Emergence of Anthrax Edema Toxin as a Master Manipulator of Macrophage and B Cell Functions

Anthrax edema toxin (ET), a powerful adenylyl cyclase, is an important virulence factor of Bacillus anthracis. Until recently, only a modest amount of research was performed to understand the role this toxin plays in the organism’s immune evasion strategy. A new wave of studies have begun to elucidate the effects this toxin has on a variety of host cells. While efforts have been made to illuminate the effect ET has on cells of the adaptive immune system, such as T cells, the greatest focus has been on cells of the innate immune system, particularly the macrophage. Here we discuss the immunoevasive activities that ET exerts on macrophages, as well as new research on the effects of this toxin on B cells

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Bacillus anthracis Edema Toxin Suppresses Human Macrophage Phagocytosis and Cytoskeletal Remodeling via the Protein Kinase A and Exchange Protein Activated by Cyclic AMP Pathways▿

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. It produces edema toxin (EdTx), a powerful adenylate cyclase that increases cyclic AMP (cAMP) levels in host cells. Because other cAMP-increasing agents inhibit key macrophage (MΦ) functions, such as phagocytosis, it was hypothesized that EdTx would exhibit similar suppressive activities. Our previous GeneChip data showed that EdTx downregulated MΦ genes involved in actin cytoskeleton remodeling, including protein kinase A (PKA). To further examine the role of EdTx during anthrax pathogenesis, we explored the hypothesis that EdTx treatment leads to deregulation of the cAMP-dependent PKA system, resulting in impaired cytoskeletal functions essential for MΦ activity. Our data revealed that EdTx significantly suppressed human MΦ phagocytosis of Ames spores. Cytoskeletal changes, such as decreased cell spreading and lowered F-actin content, were also observed for toxin-treated MΦs. Further, EdTx altered the protein levels and activity of PKA and exchange protein activated by cAMP (Epac), a recently identified cAMP-binding molecule. By using PKA- and Epac-selective cAMP analogs, we confirmed the involvement of both pathways in the inhibition of MΦ functions elicited by EdTx-generated cAMP. These results suggested that EdTx weakened the host immune response by increasing cAMP levels, which then signaled via PKA and Epac to cripple MΦ phagocytosis and interfered with cytoskeletal remodeling

Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from <i>Yersinia pestis</i> as Next Generation Plague Vaccines

<div><p>Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism <i>Yersinia pestis</i> poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH₂-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced T_H1 and T_H2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of <i>Y. pestis</i> CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.</p></div

An oligomerization deficient YscF mutant.

<p>(<b>A</b>) Schematic of native YscF and YscF35/67 mutants. (<b>B</b>) Purification of YscF and YscF35/67 mutant proteins. The gel filtration profiles showed that the native YscF eluted as a broad peak spanning the entire high molecular weight range and the mutated YscF35/67 eluted as two peaks, one as a high molecular weight aggregate near the void volume, and another at 22 kDa corresponding to the size of a dimer. (<b>C</b>) Purity of YscF and YscF35/67 proteins as analyzed by SDS-PAGE and Coomassie blue staining of the peak fractions.</p

Designing monomeric F1 mutants.

<p>(<b>A</b>) Schematic of native F1, F1mut1, and F1mut2 recombinant constructs. The donor β-strand of F1 is shown in pink, the T cell epitope region in blue, and the rest of the F1 coding sequence in green. The numbers correspond to the aa residues of F1. Native F1 has one hexa-histidine tag (orange) at the NH₂-terminus, whereas F1mut1 and F1mut2 have two hexa-histidine tags, one at the NH₂-terminus and another at the COOH-terminus. (<b>B</b>) Expression and solubility analysis. The recombinant F1 proteins were over-expressed by adding IPTG to 1 mM final concentration. The samples at 0, 1, or 2 h time points were analyzed by SDS-PAGE (15% gel) and Coomassie blue staining. The positions of F1 protein bands are marked with red arrows. The samples at 1 h or 2 h time points were analyzed for solubility using the B-PER reagent. S, soluble fraction (supernatant from 12,000 <i>g</i> centrifugation of the lysate); P, insoluble fraction (pellet); M, molecular weight standards. (<b>C</b>) Purification of F1mut1. The F1mut1 recombinant protein was purified from the cell-free lysates by HisTrap affinity chromatography followed by Hi-load 16/60 Superdex 200 gel filtration. The molecular weight of F1mut1 peak fraction was calculated from the calibration curve constructed by gel filtration on the same column of standard proteins of known molecular weight [Thyroglobulin (669 kDa), Ferritin (440 kDa), Catalase (232 kDa), aldolase (158 kDa), Ovalbumin (43 kDa), RNase A (14 kDa), and Albumin (67 kDa)]. The insert shows the purity of F1mut1 protein after SDS-PAGE and Coomassie blue staining of the peak fraction. Similar results were obtained with the F1mut2 recombinant protein. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#s4" target="_blank">Materials and Methods</a> for additional details.</p

The soluble monomeric F1 mutant protein elicits robust antibody titers and provides complete protection in a mouse model of pneumonic plague.

<p>The immunogenicity and protective efficacy of F1mut-V and other plague immunogens were evaluated in a mouse model. (<b>A</b>) Balb/c mice, twelve per group, were vaccinated with various plague antigens adjuvanted with alhydrogel. (<b>B</b>) Immunization scheme. (<b>C</b>) Antigen-specific antibody (IgG) titers were determined by ELISA, using purified V (I), F1mut2 (II), or YscF35/67 (III) as the coating antigen. No significant cross-reactivity was observed between the antibodies produced against one plague antigen versus a different plague antigen that was coated on the ELISA plate. Error bars represent S.D. “***” denotes p<0.001 (ANOVA). (<b>D</b>) Survival of immunized mice against intranasal challenge with 90 LD₅₀ of <i>Y. pestis</i> CO92. The survived mice were re-challenged with 9,800 LD₅₀ at day-48 post-first challenge. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#s4" target="_blank">Materials and Methods</a> for additional details. The animal mortality data was analyzed by Kaplan Meier's survival estimates and a p value of ≤0.05 was considered significant.</p

The T4 nanoparticle displayed plague immunogens induced robust immunogenicity and protective efficacy against pneumonic plague.

<p>The immunogenicity and protective efficacy of T4 displayed plague immunogens were evaluated in a mouse model using the same immunization scheme shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#ppat-1003495-g006" target="_blank">Figure 6B</a>. (<b>A</b>) The T4 displayed plague immunogen groups, twelve mice per group. The Soc-fused plague immunogens were displayed on T4 phage particles and were directly used for vaccination without any adjuvant. (<b>B</b>) Antigen-specific antibody (IgG) titers as determined by ELISA. (<b>C</b>) Survival of vaccinated mice against intranasal challenge with 90 LD₅₀ of <i>Y. pestis</i> CO92. The survived mice were re-challenged with 9,800 LD₅₀ at day-48 post-first challenge. The animal mortality data was analyzed by Kaplan Meier's survival estimates and a p value of ≤0.05 was considered significant.</p

Construction of mutated F1-V immunogens.

<p>(<b>A</b>) Schematic of native F1-V, F1mut-V and F1mut-V10. Cyan represents the coding sequence of V antigen, and yellow, the putative immunomodulatory sequence that is part of V sequence. Rest of the colors represents the same as described in legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#ppat-1003495-g002" target="_blank">Figure 2</a>. (<b>B</b>) Expression and solubility analysis of F1-V constructs were performed using the B-PER reagent. The samples were analyzed by SDS-PAGE and Coomassie blue staining. The positions of the F1-V protein bands are marked with red arrows. S, soluble fraction (supernatant from 12,000 <i>g</i> centrifugation of the lysate); P, insoluble fraction (pellet); M, molecular weight standards. (<b>C</b>) F1-V, F1mut-V and F1mut-V10 were purified by HisTrap column chromatography followed by Hi-load 16/60 Superdex 200 gel filtration. The calibration graph was generated by passing various molecular weight standards through the same column [Thyroglobulin (669 kDa), Ferritin (440 kDa), Catalase (232 kDa), aldolase (158 kDa), Ovalbumin (43 kDa), RNase A (14 kDa), and Albumin (67 kDa)]. The insert shows the purity of F1-V, F1mut-V, and F1mut-V10 proteins following SDS-PAGE and Coomassie blue staining of the peak fractions. The color of arrows corresponds to the color of the elution profiles of various proteins. (<b>D</b>) Stability of F1-V and F1mut-V proteins was tested by treatment with increasing amounts of trypsin at room temperature overnight. The ratios shown above the gel correspond to the ratios of F1-V or F1mut-V proteins to trypsin (wt∶wt). See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003495#s4" target="_blank">Materials and Methods</a> for additional details.</p