Asporogenic recombinant producer of anthrax protective antigen

An asporogenic recombinant strain Bacillus anthracis 55ΔTPA-1(Spo⁻) producing anthrax protective antigen (PA) was obtained. The strain contains structural gene pag as a part of a hybrid replicon pUB110PA-1 and lacks determinants encoding the synthesis of main factors of anthrax pathogenicity. The level of PA production by asporogenic genetically engineered strain is approximately 80 μg/ml that is 4–5 times more than the values determined for vaccine strains B. anthracis STI-1 and B. anthracis 55. The strain preserves asporogenicity and ability to replicate the hybrid plasmid after in vitro passages. Biologically active PA was isolated from the constructed strain B. anthracis 55ΔTPA-1(Spo⁻). Double immunization of rabbits with 50 μg of the purified recombinant product provides their 100% protection from infection with 50 LD₅₀ of a highly virulent anthrax strain.     

Acid induced unfolding of anthrax protective antigen

Acidic pH plays an important role in the membrane insertion of protective antigen (PA) of anthrax toxin leading to the translocation of the catalytic moieties. The structural transitions occurring in PA as a consequence of change in pH were investigated by fluorescence and circular dichroism measurements. Our studies revealed the presence of two intermediates on-pathway of acid induced unfolding; one at pH 2.0 and other at pH 4-5. Intrinsic fluorescence measurements of these intermediates showed a red shift in the wavelength of emission maximum with a concomitant decrease in fluorescence intensity, indicative of the exposure of tryptophan residues to the bulk solvent. Furthermore, no significant change was seen in the secondary structure of PA at a pH of 2.0, as indicated by far UV-CD spectra. The low pH intermediate of PA was characterized using the hydrophobic dye, 8-anilino-1-naphthalenesulfonate, and was found to have properties similar to those of a molten globule state.     

Characterization of the isolated 20 kDa and 50 kDa fragments of the myosin head

We have isolated two proteolytic fragments of subfragment 1 (S-1) of myosin from rabbit skeletal muscle. These fragments, identified by their molecular weights of 20 and 50 kDa, may be functional domains that, when isolated, retain their specific function. We have studied several structural and functional features of the 20 and 50 kDa fragments. Considerable secondary structure in both fragments has been observed in CD spectrum studies. Previously CD spectra showed 64% ordered structure for the 20 kDa fragment (Muhlrad and Morales, M.F. (1984) Proc. Natl. Acad. Sci. 81, 1003) and here we show 71% ordered structures for the 50 kDa fragment. Fluorescence lifetime studies of tryptophan residues in the 50 kDa fragment and 1,5-IAEDANS-labeled SH-1 in the 20 kDa fragment are used to investigate the tertiary structure of the fragments. We find the tertiary structure relating to this measurement of both fragments to be intact; however, the reaction of 1,5-IAEDANS with SH-1 on the isolated 20 kDa fragment is less specific than with S-1. Furthermore, the fragments showed a tendency to aggregate. The domain concept of S-1 was supported by the characteristic biochemical function of the isolated fragments. Both of the fragments were effective in competing with S-1 for binding to actin in acto-S-1 ATPase measurements. From these studies and in direct binding measurement the 20 kDa fragment proved to bind with higher affinity to actin than did the 50 kDa fragment.     

Interactions between anthrax toxin receptors and protective antigen

Since the anthrax mail attacks of 2001, much has been learned about the interactions between anthrax toxin and its receptors. Two distinct cellular receptors for anthrax toxin have been identified and are designated capillary morphogenesis protein 2 (CMG2) and anthrax toxin receptor/tumor endothelial marker 8 (ATR/TEM8). The molecular details of the toxin-receptor interactions have been revealed through crystallographic, biochemical and genetic studies. In addition, a novel pathway by which anthrax toxin enters cells is starting to be uncovered.     

Characterization of dominant-negative forms of anthrax protective antigen

Certain mutations within the protective antigen (PA) moiety of anthrax toxin endow the protein with a dominant-negative (DN) phenotype, converting it into a potent antitoxin. Proteolytically activated PA oligomerizes to form ring-shaped heptameric complexes that insert into the membrane of an acidic intracellular compartment and promote translocation of bound edema factor and/or lethal factor to the cytosol. DN forms of PA co-oligomerize with the wild-type protein and block the translocation process. We prepared and characterized 4 DN forms: a single, a double, a triple, and a quadruple mutant. The mutants were made by site-directed mutation of the cloned form of PA in Escherichia coli and tested by various assays conducted on CHO cells or in solution. All 4 mutant PAs were competent for heptamerization and ligand binding but were defective in the pH-dependent functions: pore formation, ability to convert to the SDS-resistant heptamer, and ability to translocate bound ligand. The single mutant (F427K) showed less attenuation than the others in the pH-dependent functions and lower DN activity in a CHO cell assay. The quadruple (K397D + D425K + F427A + 2beta2-2beta3) deletion showed the most potent DN activity at low concentrations but also gave indications of low stability in a urea-mediated unfolding assay. The double mutant (K397D + D425K) and the triple (K397D + D425K + F427A) showed strong DN activity and slight reduction in stability relative to the wild-type protein. The properties of the double and the triple mutants make these forms worthy of testing in vivo as a new type of antitoxic agent for treatment of anthrax.     

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.     

针对炭疽保护性抗原不同结构域的中和性单克隆抗体的筛选和鉴定

炭疽保护性抗原（PA）是炭疽毒素的重要组分,同时也是现有炭疽疫苗的主要有效成分,在炭疽杆菌的致病与免疫中发挥关键作用。以重组PA为免疫原,采用B淋巴细胞杂交瘤技术,结合炭疽毒素敏感细胞的毒性中和试验,大量筛选抗PA单克隆抗体,获得了9株炭疽毒素中和性单抗。进一步分析表明这些单抗以IgG1亚类为主,分别识别PA 3个结构域的4个不同中和表位区。针对结构域2的4株单抗识别同一表位区,其中3株单抗的中和活性强于抗PA多抗;针对结构域4的4株单抗识别两个不同表位区;另有1株单抗识别位于结构域3的表位。实验结果提示PA具有多个中和表位,分别位于其不同结构域,其中结构域2、4包含主要中和表位。实验中获得的针对不同表位的中和性单抗为深入研究PA的免疫保护机理提供了工具,也为研制针对炭疽毒素的被动免疫制剂和治疗药物打下基础。     

Screening and characterization of high-affinity ssDNA aptamers against anthrax protective antigen

The protective antigen (PA) of Bacillus anthracis is a secreted protein that functions as a critical virulence factor. Protective antigen has been selected as a biomarker in detecting bacterial infection. The in vitro selection method, systematic evolution of ligands by exponential enrichment (SELEX), was used to find single-stranded DNAs that were tightly bound to PA. After 8 rounds of the SELEX process with PA, 4 different oligonucleotides (referred to as aptamers) that contain a 30-residue ssDNA sequence were identified. Dissociation constant (K(d)) values with Cy3-attached aptamers were determined via fluorophotometry to be within a nanomolar range. The authors attempted to visualize the detection of PA using an aptamer-based enzyme-linked immunosorbent assay method, which has proven to be successful within a nanomolar K(d) value range. Furthermore, 2 of the 4 aptamers exhibited specificity to PA against bovine serum albumin and bovine serum. The results of this study demonstrate the analytical potential of an oligonucleotide-based biosensor for a wide variety of applications, particularly in diagnosing disease through specific protein biomarkers.     

Exchange characteristics of calcium ions bound to anthrax protective antigen

Protective antigen (PA), the receptor-binding moiety of anthrax toxin, contains two calcium atoms buried within domain 1(') (amino acid residues 168-258). We showed that these ions are stably bound and exchange with free 45Ca(2+) only slowly (t(1/2) approximately 4.0 h). Dissociation is the rate-limiting step. PA(63), the heptameric prepore form of PA, showed a slightly higher exchange rate than the monomeric intact protein. Exchange by this form was retarded by binding of the enzymatic moieties of the toxin, but was unaffected by reducing the pH to 5.0, a condition known to trigger conversion of the prepore to the pore form. These results are consistent with the hypothesis that bound Ca(2+) within PA plays primarily a structural role, maintaining domain 1(') in a conformation that allows PA(63) to oligomerize and bind the enzymatic moieties of the toxin.     

Monitoring anthrax toxin receptor dissociation from the protective antigen by NMR

The binding of the Bacillus anthracis protective antigen (PA) to the host cell receptor is the first step toward the formation of the anthrax toxin, a tripartite set of proteins that include the enzymatic moieties edema factor (EF), and lethal factor (LF). PA is cleaved by a furin‐like protease on the cell surface followed by the formation of a donut‐shaped heptameric prepore. The prepore undergoes a major structural transition at acidic pH that results in the formation of a membrane spanning pore, an event which is dictated by interactions with the receptor and necessary for entry of EF and LF into the cell. We provide direct evidence using 1‐dimensional ¹³C‐edited ¹H NMR that low pH induces dissociation of the Von‐Willebrand factor A domain of the receptor capillary morphogenesis protein 2 (CMG2) from the prepore, but not the monomeric full length PA. Receptor dissociation is also observed using a carbon‐13 labeled, 2‐fluorohistidine labeled CMG2, consistent with studies showing that protonation of His‐121 in CMG2 is not a mechanism for receptor release. Dissociation is likely caused by the structural transition upon formation of a pore from the prepore state rather than protonation of residues at the receptor PA or prepore interface.     

Insertion of anthrax protective antigen into liposomal membranes: effects of a receptor

Protective antigen (PA), the receptor-binding component of anthrax toxin, heptamerizes and inserts into the endosomal membrane at acidic pH, forming a pore that mediates translocation of the enzymic components of the toxin to the cytosol. When the heptameric pre-insertion form of PA (the prepore) is acidified in solution, it rapidly loses the ability to insert into membranes. To maximize insertion into model membranes, we examined two ways to bind the protein to large unilamellar vesicles (LUV). One involved attaching a His tag to the von Willebrand factor A domain of one of the PA receptors, ANTXR2, and using this protein as a bridge to bind PA to LUV containing a nickel-chelating lipid. The other involved using a His tag fused to the C terminus of PA to bind the protein directly to LUV containing the same lipid. Both ways enhanced pore formation at pH 5.0 strongly and about equally, as measured by the release of K+. Controls showed that pore formation in this system faithfully reproduced that in vivo. We also showed that binding unmodified ANTXR2 von Willebrand factor A to the prepore in solution enhanced its pore forming activity by slowing its inactivation at acidic pH. These findings indicate that an important role of PA receptors is to promote partitioning of PA into the bilayer by maintaining the prepore close to the target membrane and presumably in the optimal orientation as it undergoes the acidic pH-dependent conformational transition to the pore.     

Domain flexibility modulates the heterogeneous assembly mechanism of anthrax toxin protective antigen

The three protein components of anthrax toxin are nontoxic individually, but they form active holotoxin complexes upon assembly. The role of the protective antigen (PA) component of the toxin is to deliver two other enzyme components, lethal factor and edema factor, across the plasma membrane and into the cytoplasm of target cells. PA is produced as a proprotein, which must be proteolytically activated; generally, cell surface activation is mediated by a furin family protease. Activated PA can then assemble into one of two noninterconverting oligomers, a homoheptamer and a homooctamer, which have unique properties. Herein we describe molecular determinants that influence the stoichiometry of PA in toxin complexes. By tethering PA domain 4 (D4) to domain 2 with two different-length cross-links, we can control the relative proportions of PA heptamers and octamers. The longer cross-link favors octamer formation, whereas the shorter one favors formation of the heptamer. X-ray crystal structures of PA (up to 1.45 Å resolution), including these cross-linked PA constructs, reveal that a hinge-like movement of D4 correlates with the relative preference for each oligomeric architecture. Furthermore, we report the conformation of the flexible loop containing the furin cleavage site and show that, for efficient processing, the furin site cannot be moved ～ 5 or 6 residues within the loop. We propose that there are different orientations of D4 relative to the main body of PA that favor the formation of either the heptamer or the octamer.     

Lethal factor of anthrax toxin binds monomeric form of protective antigen

Anthrax toxin consists of three components: the enzymatic moieties edema factor (EF) and the lethal factor (LF) and the receptor-binding moiety protective antigen (PA). These toxin components are released from Bacillus anthracis as unassociated proteins and form complexes on the surface of host cells after proteolytic processing of PA into PA20 and PA63. The sequential order of PA heptamerization and ligand binding, as well as the exact mechanism of anthrax toxin entry into cells, are still unclear. In the present study, we provide direct evidence that PA63 monomers are sufficient for binding to the full length LF or its LF-N domain, though with lower affinity with the latter. Therefore, PA oligomerization is not a necessary condition for LF/PA complex formation. In addition, we demonstrated that the PA20 directly interacts with the LF-N domain. Our data points to an alternative process of self-assembly of anthrax toxin on the surface of host cells.     

Both PA63 and PA83 are endocytosed within an anthrax protective antigen mixed heptamer: a putative mechanism to overcome a furin deficiency

Anthrax toxin consists of protective antigen (PA), and lethal (LF) and edema (EF) factors. A 83 kDa PA monomer (PA83) precursor binds to the cell receptor. Furin-like proprotein convertases (PCs) cleave PA83 to generate cell-bound 63 kDa protein (PA63). PA63 oligomerizes to form a ring-shaped heptamer that binds LF-EF and facilitates their entry into the cells. Several additional PCs, as opposed to furin alone, are capable of processing PA83. Following the incomplete processing of the available pool of PA83, the functional heptamer includes both PA83 and PA63. The available structures of the receptor-PA complex imply that the presence of either one or two molecules of PA83 will not impose structural limitations on the formation of the heptamer and the association of either the (PA83)(1)(PA63)(6) or (PA83)(2)(PA63)(5) heteroheptamer with LF-EF. Our data point to the intriguing mechanism of anthrax that appears to facilitate entry of the toxin into the cells which express limiting amounts of PCs and an incompletely processed PA83 pool.     

pH-dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen

Protective antigen (PA) of anthrax toxin forms ion-conductive channels in planar lipid bilayers and liposomes under acidic pH conditions. We show here that PA has a similar permeabilizing action on the plasma membranes of CHO-K1 and three other mammalian cell lines (J774A.1, RAW264.7 and Vero). Changes in membrane permeability were evaluated by measuring the efflux of the K⁺ analogue, ⁸⁶Rb⁺, from prelabelled cells, and the influx of ²²Na⁺. The permeabilizing activity of PA was limited to a proteolytically activated form (PA_N) and was dependent on acidic pH for membrane insertion (optimal at pH 5.0), but not for sustained ion flux. The flux was reduced in the presence of several known channel blockers: tetrabutyl-, tetrapentyl-, and tetrahexylammonium bromides. PA_N facilitated the membrane translocation of anthrax edema factor under the same conditions that induced changes in membrane permeability to ions. These results indicate that PA_N permeabilizes cellular membranes under conditions that are believed to prevail in the endosomal compartment of toxin-sensitive cells; and they provide a basis for more detailed studies of the relationship between channel formation and translocation of toxin effector moieties in vivo.     

Mapping the anthrax protective antigen binding site on the lethal and edema factors.

Entry of anthrax edema factor (EF) and lethal factor (LF) into the cytosol of eukaryotic cells depends on their ability to translocate across the endosomal membrane in the presence of anthrax protective antigen (PA). Here we report attributes of the N-terminal domains of EF and LF (EF(N) and LF(N), respectively) that are critical for their initial interaction with PA. We found that deletion of the first 36 residues of LF(N) had no effect on its binding to PA or its ability to be translocated. To map the binding site for PA, we used the three-dimensional structure of LF and sequence similarity between EF and LF to select positions for mutagenesis. We identified seven sites in LF(N) (Asp-182, Asp-187, Leu-188, Tyr-223, His-229, Leu-235, and Tyr-236) where mutation to Ala produced significant binding defects, with H229A and Y236A almost completely eliminating binding. Homologous mutants of EF(N) displayed nearly identical defects. Cytotoxicity assays confirmed that the LF(N) mutations impact intoxication. The seven mutation-sensitive amino acids are clustered on the surface of LF and form a small convoluted patch with both hydrophobic and hydrophilic character. We propose that this patch constitutes the recognition site for PA.     

Structural determinants for the binding of anthrax lethal factor to oligomeric protective antigen

Anthrax lethal toxin assembles at the surface of mammalian cells when the lethal factor (LF) binds via its amino-terminal domain, LF(N), to oligomeric forms of activated protective antigen (PA). LF x PA complexes are then trafficked to acidified endosomes, where PA forms heptameric pores in the bounding membrane and LF translocates through these pores to the cytosol. We used enhanced peptide amide hydrogen/deuterium exchange mass spectrometry and directed mutagenesis to define the surface on LF(N) that interacts with PA. A continuous surface encompassing one face of LF(N) became protected from deuterium exchange when LF(N) was bound to a PA dimer. Directed mutational analysis demonstrated that residues within this surface on LF(N) interact with Lys-197 on two PA subunits simultaneously, thereby showing that LF(N) spans the PA subunit:subunit interface and explaining why heptameric PA binds a maximum of three LF(N) molecules. Our results elucidate the structural basis for anthrax lethal toxin assembly and may be useful in developing drugs to block toxin action.     

Mechanism of membrane translocation by anthrax toxin: insertion and pore formation by protective antigen

Proteolytic activation of receptor-bound protective antigen (PA) at the cell surface removes PA₂₀, allowing PA₆₃ to oligomerize and form a ring-shaped heptameric prepore. The prepore binds edema factor (EF) and lethal factor (LF) and, after endocytosis and trafficking of the complex to an acidic, vesicular compartment, it undergoes membrane insertion and mediates translocation of EF/LF to the cytosol. Data from membrane conductance experiments support a model of membrane insertion in which the 2β2–2β3 loop of PA, which is disordered in native PA and the prepore, forms a 14-stranded transmembrane β-barrel. Recent studies on the process of prepore-to-pore conversion and our current understanding of the mechanism of pH-dependent translocation will be described.     

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.