Cross-reactivity of anthrax and C2 toxin: protective antigen promotes the uptake of botulinum C2I toxin into human endothelial cells

Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB(7/8) type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA(63)) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA(63) binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA(63)). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.     

Complement activation is required for induction of a protective antibody response against West Nile virus infection

Infection with West Nile virus (WNV) causes a severe infection of the central nervous system (CNS) with higher levels of morbidity and mortality in the elderly and the immunocompromised. Experiments with mice have begun to define how the innate and adaptive immune responses function to limit infection. Here, we demonstrate that the complement system, a major component of innate immunity, controls WNV infection in vitro primarily in an antibody-dependent manner by neutralizing virus particles in solution and lysing WNV-infected cells. More decisively, mice that genetically lack the third component of complement or complement receptor 1 (CR1) and CR2 developed increased CNS virus burdens and were vulnerable to lethal infection at a low dose of WNV. Both C3-deficient and CR1- and CR2-deficient mice also had significant deficits in their humoral responses after infection with markedly reduced levels of specific anti-WNV immunoglobulin M (IgM) and IgG. Overall, these results suggest that complement controls WNV infection, in part through its ability to induce a protective antibody response.     

Alanine-scanning mutations in domain 4 of anthrax toxin protective antigen reveal residues important for binding to the cellular receptor and to a neutralizing monoclonal antibody

A panel of variants with alanine substitutions in the small loop of anthrax toxin protective antigen domain 4 was created to determine individual amino acid residues critical for interactions with the cellular receptor and with a neutralizing monoclonal antibody, 14B7. Substituted protective antigen proteins were analyzed by cellular cytotoxicity assays, and their interactions with antibody were measured by plasmon surface resonance and analytical ultracentrifugation. Residue Asp683 was the most critical for cell binding and toxicity, causing an approximately 1000-fold reduction in toxicity, but was not a large factor for interactions with 14B7. Substitutions in residues Tyr681, Asn682, and Pro686 also reduced toxicity significantly, by 10-100-fold. Of these, only Asn682 and Pro686 were also critical for interactions with 14B7. However, residues Lys684, Leu685, Leu687, and Tyr688 were critical for 14B7 binding without greatly affecting toxicity. The K684A and L685A variants exhibited wild type levels of toxicity in cell culture assays; the L687A and Y688A variants were reduced only 1.5- and 5-fold, respectively.     

Molecular dynamics simulations of complexes between wild-type and mutant anthrax protective antigen variants and a model anthrax toxin receptor

Bacillus anthracis, a spore-forming infectious bacterium, produces a toxin consisting of three proteins: lethal factor (LF), edema factor (EF), and protective antigen (PA). LF and EF possess intracellular enzymatic functions, the net effect of which is to severely compromise host innate immunity. During an anthrax infection PA plays the critical role of facilitating entry of both EF and LF toxins into host cell cytoplasm. Crystal structures of all three of the anthrax toxins have been determined, as well as the crystal structure of the (human) von Willebrand factor A (integrin VWA/I domain) -- an anthrax toxin receptor. A theoretical structure of the complex between VWA/I and PA has also been reported. Here we report on the results of 1,000 psec molecular dynamics (MD) simulations carried out on complexes between the Anthrax Protective Antigen Domain 4 (PA-D4) and the von Willebrand Factor A (VWA/I). MD simulations (using Insight II software) were carried out for complexes containing wild-type (WT) PA-D4, as well as for complexes containing three different mutants of PA-D4, one containing three substitutions in the PA-D4 "small loop" (residues 679-693) (D683A/L685E/Y688C), one containing a single substitution at a key site at the PA-D4 -- receptor interface (K679A) and another containing a deletion of eleven residues at the C-terminus of PA (Delta724-735). All three sets of PA mutations have been shown experimentally to result in serious deficiencies in PA function. Our MD results are consistent with these findings. Major disruptions in interactions were observed between the mutant PA-D4 domains and the anthrax receptor during the MD simulations. Many secondary structural features in PA-D4 are also severely compromised when VWA complexes with mutant variants of PA-D4 are subjected to MD simulations. These MD simulation results clearly indicate the importance of the mutated PA-D4 residues in both the "small loop" and at the carboxyl terminus in maintaining a PA conformation that is capable of effective interaction with the anthrax toxin receptor.     

Amplification of the antibody response by C3b complexed to antigen through an ester link

Complement C3 has been described as playing an important role in the cell-mediated immune response. C3b has the capacity to covalently bind Ag and then to stimulate in vitro Ag presentation to T lymphocytes. To verify this observation in vivo, we prepared and purified covalent human C3b-Ag complexes using lysozyme (HEL) as Ag. The characterization of these HEL-C3b complexes indicates that they are representative of those susceptible to be generated in physiological conditions. Mice were immunized with 0.1 to 0.6 microgram of either free HEL, HEL + C3b, HEL-C3b, or HEL + CFA. Response was assessed after two i.p. injections by quantification of specific Ab production. Immunization with either HEL-C3b complexes or HEL + CFA leads to anti-HEL IgG production whereas free HEL or HEL + C3b was ineffective. Either HEL-C3b or HEL + CFA immunizations led to a similar Ig subclass patterns, including IgG1, IgG2a, IgA, and IgM. Our experiments provide the first evidence for modulation of specific Ab response by C3b when it is bound to Ag through a physiological-like link. Taken together with previous data concerning Ab response following recombinant HEL-C3d immunization, cellular events such as processing of C3b-Ag by APC and recognition by T lymphocytes, this present result underlines the importance of C3b and its fragments in stimulation of the immune system, through the multiplicity and complementarity of its interactions.     

Gene conjugation of molecular adjuvant C3d3 to hCGbeta increased the anti-hCGbeta Th2 and humoral immune response in DNA immunization

Human chorionic gonadotropin (hCG) has been used as an anti-fertility vaccine and as a target for cancer immunotherapy. We have explored the use of three copies of C3d in DNA vaccine as molecular adjuvant to improve the immunogenicity of this hormone in previous work and found that the immune response induced by pcDNA3-hCGbeta-C3d3 has been enhanced 243-fold compared with pcDNA3-hCGbeta following DNA immunization in BALB/c mice. In the present study, a new functionally active DNA vaccine of hCGbeta-C3d3 chimera based on pCMV4 vector has been described. We compared the expression efficiency of pCMV4 and pcDNA3 eukaryotic vectors for hCGbeta and hCGbeta-C3d3 fusion protein and the immune response of mice immunized with pcDNA3-hCGbeta, pCMV4-hCGbeta, pcDNA3-hCGbeta-C3d3 and pCMV4-hCGbeta-C3d3, respectively, at 25, 50 and 100 pmol dose, and further analyzed the levels of Th1 and Th2 cytokines produced by spleen lymphocytes of the immunized mice upon hCG restimulation in vitro. It was found that pCMV4 vector achieved 1.3-1.5-fold higher protein expression and raised 1.1-1.2 (primary) and 1.2-1.3 (booster) logs higher titer of anti-hCGbeta IgG than pcDNA3. Mice vaccinated with 50 pmol of hCGbeta-C3d3-DNAs elicited the highest titer of hCGbeta-specific antibody among the serial doses and the immune response induced by pCMV4-hCGbeta-C3d3 were, respectively, 1.3, 1.3 and 1.2 logs higher than that of pcDNA3-hCGbeta-C3d3 and 2.2, 2.9 and 2.4 logs higher than that of pCMV4-hCGbeta at week 2 following the booster immunization. Moreover, we observed that the production of IL-4 and IL-10 increased in mice vaccinated with hCGbeta-C3d3-DNAs and the ratio of IL-4/IFN-(gamma) showed a Th2 bias of immune response in the mice immunized with hCGbeta-C3d3-DNAs. These findings indicated that gene fusion of C3d3 to hCGbeta, as a means of harnessing the adjuvant potential of the innate immune system, may improve the antigen-specific Th2 humoral immune response of the hCGbeta DNA vaccine and the pCMV4 vector is a more ideal eukaryotic vector for DNA vaccine than pcDNA3.     

Detection of a neoantigen on human C3bi and C3d by monoclonal antibody

A neoantigen was detected on human C3bi and C3d by using the monoclonal antibody (MoAb) 130. The antibody bound to EC3bi and EC3d cells but not to EC3b. Although highly purified C3bi or C3d strongly inhibited the binding of the antibody to EC3d, highly purified C3c had no such effect. Native C3, C3b, or C3(H2O) inhibited this binding only weakly. The neoantigen was also detected in serum after activation with zymosan or heat-aggregated IgG, and it was found bound to the aggregated IgG and zymosan particles. Plasma samples from patients with immunologic disorders were tested for this neoantigen, and 25 out of 43 samples tested were found to have levels of neoantigen corresponding to 2 to 11.5% complement activation, whereas 13 out of 14 normal donor plasmas contained amounts of neoantigen indicating much less than 1% complement activation.     

Complement component C3 promotes T-cell priming and lung migration to control acute influenza virus infection

The complement cascade defines an important link between the innate and the specific immune system. Here we show that mice deficient for the third component of complement (C3-/- mice) are highly susceptible to primary infection with influenza virus. C3-/- mice showed delayed viral clearance and increased viral titers in lung, whereas mice deficient for complement receptors CR1 and CR2 (Cr2-/- mice) cleared the infection normally. Priming of T-helper cells and cytotoxic T cells (CTLs) in lung-draining lymph nodes was reduced, and the recruitment into the lung of virus-specific CD4+ and CD8+ effector T cells producing interferon-gamma was severely impaired in C3-/- but not in Cr2-/- mice. Consequently, T-helper cell-dependent IgG responses were reduced in C3-/- mice but remained intact in Cr2-/- mice. These results demonstrate that complement induces specific immunity by promoting T-cell responses.     

The Effect of Lutein Supplementation on Blood Plasma Levels of Complement Factor D,C5a and C3d

Lutein is selectively taken up by the primate retina and plays an important role as a filter for harmful blue light and as an antioxidant. Recent studies have shown that lutein has systemic anti-inflammatory properties. Dietary lutein has been associated with reduced circulating levels of inflammatory biomarkers such as CRP and sICAM. Whether lutein also affects activation of the complement system has not yet been addressed and was the purpose of the study described here. Seventy-two subjects with signs of early macular degeneration were randomly assigned to receive either a 10 mg lutein supplement or a placebo during one year. EDTA blood samples were collected at 0, 4, 8 and 12 months. Complement factor D (CFD), a rate limiting component of the alternative pathway of complement activation and the complement activation products C5a and C3d were determined in the plasma samples by ELISA. A significant 0.11 µg/ml monthly decrease in plasma CFD concentration was observed in the lutein group (p<0.001), resulting in a 51% decrease from 2.3 µg/ml at baseline to 1.0 µg/ml at 12 months. The C5a concentration showed a significant 0.063ng/ml monthly decrease in the lutein group (p<0.001) resulting in a 36% decrease from 2.2ng/ml at baseline to 1.6ng/ml at 12 months. The C3d concentration showed a significant 0.19µg/ml monthly decrease in the lutein group (p=0.004) that gave rise to a 9% decrease from 15.4µg/ml at baseline to 14.4µg/ml at 12 months. In the placebo group we found a significant 0.04 µg/ml monthly decrease in plasma CFD concentration, whereas no changes were observed for C5a and C3d. Lutein supplementation markedly decreases circulating levels of the complement factors CFD, C5a and C3d levels, which might allow a simple method to control this inflammatory pathway of the innate immune system.     

The 2beta2-2beta3 loop of anthrax protective antigen contains a dominant neutralizing epitope

Anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. PA is the major component in the current anthrax vaccine, but the antigenic epitopes on it are not well-defined. We generated a pool of toxin-neutralizing anti-PA monoclonal antibodies (MAbs) to analyze the neutralizing epitopes of PA. Nine toxin-neutralizing MAbs obtained were found bound to three different domains of PA respectively, among which three MAbs with the strongest toxin-neutralizing activity recognized the same epitope within domain 2. This epitope was fine mapped to the chymotrypsin-sensitive site, (312)SFFD(315), in the 2beta(2)-2beta(3) loop of PA, using phage-displayed random peptide libraries and mutation analysis. The result demonstrated for the first time that the 2beta(2)-2beta(3) loop, which is involved in the transition of PA oligomers from prepore to pore, contains a dominant neutralizing epitope. This work contributes to the immunological and functional analysis of PA and offers perspective for the development of a new epitope vaccine against anthrax.     

Large-scale structural changes accompany binding of lethal factor to anthrax protective antigen: a cryo-electron microscopic study

Anthrax toxin (AT), secreted by Bacillus anthracis, is a three-protein cocktail of lethal factor (LF, 90 kDa), edema factor (EF, 89 kDa), and the protective antigen (PA, 83 kDa). Steps in anthrax toxicity involve (1) binding of ligand (EF/LF) to a heptamer of PA63 (PA63h) generated after N-terminal proteolytic cleavage of PA and, (2) following endocytosis of the complex, translocation of the ligand into the cytosol by an as yet unknown mechanism. The PA63h.LF complex was directly visualized from analysis of images of specimens suspended in vitrified buffer by cryo-electron microscopy, which revealed that the LF molecule, localized to the nonmembrane-interacting face of the oligomer, interacts with four successive PA63 monomers and partially unravels the heptamer, thereby widening the central lumen. The observed structural reorganization in PA63h likely facilitates the passage of the large 90 kDa LF molecule through the lumen en route to its eventual delivery across the membrane bilayer.     

Multimeric Interactions between Complement Factor H and Its C3d Ligand Provide New Insight on Complement Regulation

Activation of C3 to C3b signals the start of the alternative complement pathway. The C-terminal short complement regulator (SCR)-20 domain of factor H (FH), the major serum regulator of C3b, possesses a binding site for C3d, a 35-kDa physiological fragment of C3b. Size distribution analyses of mixtures of SCR-16/20 or FH with C3d by analytical ultracentrifugation in 50 and 137 mM NaCl buffer revealed a range of discrete peaks, showing that multimeric complexes had formed at physiologically relevant concentrations. Surface plasmon resonance studies showed that native FH binds C3d in two stages. An equilibrium dissociation constant K_D1 of 2.6 μM in physiological buffer was determined for the first stage. Overlay experiments indicated that C3d formed multimeric complexes with FH. X-ray scattering showed that the maximum dimension of the C3d complexes with SCR-16/20 at 29 nm was not much longer than that of the unbound SCR-16/20 dimer. Molecular modelling suggested that the ultracentrifugation and scattering data are most simply explained in terms of associating dimers of each of SCR-16/20 and C3d. We conclude that the physiological interaction between FH and C3d is not a simple 1:1 binding stoichiometry between the two proteins that is often assumed. Because the multimers involve the C-terminus of FH, which is bound to host cell surfaces, our results provide new insight on FH regulation during excessive complement activation, both in the fluid phase and at host cell surfaces decorated by C3d.     

Binding stoichiometry and kinetics of the interaction of a human anthrax toxin receptor, CMG2, with protective antigen

The protective antigen (PA) moiety of anthrax toxin binds to cellular receptors and mediates entry of the two enzymatic moieties of the toxin into the cytosol. Two PA receptors, anthrax toxin receptor (ATR)/tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2), have been identified. We expressed and purified the von Willebrand A (VWA) domain of CMG2 and examined its interactions with monomeric and heptameric forms of PA. Monomeric PA bound a stoichiometric equivalent of CMG2, whereas the heptameric prepore form bound 7 eq. The Kd of the VWA domain-PA interaction is 170 pm when liganded by Mg2+, reflecting a 1000-fold tighter interaction than most VWA domains with their endogenous ligands. The dissociation rate constant is extremely slow, indicating a 30-h lifetime for the CMG2.PA monomer complex. CMG2 metal ion-dependent adhesion site (MIDAS) was studied kinetically and thermodynamically. The association rate constant (approximately 10(5) m(-1) s(-1)) is virtually identical in the presence or absence of Mg2+ or Ca2+ , but the dissociation rate of metal ion liganded complex is up to 4 orders of magnitude slower than metal ion free complex. Residual affinity (Kd approximately 960 nm) in the absence of divalent metal ions allowed the free energy for the contribution of the metal ion to be calculated as 5 kcal mol(-1), demonstrating that the metal ion-dependent adhesion site is directly coordinated by CMG2 and PA in the binding interface. The high affinity of the VWA domain for PA supports its potency in neutralizing anthrax toxin, demonstrating its potential utility as a novel therapeutic for anthrax.     

Treatment of anthrax infection with combination of ciprofloxacin and antibodies to protective antigen of Bacillus anthracis

Currently there is no effective treatment for inhalational anthrax beyond administration of antibiotics shortly after exposure. There is need for new, safe and effective treatments to supplement traditional antibiotic therapy. Our study was based on the premise that simultaneous inhibition of lethal toxin action with antibodies and blocking of bacterial growth by antibiotics will be beneficial for the treatment of anthrax. In this study, we tested the effects of a combination treatment using purified rabbit or sheep anti-protective antigen (PA) antibodies and the antibiotic ciprofloxacin in a rodent anthrax model. In mice infected with a dose of Bacillus anthracis Sterne strain corresponding to 10 LD(50), antibiotic treatment with ciprofloxacin alone only cured 50% of infected animals. Administration of anti-PA IgG in combination with ciprofloxacin produced 90-100% survival. These data indicate that a combination of antibiotic/immunoglobulin therapy is more effective than antibiotic treatment alone in a rodent anthrax model.     

Soluble expression and purification of the anthrax protective antigen in E. coli and identification of a novel dominant-negative mutant N435C

The anthrax toxin is an AB-type bacterium toxin composed of the protective antigen (PA) as the cell-binding B component, and the lethal factor (LF) and edema toxin (EF) as the catalytic A components. The PA component is a key factor in anthrax-related research and recombinant PA can be produced in general in Escherichia coli. However, such recombinant PA always forms inclusion bodies in the cytoplasm of E. coli, making difficult the procedure of its purification. In this study, we found that the solubility of recombinant PA was dramatically enhanced by fusion with glutathione S-transferase (GST) and an induction of its expression at 28°C. The PA was purified to high homogeneity and a yield of 3 mg protein was obtained from 1 l culture by an affinity-chromatography approach. Moreover, we expressed and purified three PA mutants, I394C, A396C, and N435C, which were impaired in expression in previous study. Among them, a novel mutant N435C which conferred dominant-negative inhibitory activity on PA was identified. This new mutant may be useful in designing new antitoxin for anthrax prophylaxis and therapy.     

Complement component C3 is required for protective innate and adaptive immunity to larval strongyloides stercoralis in mice

This study examines the role of complement components C3 and C5 in innate and adaptive protective immunity to larval Strongyloides stercoralis in mice. Larval survival in naive C3(-/-) mice was increased as compared with survival in wild-type mice, whereas C3aR(-/-) and wild-type mice had equivalent levels of larval killing. Larval killing in naive mice was shown to be a coordinated effort between effector cells and C3. There was no difference between survival in wild-type and naive C5(-/-) mice, indicating that C5 was not required during the innate immune response. Naive B cell-deficient and wild-type mice killed larvae at comparable levels, suggesting that activation of the classical complement pathway was not required for innate immunity. Adaptive immunity was equivalent in wild-type and C5(-/-) mice; thus, C5 was also not required during the adaptive immune response. Larval killing was completely ablated in immunized C3(-/-) mice, even though the protective parasite-specific IgM response developed and effector cells were recruited. Protective immunity was restored to immunized C3(-/-) mice by transferring untreated naive serum, but not C3-depleted heat-inactivated serum to the location of the parasites. Finally, immunized C3aR(-/-) mice killed larvae during the adaptive immune response as efficiently as wild-type mice. Therefore, C3 was not required for the development of adaptive immunity, but was required for the larval killing process during both protective innate and adaptive immune responses in mice against larval S. stercoralis.     

Effect of 2-fluorohistidine labeling of the anthrax protective antigen on stability, pore formation, and translocation

The action of anthrax toxin relies in part upon the ability of the protective antigen (PA) moiety to form a heptameric pore in the endosomal membrane, providing a portal for entry of the enzymic moieties of the toxin into the cytosol. Pore formation is dependent on a conformational change in the heptameric prepore that occurs in the neutral to mildly acidic pH range, and it has been hypothesized that protonation of one or more histidine residues triggers this transition. To test this hypothesis, we used biosynthetic methods to incorporate the unnatural amino acid analogue 2-fluorohistidine (2-FHis) into PA. 2-FHis is isosteric with histidine but resists protonation at physiological pH values due to a dramatically reduced side-chain pKa ( approximately 1). We found that 2-FHis-labeled PA was biologically inactive, as judged by its inability to deliver a model intracellular effector, LFN-DTA, to the cytosol of CHO-K1 cells. However, whereas 2-FHis blocked a conformational transition in the full-length PA83 protein in the pH 5-6 range, the pH dependence of prepore-to-pore conversion of (PA63)7 was unchanged from the wild-type protein, implying that this conversion is not dependent on His protonation. Consistent with this result, the labeled, trypsin-activated PA was able to permeabilize liposomes to K+ and retained pore-forming activity in planar phospholipid bilayers. The pores in planar bilayers were incapable, however, of translocating a model ligand in response to a transmembrane pH gradient or elevated voltage. The results indicate that protonation of residues other than His, presumably Glu and/or Asp side chains, triggers pore formation in vitro, but His residues are nonetheless important for PA functioning in vivo.