scholarly journals Membrane insertion of anthrax protective antigen and cytoplasmic delivery of lethal factor occur at different stages of the endocytic pathway

2004 ◽  
Vol 166 (5) ◽  
pp. 645-651 ◽  
Author(s):  
Laurence Abrami ◽  
Margaret Lindsay ◽  
Robert G. Parton ◽  
Stephen H. Leppla ◽  
F. Gisou van der Goot
Keyword(s):  
Protective Antigen ◽  
Lethal Factor ◽  
Endocytic Pathway ◽  
Cell Surface Receptor ◽  
Membrane Insertion ◽  
Edema Factor ◽  
Early Endosomes ◽  
Surface Receptor ◽  
A Cell ◽  
Anthrax Protective Antigen

The protective antigen (PA) of anthrax toxin binds to a cell surface receptor, undergoes heptamerization, and binds the enzymatic subunits, the lethal factor (LF) and the edema factor (EF). The resulting complex is then endocytosed. Via mechanisms that depend on the vacuolar ATPase and require membrane insertion of PA, LF and EF are ultimately delivered to the cytoplasm where their targets reside. Here, we show that membrane insertion of PA already occurs in early endosomes, possibly only in the multivesicular regions, but that subsequent delivery of LF to the cytoplasm occurs preferentially later in the endocytic pathway and relies on the dynamics of internal vesicles of multivesicular late endosomes.

scholarly journals Identification of Amino Acid Residues of Anthrax Protective Antigen Involved in Binding with Lethal Factor

2002 ◽  
Vol 70 (8) ◽  
pp. 4477-4484 ◽  
Author(s):  
Vibha Chauhan ◽  
Rakesh Bhatnagar
Keyword(s):  
Cell Surface ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Lethal Toxin ◽  
Cell Surface Receptor ◽  
Amino Acid Residues ◽  
Anthrax Lethal Toxin ◽  
Mutant Proteins ◽  
Surface Receptors ◽  
Surface Receptor

ABSTRACT Protective antigen (PA) and lethal factor (LF) are the two components of anthrax lethal toxin. PA is responsible for the translocation of LF to the cytosol. The binding of LF to cell surface receptor-bound PA is a prerequisite for the formation of lethal toxin. It has been hypothesized that hydrophobic residues P184, L187, F202, L203, P205, I207, I210, W226, and F236 of domain 1b of PA play an important role in the binding of PA to LF. These residues are normally buried in the 83-kDA version of PA, PA83, as determined by the crystal structure of PA. However, they become exposed due to the conformational change brought about by the cleavage of PA83 to PA63 by a cell surface protease. Mutation of the above-mentioned residues to alanine resulted in mutant proteins that were able to bind to the cell surface receptors and also to be specifically cleaved by the cellular proteases. All the mutant proteins except the F202A, L203A, P205A, and I207A mutants were able to bind to LF and were also toxic to macrophage cells in combination with LF. It was concluded that residues 202, 203, 205, and 207 of PA are essential for the binding of LF to PA.

scholarly journals Structure of a dominant negative mutant reveals a stalled intermediate state of anthrax protective antigen pore maturation

2020 ◽  
Author(s):  
Harry Scott ◽  
Wei Huang ◽  
Srinivas Gonti ◽  
Kaiming Zhang ◽  
Nurjahan Mehzabeen ◽  
...  
Keyword(s):  
Pore Formation ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Edema Factor ◽  
Beta Barrel ◽  
Anthrax Protective Antigen ◽  
Maturation State ◽  
Membrane Spanning

AbstractAnthrax is a severe bacterial infection caused by Bacillus anthracis, which produces a tripartite toxin that includes protective antigen (PA), lethal factor (LF) and edema factor (EF). A series of dominant-negative mutations have been previously identified that prevent the heptameric PA prepore from forming the pH-induced, membrane spanning beta-barrel pore that is required for translocation of EF and LF to the cytoplasm of the infected cell. Here we show that the dominant negative D425A mutation stalls the formation of the pore at a reversible intermediate maturation state, which exhibits many of the structural aspects of the pore but fails to form the phi(ϕ)-clamp and beta-barrel structure needed for full pore maturation. Overall, this structure reveals that ϕ-clamp and beta-barrel pore formation are later steps in the pathway to pore formation, thereby providing a regulatory mechanism to prevent premature translocation of EF and LF.

scholarly journals Purification of Anthrax Edema Factor fromEscherichia coli and Identification of Residues Required for Binding to Anthrax Protective Antigen

2001 ◽  
Vol 69 (10) ◽  
pp. 6532-6536 ◽  
Author(s):  
Praveen Kumar ◽  
Nidhi Ahuja ◽  
Rakesh Bhatnagar
Keyword(s):  
Recombinant Protein ◽  
Protective Antigen ◽  
Metal Chelate ◽  
Lethal Factor ◽  
Exchange Chromatography ◽  
Biologically Active ◽  
E Coli ◽  
Edema Factor ◽  
Step Procedure ◽  
Anthrax Protective Antigen

ABSTRACT The structural gene for anthrax edema factor (EF) was expressed in Escherichia coli under the control of a powerful T5 promoter to yield the 89-kDa recombinant protein that reacted with anti-EF antibodies. Recombinant EF was purified to homogeneity by a two-step procedure involving metal chelate affinity chromatography and cation-exchange chromatography. From 1 liter of culture, 2.5 mg of biologically active EF was easily purified. This is the first report of purification of anthrax EF from E. coli. EF purified from E. coli was biologically and functionally as active as its Bacillus anthracis counterpart. The recombinant protein could compete with lethal factor for binding to protective antigen. Sequence analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile Gly Lys, present both in EF (residues 136 to 142) and lethal factor (residues 147 to 153). To investigate the role of these seven residues in binding to protective antigen, the residues were individually mutated to alanine in EF. Mutations in residues Tyr137, Tyr138, Ile140, and Lys142 of EF specifically blocked its interaction with anthrax protective antigen. The adenylate cyclase activity of the mutants remained unaffected. The results suggested that residues Tyr137, Tyr138, Ile140, and Lys142 are required for binding of EF to anthrax protective antigen, which facilitates its entry into susceptible cells.

scholarly journals Anthrax toxin triggers endocytosis of its receptor via a lipid raft–mediated clathrin-dependent process

2003 ◽  
Vol 160 (3) ◽  
pp. 321-328 ◽  
Author(s):  
Laurence Abrami ◽  
Shihui Liu ◽  
Pierre Cosson ◽  
Stephen H. Leppla ◽  
F. Gisou van der Goot
Keyword(s):  
Lipid Rafts ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Physiological Role ◽  
Membrane Lipid ◽  
Anthrax Toxin ◽  
Cell Surface Receptor ◽  
Surface Receptor ◽  
Toxin Receptor ◽  
Major Route

The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.

The transfer of transthyretin and receptor-binding properties from the plasma retinol-binding protein to the epididymal retinoic acid-binding protein

2002 ◽  
Vol 362 (2) ◽  
pp. 265-271 ◽  
Author(s):  
Manickavasagam SUNDARAM ◽  
Daan M. F. van AALTEN ◽  
John B. C. FINDLAY ◽  
Asipu SIVAPRASADARAO
Keyword(s):  
Retinoic Acid ◽  
Binding Protein ◽  
Binding Pocket ◽  
Retinol Binding Protein ◽  
Cell Surface Receptor ◽  
Acid Binding Protein ◽  
The Family ◽  
Surface Receptor ◽  
A Cell ◽  
Plasma Retinol

Members of the lipocalin superfamily share a common structural fold, but differ from each other with respect to the molecules with which they interact. They all contain eight β-strands (A—H) that fold to form a well-defined β-barrel, which harbours a binding pocket for hydrophobic ligands. These strands are connected by loops that vary in size and structure and make up the closed and open ends of the pocket. In addition to binding ligands, some members of the family interact with other macromolecules, the specificity of which is thought to be associated with the variable loop regions. Here, we have investigated whether the macromolecular-recognition properties can be transferred from one member of the family to another. For this, we chose the prototypical lipocalin, the plasma retinol-binding protein (RBP) and its close structural homologue the epididymal retinoic acid-binding protein (ERABP). RBP exhibits three molecular-recognition properties: it binds to retinol, to transthyretin (TTR) and to a cell-surface receptor. ERABP binds retinoic acid, but whether it interacts with other macromolecules is not known. Here, we show that ERABP does not bind to TTR and the RBP receptor, but when the loops of RBP near the open end of the pocket (L-1, L-2 and L-3, connecting β-strands A—B, C—D and E—F, respectively) were substituted into the corresponding regions of ERABP, the resulting chimaera acquired the ability to bind TTR and the receptor. L-2 and L-3 were found to be the major determinants of the receptor- and TTR-binding specificities respectively. Thus we demonstrate that lipocalins serve as excellent scaffolds for engineering novel biological functions.

scholarly journals A Chimeric Protein That Functions as both an Anthrax Dual-Target Antitoxin and a Trivalent Vaccine

2010 ◽  
Vol 54 (11) ◽  
pp. 4750-4757 ◽  
Author(s):  
Gaobing Wu ◽  
Yuzhi Hong ◽  
Aizhen Guo ◽  
Chunfang Feng ◽  
Sha Cao ◽  
...  
Keyword(s):  
Protective Antigen ◽  
Vaccine Candidate ◽  
Lethal Factor ◽  
Lethal Dose ◽  
Chimeric Protein ◽  
Dominant Negative ◽  
Lethal Challenge ◽  
Edema Factor ◽  
Trivalent Vaccine

ABSTRACT Effective measures for the prophylaxis and treatment of anthrax are still required for counteracting the threat posed by inhalation anthrax. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing the protective antigen (PA)-binding domain of lethal factor (LFn) to PA, retained the functions of the respective molecules. On the basis of this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF) to PA and the transportation of LF/EF. Therefore, we replaced PA in LFn-PA with a dominant-negative inhibitory PA (DPA), i.e., PAF427D. In in vitro models of anthrax intoxication, the LFn-DPA chimera showed 3-fold and 2-fold higher potencies than DPA in protecting sensitive cells against anthrax lethal toxin (LeTx) and edema toxin (EdTx), respectively. In animal models, LFn-DPA exhibited strong potency in rescuing mice from lethal challenge with LeTx. We also evaluated the immunogenicity and immunoprotective efficacy of LFn-DPA as an anthrax vaccine candidate. In comparison with recombinant PA, LFn-DPA induced significantly higher levels of the anti-PA immune response. Moreover, LFn-DPA elicited an anti-LF antibody response that could cross-react with EF. Mice immunized with LFn-DPA tolerated a LeTx challenge that was 5 times its 50% lethal dose. Thus, LFn-DPA represents a highly effective trivalent vaccine candidate for both preexposure and postexposure vaccination. Overall, we have developed a novel and dually functional reagent for the prophylaxis and treatment of anthrax.

scholarly journals Differential Contribution of Bacillus anthracis Toxins to Pathogenicity in Two Animal Models

2012 ◽  
Vol 80 (8) ◽  
pp. 2623-2631 ◽  
Author(s):  
Haim Levy ◽  
Shay Weiss ◽  
Zeev Altboum ◽  
Josef Schlomovitz ◽  
Itai Glinert ◽  
...  
Keyword(s):  
Animal Models ◽  
Guinea Pigs ◽  
Protective Antigen ◽  
Comprehensive Evaluation ◽  
Lethal Factor ◽  
Rabbit Model ◽  
Content Type ◽  
Edema Factor ◽  
Genes Encoding ◽  
Guinea Pig Model

ABSTRACTThe virulence ofBacillus anthracis, the causative agent of anthrax, stems from its antiphagocytic capsule, encoded by pXO2, and the tripartite toxins encoded by pXO1. The accepted paradigm states that anthrax is both an invasive and toxinogenic disease and that the toxins play major roles in pathogenicity. We tested this assumption by a systematic study of mutants with combined deletions of thepag,lef, andcyagenes, encoding protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively. The resulting seven mutants (single, double, and triple) were evaluated following subcutaneous (s.c.) and intranasal (i.n.) inoculation in rabbits and guinea pigs. In the rabbit model, virulence is completely dependent on the presence of PA. Any mutant bearing apagdeletion behaved like a pXO1-cured mutant, exhibiting complete loss of virulence with attenuation indices of over 2,500,000 or 1,250 in the s.c. or i.n. route of infection, respectively. In marked contrast, in guinea pigs, deletion ofpagor even of all three toxin components resulted in relatively moderate attenuation, whereas the pXO1-cured bacteria showed complete attenuation. The results indicate that a pXO1-encoded factor(s), other than the toxins, has a major contribution to the virulence mechanism ofB. anthracisin the guinea pig model. These unexpected toxin-dependent and toxin-independent manifestations of pathogenicity in different animal models emphasize the importance and need for a comprehensive evaluation ofB. anthracisvirulence in general and in particular for the design of relevant next-generation anthrax vaccines.

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