The capsule of Bacillus anthracis protects it from the bactericidal activity of human defensins and other cationic antimicrobial peptides.

During infection, Bacillus anthracis bacilli encounter potent antimicrobial peptides (AMPs) such as defensins. We examined the role that B. anthracis capsule plays in protecting bacilli from defensins and other cationic AMPs by comparing their effects on a fully virulent encapsulated wild type (WT) strain and an isogenic capsule-deficient capA mutant strain. We identified several human defensins and non-human AMPs that were capable of killing B. anthracis. The human alpha defensins 1-6 (HNP-1-4, HD-5-6), the human beta defensins 1-4 (HBD-1-4), and the non-human AMPs, protegrin, gramicidin D, polymyxin B, nisin, and melittin were all capable of killing both encapsulated WT and non-encapsulated capA mutant B. anthracis. However, non-encapsulated capA mutant bacilli were significantly more susceptible than encapsulated WT bacilli to killing by nearly all of the AMPs tested. We demonstrated that purified capsule bound HBD-2, HBD-3, and HNP-1 in an electrophoretic mobility shift assay. Furthermore, we determined that the capsule layer enveloping WT bacilli bound and trapped HBD-3, substantially reducing the amount reaching the cell wall. To assess whether released capsule might also play a protective role, we pre-incubated HBD-2, HBD-3, or HNP-1 with purified capsule before their addition to non-encapsulated capA mutant bacilli. We found that free capsule completely rescued the capA mutant bacilli from killing by HBD-2 and -3 while killing by HNP-1 was reduced to the level observed with WT bacilli. Together, these results suggest an immune evasion mechanism by which the capsule, both that enveloping the bacilli and released fragments, contributes to virulence by binding to and inhibiting the antimicrobial activity of cationic AMPs.

Burkholderia pseudomallei JW270 Is Lethal in the Madagascar Hissing Cockroach Infection Model and Can Be Utilized at Biosafety Level 2 to Identify Putative Virulence Factors.

Burkholderia pseudomallei, the causative agent of melioidosis, is classified by the CDC as a tier 1 select agent, and work involving it must be performed in a biosafety level 3 (BSL-3) laboratory. Three BSL-2 surrogate strains derived from B. pseudomallei 1026b, a virulent clinical isolate, have been removed from the CDC select agent list. These strains, Bp82, B0011, and JW270, are highly attenuated in rodent models of melioidosis and cannot be utilized to identify virulence determinants because of their high 50% lethal dose (LD50). We previously demonstrated that the Madagascar hissing cockroach (MHC) is a tractable surrogate host to study the innate immune response against Burkholderia. In this study, we found that JW270 maintains its virulence in MHCs. This surprising result indicates that it may be possible to identify potential virulence genes in JW270 by using MHCs at BSL-2. We tested this hypothesis by constructing JW270 mutations in genes that are required (hcp1) or dispensable (hcp2) for B. pseudomallei virulence in rodents. JW270 Δhcp1 was avirulent in MHCs and JW270 Δhcp2 was virulent, suggesting that MHCs can be used at BSL-2 for the discovery of important virulence factors. JW270 ΔBPSS2185, a strain harboring a mutation in a type IV pilin locus (TFP8) required for full virulence in BALB/c mice, was also found to be attenuated in MHCs. Finally, we demonstrate that the hmqA-G locus, which encodes the production of a family of secondary metabolites called 4-hydroxy-3-methyl-2-alkylquinolines, is important for JW270 virulence in MHCs and may represent a novel virulence determinant.

Clinical Features of Patients Hospitalized for All Routes of Anthrax, 1880-2018: A Systematic Review.

BACKGROUND: Anthrax is a toxin-mediated zoonotic disease caused by Bacillus anthracis, with a worldwide distribution recognized for millennia. Bacillus anthracis is considered a potential biowarfare agent. METHODS: We completed a systematic review for clinical and demographic characteristics of adults and children hospitalized with anthrax (cutaneous, inhalation, ingestion, injection [from contaminated heroin], primary meningitis) abstracted from published case reports, case series, and line lists in English from 1880 through 2018, assessing treatment impact by type and severity of disease. We analyzed geographic distribution, route of infection, exposure to anthrax, and incubation period. RESULTS: Data on 764 adults and 167 children were reviewed. Most cases reported for 1880 through 1915 were from Europe; those for 1916 through 1950 were from North America; and from 1951 on, cases were from Asia. Cutaneous was the most common form of anthrax for all populations. Since 1960, adult anthrax mortality has ranged from 31% for cutaneous to 90% for primary meningitis. Median incubation periods ranged from 1 day (interquartile range [IQR], 0-4) for injection to 7 days (IQR, 4-9) for inhalation anthrax. Most patients with inhalation anthrax developed pleural effusions and more than half with ingestion anthrax developed ascites. Treatment and critical care advances have improved survival for those with systemic symptoms, from approximately 30% in those untreated to approximately 70% in those receiving antimicrobials or antiserum/antitoxin. CONCLUSIONS: This review provides an improved evidence base for both clinical care of individual anthrax patients and public health planning for wide-area aerosol releases of B. anthracis spores.

Human Innate Immune Cells Respond Differentially to Poly-γ-Glutamic Acid Polymers from Bacillus anthracis and Nonpathogenic Bacillus Species.

The poly-γ-glutamic acid (PGA) capsule produced by Bacillus anthracis is composed entirely of d-isomer glutamic acid, whereas nonpathogenic Bacillus species produce mixed d-, l-isomer PGAs. To determine if B. anthracis PGA confers a pathogenic advantage over other PGAs, we compared the responses of human innate immune cells to B. anthracis PGA and PGAs from nonpathogenic B. subtilis subsp. chungkookjang and B. licheniformis Monocytes and immature dendritic cells (iDCs) responded differentially to the PGAs, with B. anthracis PGA being least stimulatory and B. licheniformis PGA most stimulatory. All three elicited IL-8 and IL-6 from monocytes, but B. subtilis PGA also elicited IL-10 and TNF-α, whereas B. licheniformis PGA elicited all those plus IL-1ß. Similarly, all three PGAs elicited IL-8 from iDCs, but B. subtilis PGA also elicited IL-6, and B. licheniformis PGA elicited those plus IL-12p70, IL-10, IL-1ß, and TNF-α. Only B. licheniformis PGA induced dendritic cell maturation. TLR assays also yielded differential results. B. subtilis PGA and B. licheniformis PGA both elicited more TLR2 signal than B. anthracis PGA, but only responses to B. subtilis PGA were affected by a TLR6 neutralizing Ab. B. licheniformis PGA elicited more TLR4 signal than B. anthracis PGA, whereas B. subtilis PGA elicited none. B. anthracis PGA persisted longer in high m.w. form in monocyte and iDC cultures than the other PGAs. Reducing the m.w. of B. anthracis PGA reduced monocytes' cytokine responses. We conclude that B. anthracis PGA is recognized less effectively by innate immune cells than PGAs from nonpathogenic Bacillus species, resulting in failure to induce a robust host response, which may contribute to anthrax pathogenesis.

Clindamycin Protects Nonhuman Primates Against Inhalational Anthrax But Does Not Enhance Reduction of Circulating Toxin Levels When Combined With Ciprofloxacin.

BACKGROUND: Inhalational anthrax is rare and clinical experience limited. Expert guidelines recommend treatment with combination antibiotics including protein synthesis-inhibitors to decrease toxin production and increase survival, although evidence is lacking. METHODS: Rhesus macaques exposed to an aerosol of Bacillus anthracis spores were treated with ciprofloxacin, clindamycin, or ciprofloxacin + clindamycin after becoming bacteremic. Circulating anthrax lethal factor and protective antigen were quantitated pretreatment and 1.5 and 12 hours after beginning antibiotics. RESULTS: In the clindamycin group, 8 of 11 (73%) survived demonstrating its efficacy for the first time in inhalational anthrax, compared to 9 of 9 (100%) with ciprofloxacin, and 8 of 11 (73%) with ciprofloxacin + clindamycin. These differences were not statistically significant. There were no significant differences between groups in lethal factor or protective antigen levels from pretreatment to 12 hours after starting antibiotics. Animals that died after clindamycin had a greater incidence of meningitis compared to those given ciprofloxacin or ciprofloxacin + clindamycin, but numbers of animals were very low and no definitive conclusion could be reached. CONCLUSION: Treatment of inhalational anthrax with clindamycin was as effective as ciprofloxacin in the nonhuman primate. Addition of clindamycin to ciprofloxacin did not enhance reduction of circulating toxin levels.

Use of Anthrax Vaccine in the United States: Recommendations of the Advisory Committee on Immunization Practices, 2019.

This report updates the 2009 recommendations from the CDC Advisory Committee on Immunization Practices (ACIP) regarding use of anthrax vaccine in the United States (Wright JG, Quinn CP, Shadomy S, Messonnier N. Use of anthrax vaccine in the United States: recommendations of the Advisory Committee on Immunization Practices [ACIP)], 2009. MMWR Recomm Rep 2010;59[No. RR-6]). The report 1) summarizes data on estimated efficacy in humans using a correlates of protection model and safety data published since the last ACIP review, 2) provides updated guidance for use of anthrax vaccine adsorbed (AVA) for preexposure prophylaxis (PrEP) and in conjunction with antimicrobials for postexposure prophylaxis (PEP), 3) provides updated guidance regarding PrEP vaccination of emergency and other responders, 4) summarizes the available data on an investigational anthrax vaccine (AV7909), and 5) discusses the use of anthrax antitoxins for PEP. Changes from previous guidance in this report include the following: 1) a booster dose of AVA for PrEP can be given every 3 years instead of annually to persons not at high risk for exposure to Bacillus anthracis who have previously received the initial AVA 3-dose priming and 2-dose booster series and want to maintain protection; 2) during a large-scale emergency response, AVA for PEP can be administered using an intramuscular route if the subcutaneous route of administration poses significant materiel, personnel, or clinical challenges that might delay or preclude vaccination; 3) recommendations on dose-sparing AVA PEP regimens if the anthrax vaccine supply is insufficient to vaccinate all potentially exposed persons; and 4) clarification on the duration of antimicrobial therapy when used in conjunction with vaccine for PEP.These updated recommendations can be used by health care providers and guide emergency preparedness officials and planners who are developing plans to provide anthrax vaccine, including preparations for a wide-area aerosol release of B. anthracis spores. The recommendations also provide guidance on dose-sparing options, if needed, to extend the supply of vaccine to increase the number of persons receiving PEP in a mass casualty event.

Formaldehyde and Glutaraldehyde Inactivation of Bacterial Tier 1 Select Agents in Tissues.

For safety, designated Select Agents in tissues must be inactivated and viability tested before the tissue undergoes further processing and analysis. In response to the shipping of samples of "inactivated" Bacillus anthracis that inadvertently contained live spores to nonregulated entities and partners worldwide, the Federal Register now mandates in-house validation of inactivation procedures and standardization of viability testing to detect live organisms in samples containing Select Agents that have undergone an inactivation process. We tested and validated formaldehyde and glutaraldehyde inactivation procedures for animal tissues infected with virulent B. anthracis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis. We confirmed that our fixation procedures for tissues containing these Tier 1 Select Agents resulted in complete inactivation and that our validated viability testing methods do not interfere with detection of live organisms. Institutions may use this work as a guide to develop and conduct their own testing to comply with the policy.

pH Alkalinization by Chloroquine Suppresses Pathogenic Burkholderia Type 6 Secretion System 1 and Multinucleated Giant Cells.

Burkholderia mallei and B. pseudomallei cause glanders and melioidosis, respectively, in humans and animals. A hallmark of pathogenesis is the formation of granulomas containing multinucleated giant cells (MNGCs) and cell death. These processes depend on type 6 secretion system 1 (T6SS-1), which is required for virulence in animals. We examined the cell biology of MNGC formation and cell death. We found that chloroquine diphosphate (CLQ), an antimalarial drug, inhibits Burkholderia growth, phagosomal escape, and subsequent MNGC formation. This depends on CLQ's ability to neutralize the acid pH because other alkalinizing compounds similarly inhibit escape and MNGC formation. CLQ inhibits bacterial virulence protein expression because T6SS-1 and some effectors of type 3 secretion system 3 (T3SS-3), which is also required for virulence, are expressed at acid pH. We show that acid pH upregulates the expression of Hcp1 of T6SS-1 and TssM, a protein coregulated with T6SS-1. Finally, we demonstrate that CLQ treatment of Burkholderia-infected Madagascar hissing cockroaches (HCs) increases their survival. This study highlights the multiple mechanisms by which CLQ inhibits growth and virulence and suggests that CLQ be further tested and considered, in conjunction with antibiotic use, for the treatment of diseases caused by Burkholderia.

A reaction path study of the catalysis and inhibition of the Bacillus anthracis CapD γ-glutamyl transpeptidase.

The CapD enzyme of Bacillus anthracis is a γ-glutamyl transpeptidase from the N-terminal nucleophile hydrolase superfamily that covalently anchors the poly-γ-D-glutamic acid (pDGA) capsule to the peptidoglycan. The capsule hinders phagocytosis of B. anthracis by host cells and is essential for virulence. The role CapD plays in capsule anchoring and remodeling makes the enzyme a promising target for anthrax medical countermeasures. Although the structure of CapD is known, and a covalent inhibitor, capsidin, has been identified, the mechanisms of CapD catalysis and inhibition are poorly understood. Here, we used a computational approach to map out the reaction steps involved in CapD catalysis and inhibition. We found that the rate-limiting step of either CapD catalysis or inhibition was a concerted asynchronous formation of the tetrahedral intermediate with a barrier of 22-23 kcal/mol. However, the mechanisms of these reactions differed for the two amides. The formation of the tetrahedral intermediate with pDGA was substrate-assisted with two proton transfers. In contrast, capsidin formed the tetrahedral intermediate in a conventional way with one proton transfer. Interestingly, capsidin coupled a conformational change in the catalytic residue of the tetrahedral intermediate to stretching of the scissile amide bond. Furthermore, capsidin took advantage of iminol-amide tautomerism of its diacetamide moiety to convert the tetrahedral intermediate to the acetylated CapD. As evidence of the promiscuous nature of CapD, the enzyme cleaved the amide bond of capsidin by attacking it on the opposite side compared to pDGA.

Exposure to Bacillus anthracis capsule results in suppression of human monocyte-derived dendritic cells.

The antiphagocytic capsule of Bacillus anthracis is a major virulence factor. We hypothesized that it may also mediate virulence through inhibition of the host's immune responses. During an infection, the capsule exists attached to the bacterial surface but also free in the host tissues. We sought to examine the impact of free capsule by assessing its effects on human monocytes and immature dendritic cells (iDCs). Human monocytes were differentiated into iDCs by interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) over 7 days in the presence of capsule derived from wild-type encapsulated B. anthracis Ames (WT) or a control preparation from an isogenic B. anthracis Ames strain that produces only 2% of the capsule of the WT (capA mutant). WT capsule consistently induced release of IL-8 and IL-6 while the capA mutant control preparation elicited either no response or only a minimal release of IL-8. iDCs that were differentiated in the presence of WT capsule had increased side scatter (SSC), a measure of cellular complexity, when assessed by flow cytometry. iDCs differentiated in the presence of WT capsule also matured less well in response to subsequent B. anthracis peptidoglycan (Ba PGN) exposure, with reduced upregulation of the chemokine receptor CCR7, reduced CCR7-dependent chemotaxis, and reduced release of certain cytokines. Exposure of naive differentiated control iDCs to WT capsule did not alter cell surface marker expression but did elicit IL-8. These results indicate that free capsule may contribute to the pathogenesis of anthrax by suppressing the responses of immune cells and interfering with the maturation of iDCs.

Probing the donor and acceptor substrate specificity of the γ-glutamyl transpeptidase.

γ-Glutamyl transpeptidase (GGT) is a two-substrate enzyme that plays a central role in glutathione metabolism and is a potential target for drug design. GGT catalyzes the cleavage of γ-glutamyl donor substrates and the transfer of the γ-glutamyl moiety to an amine of an acceptor substrate or water. Although structures of bacterial GGT have revealed details of the protein-ligand interactions at the donor site, the acceptor substrate site is relatively undefined. The recent identification of a species-specific acceptor site inhibitor, OU749, suggests that these inhibitors may be less toxic than glutamine analogues. Here we investigated the donor and acceptor substrate preferences of Bacillus anthracis GGT (CapD) and applied computational approaches in combination with kinetics to probe the structural basis of the enzyme's substrate and inhibitor binding specificities and compare them with human GGT. Site-directed mutagenesis studies showed that the R432A and R520S variants exhibited 6- and 95-fold decreases in hydrolase activity, respectively, and that their activity was not stimulated by the addition of the l-Cys acceptor substrate, suggesting an additional role in acceptor binding and/or catalysis of transpeptidation. Rat GGT (and presumably HuGGT) has strict stereospecificity for L-amino acid acceptor substrates, while CapD can utilize both L- and D-acceptor substrates comparably. Modeling and kinetic analysis suggest that R520 and R432 allow two alternate acceptor substrate binding modes for L- and D-acceptors. R432 is conserved in Francisella tularensis, Yersinia pestis, Burkholderia mallei, Helicobacter pylori and Escherichia coli, but not in human GGT. Docking and MD simulations point toward key residues that contribute to inhibitor and acceptor substrate binding, providing a guide to designing novel and specific GGT inhibitors.

Improvement of a potential anthrax therapeutic by computational protein design.

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.

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.

Human transferrin confers serum resistance against Bacillus anthracis.

The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the identification of human transferrin as the inhibiting factor. Purified transferrin blocks growth of both the fully virulent encapsulated B. anthracis Ames and the non-encapsulated Sterne strain. Growth inhibition was also observed in serum of wild-type mice but not of hypotransferrinemic mice that only have approximately 1% circulating transferrin levels. We were able to definitely assign the bacteriostatic activity of transferrin to its iron-binding function: neither iron-saturated transferrin nor a recombinant transferrin mutant unable to bind iron could inhibit growth of B. anthracis. Additional iron could restore bacterial growth in human serum. The observation that other important gram-positive pathogens are not inhibited by transferrin suggests they have evolved effective mechanisms to circumvent serum iron deprivation. These findings provide a better understanding of human host defense mechanisms against anthrax and provide a mechanistic basis for the antimicrobial activity of human transferrin.

Poly-γ-Glutamic Acid Encapsulation of Bacillus anthracis Inhibits Human Dendritic Cell Responses.

The capsule of Bacillus anthracis is composed of a d isomer poly-γ-glutamic acid polymer, which is especially nonstimulatory to dendritic cells, even more so than similar mixed d, l isomer polymers from nonpathogenic Bacillus species. Capsule is an essential virulence factor for B. anthracis, protecting the bacilli from phagocytosis by innate immune cells. In this study, we demonstrate that encapsulation provides a further pathogenic advantage by shielding more inflammatory Ags on the bacillus surface, thereby reducing dendritic cell responses. We exposed human immature dendritic cells (DCs) to increasing multiplicities of infection (MOIs) of killed B. anthracis bacilli from the fully encapsulated wild-type Ames strain (WT) and an isogenic capsule-deficient strain (capA mutant). Both strains elicited robust cytokine responses, but IL-23, TNF-α, and IL-10 were significantly reduced in response to the encapsulated WT compared with capA mutant up to an MOI of 15. capA mutant bacilli could induce phenotypic maturation of immature DCs with upregulation of MHC classes I and II, CD83, and CCR7 at an MOI of 3.75, whereas encapsulated WT bacilli still did not induce significant upregulation of MHC classes I and II at an MOI of 15. DCs exposed to capA mutant bacilli (MOI 3.75) exhibited CCR7-dependent chemotaxis that was comparable to that of LPS-stimulated controls, whereas DCs exposed to encapsulated WT bacilli exhibited significantly less chemotaxis. We conclude that capsule shields more inflammatory surface Ags, delaying development of an adaptive immune response by reducing TNF-α, thereby inhibiting DC maturation.

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.

Capsule depolymerase overexpression reduces Bacillus anthracis virulence.

Capsule depolymerase (CapD) is a gamma-glutamyl transpeptidase and a product of the Bacillus anthracis capsule biosynthesis operon. In this study, we examined the effect of modulating capD expression on B. anthracis capsule phenotype, interaction with phagocytic cells and virulence in guinea pigs. Transcriptional fusions of capD were made to the genes encoding heat-shock protein 60 (hsp60) and elongation factor Tu (EFTu), and to capA, a B. anthracis capsule biosynthesis gene. Translation signals were altered to improve expression of capD, including replacing the putative ribosome-binding site with a consensus sequence and the TTG start codon with ATG. CapD was not detected by immunoblotting in lysates from wild-type B. anthracis Ames but was detected in strains engineered with a consensus ribosome-binding site for capD. Strains overexpressing capD at amounts detected by immunoblotting were found to have less surface-associated capsule and released primarily lower-molecular-mass capsule into culture supernatants. Overexpression of capD increased susceptibility to neutrophil phagocytic killing and adherence to macrophages and resulted in reduced fitness in a guinea pig model of infection. These data suggest that B. anthracis may have evolved weak capD expression resulting in optimized capsule-mediated virulence.

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens.

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to opsono-phagocytic killing assays. An advantage of the opsono-adherence assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.

Green tea and epigallocatechin-3-gallate are bactericidal against Bacillus anthracis.

Bacillus anthracis, the etiological agent of anthrax, is listed as a category A biothreat agent by the United States Centers for Disease Control and Prevention. The virulence of the organism is due to expression of two exotoxins and capsule, which interfere with host cellular signaling, alter host water homeostasis and inhibit phagocytosis of the pathogen, respectively. Concerns regarding the past and possible future use of B. anthracis as a bioterrorism agent have resulted in an impetus to develop more effective protective measures and therapeutics. In this study, green tea was found to inhibit the in vitro growth of B. anthracis. Epigallocatechin-3-gallate (EGCG), a compound found abundantly in green tea, was shown to be responsible for this activity. EGCG was bactericidal against both the attenuated B. anthracis ANR and the virulent encapsulated B. anthracis Ames strain. This study highlights the antimicrobial activity of green tea and EGCG against anthrax and suggests the need for further investigation of EGCG as a therapeutic candidate against B. anthracis.