Anthrax toxin: a tripartite lethal combination

Anthrax is a severe bacterial infection that occurs when Bacillus anthracis spores gain access into the body and germinate in macrophages, causing septicemia and toxemia. Anthrax toxin is a binary A-B toxin composed of protective antigen (PA), lethal factor (LF), and edema factor (EF). PA mediates the entry of either LF or EF into the cytosol of host cells. LF is a zinc metalloprotease that inactivates mitogen-activated protein kinase kinase inducing cell death, and EF is an adenylyl cyclase impairing host defences. Inhibitors targeting different steps of toxin activity have recently been developed. Anthrax toxin has also been exploited as a therapeutic agent against cancer.     

Construction and characterization of a protective antigen-deficient Bacillus anthracis strain

The pag gene coding for protective antigen (PA), one of the three toxin components of Bacillus anthracis, has been cloned into the mobilizable shuttle vector pAT187 and transferred by conjugation from Escherichia coli to B. anthracis. Using this strategy, an insertionally mutated pag gene constructed and characterized in E. coli, was introduced into B. anthracis Sterne strain. This transconjugant was used to select a recombinant clone (RP8) carrying the inactivated pag gene on the toxin-encoding plasmid, pXO1. Strain RP8 was deficient for PA while still producing the two other toxin components, i.e. lethal factor (LF) and edema factor (EF). In contrast to spores from the wild-type Sterne strain, spores prepared from RP8 were totally non-lethal in mice. These results clearly establish the central role played by PA in B. anthracis pathogenicity.     

Anthrax toxin components stimulate chemotaxis of human polymorphonuclear neutrophils

Effects of the three-component toxin of Bacillus anthracis on chemotaxis of human polymorphonuclear leukocytes (PMN) were investigated in an effort to determine the basis of the reported antiphagocytic effect of the toxin. The three toxin components, edema factor (EF), protective antigen (PA), and lethal factor (LF), were tested alone and in various combinations for their effect on PMN chemotaxis under agarose to formyl peptides and zymosan-activated serum. No component was active alone; combinations of EF + PA, LF + PA, and EF + LF + PA markedly stimulated chemotaxis (directed migration), but had little or no effect on unstimulated random migration. The toxin components were not themselves chemoattractants. EF in combination with PA had previously been identified as an adenylate cyclase in Chinese hamster ovary (CHO) cells. We found that EF + PA produced detectable cyclic adenosine 3'-5'monophosphate (cAMP) in PMN, but the level of cAMP was less than 1% of that produced in CHO cells by EF + PA, and in PMN by other bacterial adenylate cyclases. LF + PA (which stimulated chemotaxis to an equivalent extent) had no effect on cAMP levels. Thus, the enhancement of chemotaxis by anthrax toxin (at least by LF + PA) does not seem to be related to adenylate cyclase activity.     

Effect of pH on stability of anthrax lethal factor: correlation between denaturation and activity

Anthrax is caused by Gram positive bacterium Bacillus anthracis. Pathogenesis is result of production of three protein components, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA in combination with LF (lethal toxin) is lethal to animals, while PA in combination with EF (edema toxin), causes edema. PA, LF, and EF are very thermolabile. Differential scanning calorimetry (DSC) was used to unravel the energetics of LF denaturation as a function of pH ranging from 7.8 to 5.5. Transition temperature (T(m)) of LF was found to be approximately equal to 42 degrees C and onset of denaturation occurs at approximately equal to 30 degrees C. The ratio of calorimetric to van't Hoff's enthalpy was nearly equal to unity at pH 7.0, indicative of presence of single structural domain in LF at pH 7.0, unlike PA which has been structurally observed to consist of 4 domains. It was found by cytotoxicity studies using J774A.1 macrophage like cells that LF was most stable at pH approximately 6.5. This paper reports for the first time the denaturation of LF at different pH values at 37 degrees C and tries to establish a correlation between denaturation and loss of LF activity at different pH values.     

利用转基因小鼠筛选全人源炭疽致死因子中和抗体

炭疽是由炭疽芽孢杆菌引起的严重威胁人类健康的传染病。炭疽毒素包括3种蛋白质成分:保护性抗原(PA)、致死因子(LF)和水肿因子(EF)。PA与LF形成致死毒素(LT),与EF形成水肿毒素(ET)。由于致死毒素(LT)在感染者损伤及死亡中发挥主要作用,因此在炭疽感染晚期单纯使用抗生素治疗难以发挥疗效,治疗性中和抗体成为目前最有效的炭疽治疗药物。目前国外获得的炭疽毒素抗体多为炭疽PA抗体,美国FDA已批准瑞西巴库(人源PA单抗)用于吸入性炭疽的治疗。一旦炭疽芽孢杆菌被人为改构或PA中和表位发生突变,针对PA单一表位的抗体将可能失效,因此针对LF的抗体将成为炭疽治疗的有效补充。目前国外已有的LF抗体多为鼠源抗体和嵌合抗体,而全人源抗体可以避免鼠源抗体免疫原性高等缺点。本研究首先用LF抗原免疫人抗体转基因小鼠,利用流式细胞仪从小鼠脾淋巴细胞中分选抗原特异的记忆B细胞,通过单细胞PCR方法快速获得两株具有结合活性的抗LF单抗1D7和2B9。瞬时转染Expi 293F细胞制备抗体,通过毒素中和实验(TNA)发现1D7和2B9在细胞模型中均显示较好的中和活性,并且与PA单抗联合使用时,表现出较好的协同作用。总之,本文利用转基因小鼠、流式分选技术和单细胞PCR技术的优势,快速筛选到全人源LF抗体,为快速筛选全人源单克隆抗体开辟了新的思路与方法。     

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.     

A Bacillus anthracis strain deleted for six proteases serves as an effective host for production of recombinant proteins

Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. In this study we show that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pXO1?, pXO2?), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (InhA1, InhA2, camelysin, TasA, NprB, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pXO1? A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXO1-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or Escherichia coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10mg pure protein per liter of culture.     

Anthrax toxin evades Toll-like receptor recognition, whereas its cell wall components trigger activation via TLR2/6 heterodimers

Bacillus anthracis is a Gram-positive bacillus that is the causative agent of anthrax. The virulence of the bacillus is partly due to the production of a tripartite virulence factor: protective antigen (PA), lethal factor (LF) and edema factor (EF). Recognition of the bacillus and its toxins by the innate immune system is likely to play a key role following infection. In this study we set out to investigate whether anthrax cell wall (ACW) components as well as the lethal toxin are sensed by Toll-like receptors (TLRs). Our data suggest that ACW components as well as PA are sensed by TLR2/6 heterodimers triggering an inflammatory response. This recognition takes place on the cell surface within specialized microdomains for ACW, whereas PA seems to trigger responses intracellularly. Interestingly, LF does not trigger a pro-inflammatory response, and when combined with PA, the complex is not sensed by the innate immune system. Overall our data suggest that TLR2/6 heterodimers are responsible for sensing the ACW and PA, whereas the formation of the subsequent toxin (LF + PA) seems to evade detection by the innate immune system contributing to the virulence of the toxin.     

Proteome analysis of mouse macrophages treated with anthrax lethal toxin

Anthrax toxin produced by Bacillus anthracis is a tripartite toxin comprising of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the receptor-binding component, which facilitates the entry of LF or EF into the cytosol. EF is a calmodulin-dependent adenylate cyclase that causes edema whereas LF is a zinc metalloprotease and leads to necrosis of macrophages. It is also important to note that the exact mechanism of LF action is still unclear. With this view in mind, in the present study, we investigated a proteome wide effect of anthrax lethal toxin (LT) on mouse macrophage cells (J774A.1). Proteome analysis of LT-treated and control macrophages revealed 41 differentially expressed protein spots, among which phosphoglycerate kinase I, enolase I, ATP synthase (beta subunit), tubulin beta2, gamma-actin, Hsp70, 14-3-3 zeta protein and tyrosine/tryptophan-3-monooxygenase were found to be down-regulated, while T-complex protein-1, vimentin, ERp29 and GRP78 were found to be up-regulated in the LT-treated macrophages. Analysis of up- and down-regulated proteins revealed that primarily the stress response and energy generation proteins play an important role in the LT-mediated macrophage cell death.     

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。     

Purification and Properties of In Vitro–produced Anthrax Toxin Components

The three components of the toxin of Bacillus anthracis, edema factor (EF), protective antigen (PA), and lethal factor (LF), were purified 197-, 156-, and 1,025- fold, with 38, 78, and 11% recovery, respectively. Each purified component was serologically active, distinct, and free from the other components. The purified EF produced edema when mixed with PA, and the purified PA was an active immunogen. The components did not appear to be simple proteins by spectrophotometric analysis. As they were purified, the pH range in which they were most stable narrowed, centering between pH 7.4 and 7.8. Heat readily destroyed the biological activity of the components but not their serological activity. The rat lethality test showed that, with a constant amount of LF and an increasing amount of PA, the time to death reached a minimum and then was extended. When an increasing amount of LF was added to a constant amount of PA, the time to death became shorter as more LF was added. The biological, immunological, and serological properties of the components were shown to vary independently with storage and extent of purification so that serological activity was not always directly correlated with biological activity. Evidence is presented that the components can exist in different molecular configurations or as aggregates, and that this property is influenced by the state of component purity and by the environment.     

Anthrax toxin: the long and winding road that leads to the kill

The past five years have led to a tremendous increase in our molecular understanding of the mode of action of the anthrax toxin, one of the two main virulence factors produced by Bacillus anthracis. The structures of each of the three components of the toxin--lethal factor (LF), edema factor (EF) and protective antigen (PA)--have been solved not only in their monomeric forms but, depending on the subunit, in a heptameric form, bound to their substrate, co-factor or receptor. The endocytic route followed by the toxin has also been unraveled and the enzymatic mechanisms of EF and LF elucidated.     

The early humoral immune response to Bacillus anthracis toxins in patients infected with cutaneous anthrax

Bacillus anthracis, the causative agent of anthrax, produces a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF), which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin and edema toxin, respectively. In this preliminary study, we characterized the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody responses observed following infection were directed against LF, with immunoglobulin G (IgG) detected as early as 4 days after the onset of symptoms in contrast to the later and lower EF- and PA-specific IgG responses. Unlike the case with infection, the predominant toxin-specific antibody response of those immunized with the US anthrax vaccine absorbed and UK anthrax vaccine precipitated licensed anthrax vaccines was directed against PA. We observed that the LF-specific human antibodies were, like anti-PA antibodies, able to neutralize toxin activity, suggesting the possibility that they may contribute to protection. We conclude that an antibody response to LF might be a more sensitive diagnostic marker of anthrax than to PA. The ability of human LF-specific antibodies to neutralize toxin activity supports the possible inclusion of LF in future anthrax vaccines.     

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.     

Cross-inhibition between furin and lethal factor inhibitors

Bacillus anthracis synthesizes two toxins composed of the three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). The cleavage of PA on the cell surface by the convertase furin leads to the translocation of LF and EF into the cytosol. We have investigated the cross-inhibitory activities of the furin inhibitors hexa-d-arginine amide (D6R) and nona-d-arginine amide (D9R), which block the proteolytic activation of PA; and of the LF inhibitor In-2-LF, a peptide hydroxamate. D6R and D9R inhibit LF with IC(50s) of 300 and 10microM, respectively; conversely, In-2-LF also inhibits furin (IC(50) 2microM). In-2-LF was efficiently cleaved by furin with the concomitant loss of inhibitory activity on both LF and furin. Incubation of In-2-LF with LF however generated a product that retained partial inhibitory activity against LF. Combined treatment of cells with D6R and In-2-LF enhanced protection against anthrax lethal toxin, indicating that combined administration of inhibitors could represent an effective therapeutic approach.     

Expression of anthrax lethal factor gene by osmolyte induction

The anthrax toxin consists of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA mediates the entry of LF and EF to the cytosol where they exert their effects. Although PA is the major component of the vaccines against anthrax, LF has also been found to play an important role in enhancing protective immunity. We have developed an osmolyte-inducible LF expression system. The protein expression system contributed no additional amino acids to the recombinant LF making it suitable for the human vaccine trials.     

Production and proteolytic assay of lethal factor from Bacillus anthracis

Bacillus anthracis is the causative agent of anthrax. The major virulence factors are a poly-D-glutamic acid capsule and three-protein component exotoxin, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa), respectively. These three proteins individually have no known toxic activities, but in combination with PA form two toxins (lethal toxin or edema toxin), causing different pathogenic responses in animals and cultured cells. In this study, we constructed and produced rLF as a form of GST fusion protein in Escherichia coli. rLF was rapidly purified through a single affinity purification step to near homogeneity. Furthermore, we developed an in vitro immobilized proteolytic assay of LF under the condition containing full-length native substrate, MEK1, rather than short synthetic peptide. The availability of full-length substrate and of an immobilized LF assay could facilitate not only the in-depth investigation of structure-function relationship of the enzyme toward its substrate but also wide spectrum screening of inhibitor collections based on the 96-well plate system.     

Production of biologically active Bacillus anthracis edema factor in Escherichia coli

Anthrax is caused by the gram-positive spore-forming bacterium Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA facilitates the translocation of LF and EF into the cytosol of mammalian cells. LF is thought to be a zinc-dependent metalloprotease that results in death. EF is a calmodulin- and calcium-dependent adenylate cyclase that causes edema upon entrance into the cytosol by elevating the cAMP levels in cells. Previous efforts to produce recombinant EF (rEF) in Escherichia coli yielded only 2.5 mg of rEF per liter of culture. In this work, we produced soluble rEF in large quantities in both the periplasm and cytoplasm of E. coli from shake flasks and fermentors. The rEF protein was purified by standard chromatography and yielded >97% pure, biologically active rEF. Yields of purified rEF from medium cell density fermentations resulted in up to 2.38 g/L of highly pure, biologically active rEF protein. These results exhibit the ability to generate gram quantities of active rEF from E. coli.     

炭疽毒素及其细胞受体的研究进展

炭疽毒素由 3种蛋白组成 :保护性抗原 (protectiveantigen ,PA)、致死因子 (lethalfactor,LF)和水肿因子 (edemafactor ,EF) .综述炭疽毒素研究的最新进展 .主要介绍炭疽毒素的关键致病因子———LF的结构与功能 ,炭疽毒素膜转运成分PA的结构及其受体 (anthraxtoxinreceptor ,ATR)和其cDNA克隆的结构 ,并讨论了在炭疽的治疗、预防和毒素在肿瘤治疗中的可能应用 .     

The carboxyl-terminal end of protective antigen is required for receptor binding and anthrax toxin activity.

Anthrax toxin consists of three separate proteins produced by Bacillus anthracis: protective antigen (PA), lethal factor (LF), and edema factor (EF). Previous work showed that the process by which these proteins damage eukaryotic cells begins with binding of PA (83 kDa) to cell surface receptors. PA is then cleaved by a cell surface protease so as to expose a high-affinity binding site for LF or EF on the COOH-terminal, receptor-bound, 63-kilodalton fragment. In this report we more closely define a region of PA involved in receptor binding. The gene encoding PA was mutagenized so as to delete 3, 5, 7, 12, or 14 amino acids from the carboxyl terminus of the protein, and the truncated PA variants were purified from Bacillus subtilis or Escherichia coli. Deletion of 3, 5, or 7 amino acids reduced the binding of PA to cells and the subsequent toxicity of the PA.LF complex to J774A.1 cells and also the ability to cause EF binding to cells. Deletion of 12 or 14 amino acids completely eliminated all these activities. These results show that the carboxy terminus comprises or is part of the receptor-binding domain of PA.