Poly-γ-Glutamate Capsule-Degrading Enzyme Treatment Enhances Phagocytosis and Killing of EncapsulatedBacillus anthracis

Abstract

The poly-␥-D-glutamic acid capsule confers antiphagocytic properties on Bacillus anthracis and is essential for virulence. In this study, we showed that CapD, a ␥-polyglutamic acid depolymerase encoded on the B. anthracis capsule plasmid, degraded purified capsule and removed the capsule from the surface of anthrax bacilli. Treatment with CapD induced macrophage phagocytosis of encapsulated B. anthracis and enabled human neutrophils to kill encapsulated organisms. A second glutamylase, PghP, a ␥-polyglutamic acid hydrolase encoded by Bacillus subtilis bacteriophage ⌽NIT1, had minimal activity in degrading B. anthracis capsule, no effect on macrophage phagocytosis, and only minimal enhancement of neutrophil killing. Thus, the levels of both phagocytosis and killing corresponded to the degree of enzyme-mediated capsule degradation. The use of enzymes to degrade the capsule and enable phagocytic killing of B. anthracis offers a new approach to the therapy of anthrax.

Treatment of Experimental Anthrax with Recombinant Capsule Depolymerase

“…The capD gene encoding amino acids 28 to 528 was expressed in pET15b (EMD Biosciences, San Diego, CA) as previously described (39). An additional construct of the capD gene encoding amino acids 43 to 528 was cloned into pTYB12 and expressed using the IMPACT protein purification system (New England BioLabs, Beverly, MA).…”

“…Endotoxin was removed from protein preparations with EndoTrap Red columns (Cambrex Corp., Walkersville, MD) or Detoxi-Gel endotoxin removal columns (Pierce Biotechnology, Inc., Rockford, IL). To evaluate the polyglutamate capsule-degrading activity of recombinant CapD proteins, purified B. anthracis capsule (500 g/ml) prepared as previously described (6) was incubated for 1 h at 37°C in Dulbecco's modified Eagle's medium containing CapD amino acids 28 to 528 (CapD 28-528; 50 g/ml) or CapD amino acids 43 to 528 (CapD 43-528; 50 g/ml), and the degraded capsule products were analyzed on a 1% agarose gel and visualized with StainsAll (Sigma, St. Louis, MO) as described previously (39). In some experiments CapD was inactivated by incubation at 75°C for 30 min.…”

“…Human neutrophils were purified from unvaccinated volunteers by Ficoll-Hypaque density gradient centrifugation followed by dextran sedimentation and used to examine CapD-mediated neutrophil killing as previously described (39). Briefly, purified human neutrophils were resuspended in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum (DF10), recombinant CapD (20 g/ml), and 10% human AB serum as a source of complement.…”

“…While neutrophils are reported to have a variable role in resistance to anthrax spore infection in mice (7), mouse neutrophils have been shown to kill unencapsulated B. anthracis (42). It has recently been demonstrated that human neutrophils kill encapsulated B. anthracis, although the killing was modest and predominantly extracellular (28), and we have previously reported that they can reduce viability of decapsulated bacilli by up to 3 logs while having minimal activity against encapsulated bacilli (39). The B. anthracis capsule (pX02) and toxin (pX01) plasmids encode factors that enhance survival in the host, likely in part by subverting the bactericidal activity of neutrophils.…”

“…The enzyme is thought to contribute to the virulence of B. anthracis by releasing low-molecular-weight capsule from the surface of bacilli (27) and was recently reported to anchor polyglutamate to the peptidoglycan layer (5). In our previous work we demonstrated that CapD treatment of encapsulated anthrax bacilli efficiently degrades the capsule, removing it from the surface of the bacilli and thereby rendering the bacteria susceptible to phagocytic killing (39). In this study, using two mouse models of infection we show that administration of CapD protected mice from anthrax, likely by promoting phagocytic killing of bacilli in vivo.…”