased level of the activation of the proform of caspase-1 to its processed form compared to the effects of LT alone. In addition, mice that received a tail vein injection of both PGA and LT had a significantly increased rate of death compared to that of mice injected with LT alone. PGA had no effect when added to cultures or administered to mice in the absence of LT. These results emphasize the importance of PGA in the pathogenesis of anthrax infection.

Anthrax toxin protective antigen integrates poly-γ- d -glutamate and pH signals to sense the optimal environment for channel formation

“…The γ-DPGA was filtered, lyophilized, dissolved in water, adjusted to pH 7.4, and stored at −80°C. Fifteen-to 50 kDa-fragments of γ-DPGA were produced by CapD- (25) or HCl-catalyzed hydrolysis (29). Synthetic HPLC-purified γ-DPGA 2-mer and 10-mer were obtained from Bachem and Biopeptide, respectively.…”

“…A recent report revealed that γ-DPGA and lethal toxin may interact (27). Also, 10-50-kDa fragments of γ-DPGA were reported to enhance LT-mediated killing of J774A.1 cells (29). Whereas the PA monomer, LF, and EF can bind to capsule, the PA prechannel oligomer binds most avidly to the capsule in the nanomolar range.…”

“…The weak 0.3-mM K inhibit value observed for EDTA rules out the involvement of trace metal ions, as EDTA has submicromolar affinity for metal ions. Hydrolysis of γ-DPGA into 10-50-kDa fragments by CapD [from B. anthracis (25)] or HCl (29) [estimated by SDS/PAGE ( Fig. S1D)] reduces the K inhibit value to 100 (±7) μM and 1 (±1) mM, respectively.…”

The Poly-γ-d-Glutamic Acid Capsule Surrogate of the Bacillus anthracis Capsule Is a Novel Toll-Like Receptor 2 Agonist

“…Thus, we maximized the production of the D-glutamate capsule by B. licheniformis using E medium. The PGA from B. licheniformis, which is a nonpathogenic bacterium treated in a biosafety level 1 facility, has been used as a surrogate for PGA from B. anthracis in several studies, including ours (13,27,46,47). In addition, it has been known that B. anthracis PGA capsule was first polymerized on the cell surface to the high-molecular-mass form (Ͼ100 kDa) and then degraded by capsule depolymerase to the lower-molecular-mass capsule, concomitantly releasing from the cell surface (48).…”

“…PGA capsule released from B. anthracis is associated with lethal toxin (LT) in experimental infection models (12). PGA enhances LT-mediated macrophage death (13), indicating that PGA can intensify the LT-induced toxemia that occurs at the terminal stage of anthrax infection.…”