Anthrax toxin-induced rupture of artificial lipid bilayer membranes.

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

Engineering an Fc-Fusion of a Capsule Degrading Enzyme for the Treatment of Anthrax.

Polymers of d-glutamic acid (PDGA) form the capsule of the highly virulent Ames strain of B. anthracis. PDGA is antiphagocytic and weakly immunogenic; it enables the bacteria to evade the innate immune responses. CapD is an enzyme that catalyzes the covalent anchoring of PDGA. CapD is an Ntn-amido hydrolase that utilizes an internal Thr-352 as its nucleophile and general acid and base. An internal cleavage produces a free N-terminal Thr-352 and a short and long polypeptide chain. The chains were circularly permuted (CP) to move Thr-352 to the N-terminus of the polypeptide. We previously showed that a branched PEG-CapDS334C-CP could protect mice (80% survival) against a 5 LD50 challenge with B. anthracis Ames without the use of antibiotics, monoclonals, or vaccines. In attempts to improve the in vivo circulation time of CapD and enhance its avidity to its polymeric substrate, an Fc-domain of a mouse IgG1 was fused to CapDS334C-CP and the linker length and sequence were optimized. The resulting construct, Fc-CapDS334C-CP, then was pegylated with a linear 2 kDa mPEG at S334C to produce mPEG-Fc-CapDS334C-CP. Interestingly, the fusion of the Fc-domain and incorporation of the S334C mutation imparted acid stability, but slightly reduced the kcat (∼ 2-fold lower). In vivo, the measured protein concentration in sera was higher for the Fc-fusion constructs compared to the mPEG-Fc-CapDS334C-CP. However, the exposure calculated from measured sera enzymatic activity was higher for the mPEG-CapDS334C-CP. The pegylated Fc-fusion was less active than the PEG-CapDS334C-CP, but detectable in sera at 24 h by immunoblot. Here we describe the engineering of a soluble, active, pegylated Fc-fusion of B. anthracis CapD (mPEG-Fc-CapD-CP) with activity in vitro, in serum, and on encapsulated bacteria.

Treatment of experimental anthrax with pegylated circularly permuted capsule depolymerase.

Anthrax is considered one of the most dangerous bioweapon agents, and concern about multidrug-resistant strains has led to the development of alternative therapeutic approaches that target the antiphagocytic capsule, an essential virulence determinant of Bacillus anthracis, the causative agent. Capsule depolymerase is a γ-glutamyltransferase that anchors the capsule to the cell wall of B. anthracis. Encapsulated strains of B. anthracis can be treated with recombinant capsule depolymerase to enzymatically remove the capsule and promote phagocytosis and killing by human neutrophils. Here, we show that pegylation improved the pharmacokinetic and therapeutic properties of a previously described variant of capsule depolymerase, CapD-CP, when delivered 24 hours after exposure every 8 hours for 2 days for the treatment of mice infected with B. anthracis. Mice infected with 382 LD50 of B. anthracis spores from a nontoxigenic encapsulated strain were completely protected (10 of 10) after treatment with the pegylated PEG-CapD-CPS334C, whereas 10% of control mice (1 of 10) survived with control treatment using bovine serum albumin (P < 0.0001, log-rank analysis). Treatment of mice infected with five LD50 of a fully virulent toxigenic, encapsulated B. anthracis strain with PEG-CapD-CPS334C protected 80% (8 of 10) of the animals, whereas 20% of controls (2 of 10) survived (P = 0.0125, log-rank analysis). This strategy renders B. anthracis susceptible to innate immune responses and does not rely on antibiotics. These findings suggest that enzyme-catalyzed removal of the capsule may be a potential therapeutic strategy for the treatment of multidrug- or vaccine-resistant anthrax and other bacterial infections.

Immunogenicity and protective efficacy of Bacillus anthracis poly-gamma-D-glutamic acid capsule covalently coupled to a protein carrier using a novel triazine-based conjugation strategy.

The capsular polypeptide of Bacillus anthracis is composed of a unique polyglutamic acid polymer in which D-glutamate monomers are joined by gamma-peptidyl bonds. The capsule is poorly immunogenic, and efforts at exploiting the polymer for vaccine development have focused on increasing its inherent immunogenicity through chemical coupling to immune-stimulating protein carriers. The usual strategy has employed carbodiimide-based condensing reagents for activation of free alpha-carboxyl groups, despite reports that this chemistry may lead to chain scission. We have purified the high molecular mass capsule to >95% homogeneity and have demonstrated that the polymer contains >99% poly-gamma-D-glutamic acid. The predominant structure of the polymer as assessed by circular dichroism and multiangle laser light scattering was unordered at near-neutral pH. We investigated the effects of various activation chemistries, and we demonstrated that carbodiimide treatment under aqueous conditions results in significant cleavage of the gamma-peptidyl bond, whereas scission is significantly reduced in nonaqueous polar solvents, although undesired side chain modification was still observed. An activation chemistry was developed using the triazine-based reagent 4-(4,6-dimethoxy (1,3,5)triazin-2-yl)-4-methylmorpholinium chloride, which allowed for controlled and reproducible derivatization of alpha-carbonyls. In a two-pot reaction scheme, activated capsule was derivatized with a sulfhydryl-reactive heterobifunctional moiety and was subsequently coupled to thiolated carrier protein. This conjugate elicited very high capsule-specific immune titers in mice. More importantly, mice immunized with conjugated capsule exhibited good protection against lethal challenge from a virulent B. anthracis strain in two models of infection. We also showed, for the first time, that treatment of capsule with carbodiimide significantly reduced recognition by capsule-specific antisera concurrent with the reagent-induced reduction of polymer mass. The data suggested that for vaccine development, maintenance of the high mass of the polymer may be important.