Person: Collier, Robert
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Collier
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Collier, Robert
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Publication Changing the Receptor Specificity of Anthrax Toxin(American Society of Microbiology, 2012) Mechaly, Adva; McCluskey, Andrew Jeffrey; Collier, RobertThe actions of many bacterial toxins depend on their ability to bind to one or more cell-surface receptors. Anthrax toxin acts by a sequence of events that begins when the protective-antigen (PA) moiety of the toxin binds to either one of two cell-surface proteins, ANTXR1 and ANTXR2, and is proteolytically activated. The activated PA self-associates to form oligomeric pore precursors, which, in turn, bind the enzymatic moieties of the toxin and transport them to the cytosol. We introduced a double mutation into domain 4 of PA to ablate its native receptor-binding function and fused epidermal growth factor (EGF) to the C terminus of the mutated protein. The resulting fusion protein transported enzymatic effector proteins into a cell line that expressed the EGF receptor (A431 cells), but not into a line lacking this receptor (CHO-K1 cells). Addition of excess free EGF blocked transport of effector proteins into A431 cells via the fusion protein, but not via native PA. We also showed that fusing the diphtheria toxin receptor-binding domain to the C terminus of the mutated PA channeled effector-protein transport through the diphtheria toxin receptor. PA fusion proteins with altered receptor specificity may be useful in biological research and could have practical applications, including ablation or perturbation of selected populations of cells in vivo.Publication Evidence for a Proton–Protein Symport Mechanism in the Anthrax Toxin Channel(Rockefeller University Press, 2009) Basilio, Daniel; Juris, Stephen J.; Collier, Robert; Finkelstein, AlanThe toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA\(_{63})_7\), formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA\(_{63})_7\) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LFN (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA\(_{63})_7\) channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LF\(_\text{N}\) enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton–protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO\(_3^−\), which is essentially not titratable, is introduced at most positions in LF\(_\text{N}\), through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF\(_\text{N}\) translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA\(_{63})_7\) channel resides at or near its Φ-clamp, the ring of seven phenylalanines near the channel's entrance.Publication Anthrax Toxin Receptor 2 Determinants that Dictate the pH Threshold of Toxin Pore Formation(Public Library of Science, 2007) Scobie, Heather M.; Marlett, John M.; Rainey, G. Jonah A.; Lacy, D. Borden; Collier, Robert; Young, John A.T.The anthrax toxin receptors, ANTXR1 and ANTXR2, act as molecular clamps to prevent the protective antigen (PA) toxin subunit from forming pores until exposure to low pH. PA forms pores at pH ∼6.0 or below when it is bound to ANTXR1, but only at pH ∼5.0 or below when it is bound to ANTXR2. Here, structure-based mutagenesis was used to identify non-conserved ANTXR2 residues responsible for this striking 1.0 pH unit difference in pH threshold. Residues conserved between ANTXR2 and ANTXR1 that influence the ANTXR2-associated pH threshold of pore formation were also identified. All of these residues contact either PA domain 2 or the neighboring edge of PA domain 4. These results provide genetic evidence for receptor release of these regions of PA as being necessary for the protein rearrangements that accompany anthrax toxin pore formation.Publication Anthrax Toxin Receptor 2-Dependent Lethal Toxin Killing In Vivo(Public Library of Science, 2006) Scobie, Heather M; Wigelsworth, Darran J; Marlett, John M; Thomas, Diane; Rainey, G. Jonah A; Lacy, D. Borden; Manchester, Marianne; Collier, Robert; Young, John A. TAnthrax toxin receptors 1 and 2 (ANTXR1 and ANTXR2) have a related integrin-like inserted (I) domain which interacts with a metal cation that is coordinated by residue D683 of the protective antigen (PA) subunit of anthrax toxin. The receptor-bound metal ion and PA residue D683 are critical for ANTXR1-PA binding. Since PA can bind to ANTXR2 with reduced affinity in the absence of metal ions, we reasoned that D683 mutant forms of PA might specifically interact with ANTXR2. We show here that this is the case. The differential ability of ANTXR1 and ANTXR2 to bind D683 mutant PA proteins was mapped to nonconserved receptor residues at the binding interface with PA domain 2. Moreover, a D683K mutant form of PA that bound specifically to human and rat ANTXR2 mediated killing of rats by anthrax lethal toxin, providing strong evidence for the physiological importance of ANTXR2 in anthrax disease pathogenesis.Publication Anthrax Toxins Regulate Pain Signaling and Can Deliver Molecular Cargoes Into ANTXR2+ DRG Sensory Neurons(Springer Science and Business Media LLC, 2021-12-20) Yang, Nicole J.; Isensee, Jörg; Neel, Dylan V.; Quadros, Andreza U.; Zhang, Han-Xiong Bear; Lauzadis, Justas; Liu, Sai Man; Shiers, Stephanie; Belu, Andreea; Palan, Shilpa; Marlin, Sandra; Maignel, Jacquie; Kennedy-Curran, Angela; Tong, Victoria S.; Moayeri, Mahtab; Röderer, Pascal; Nitzsche, Anja; Lu, Mike; Pentelute, Bradley L.; Brüstle, Oliver; Tripathi, Vineeta; Foster, Keith A.; Price, Theodore J.; Collier, Robert; Leppla, Stephen H.; Puopolo, Michelino; Bean, Bruce; Cunha, Thiago M.; Hucho, Tim; Chiu, IsaacBacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET); (PA + EF) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan and nerve injury. Analgesia depended on ANTXR2 expressed by Nav1.8+ or Advillin+ neurons. ET modulated protein kinase A signaling in mouse sensory and human induced pluripotent stem cell-derived sensory neurons, and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors, which may lead to the development of new pain therapeutics.