Anthrax toxin, a major virulence factor of Bacillus anthracis, consists of the cellular binding moiety protective antigen (PA) and the enzymatic moieties lethal factor (LF) and edema factor (EF). To intoxicate host organisms, PA binds to the cellular receptors TEM8 and CMG2 and is proteolytically activated by the ubiquitously expressed cell surface furin protease, resulting in the formation of active PA heptamer, which in turn translocates LF and EF into the cytosol of cells. LF cleaves several MEKs, thereby inactivating the ERK, p38, and JUNK MAPK pathways. EF is an adenylate cyclase that generates abnormally high concentrations of cAMP. PA is a key virulence determinant of anthrax disease, and antibodies to PA protect against infection. Thus, PA is the focus of existing and new vaccines, and structure-function studies of the toxin proteins provide a rational basis on which to improve vaccines and therapeutics. Recent work by others provided evidence that the oligomer PA channel can exist in both heptameric and octameric forms. It was therefore important to assess the relative functional activities of these alternative forms. In 2013 we constructed and screened a highly directed library of PA mutants, and identified variants that complement each other to exclusively form octamers. These PA variants were individually nontoxic and demonstrated toxicity only when combined with their complementary partner. This work provided convincing proof that the octameric form of PA is active. We then engineered requirements for activation by matrix metalloproteases and urokinase plasminogen activator into two of these variants. The resulting therapeutic toxin specifically targeted cells expressing both tumor associated proteases and completely stopped tumor growth in mice when used at a dose far below that which caused toxicity. This scheme for obtaining intercomplementing subunits can be employed with other oligomeric proteins and potentially has wide application. Other bacterial protein toxins in the same family as anthrax toxin also form oligomeric channels to internalize catalytic effector domains. We extended prior work on the ability of cationic b-cyclodextrin derivatives to block PA activity and in 2013 showed that these compounds inhibit two other toxin, the C2 toxin of Clostridium botulinum and iota toxin of Clostridium perfringens. Studies were done in artificial lipid membranes, which provide detailed information about the properties of the oligomeric channels. We studied the voltage and salt dependence of the rate constants of binding and dissociation reactions of two structurally different b-cyclodextrins (AmPrbCD and AMBnTbCD) in the PA, C2, and iota toxin channels. With all three channels, the blocker carrying extra hydrophobic aromatic groups on the thio-alkyl linkers of positively charged amino groups, AMBnTbCD, demonstrated significantly stronger binding compared with AmPrbCD. The more-effective AMBnTbCD blocker shows weaker salt dependence of the binding and dissociation rate constants compared with AmPrbCD. In a search for the putative groups in the channel lumen that are responsible for the short-range forces, we performed measurements with the F427A mutant of PA, which lacks the functionally important phenylalanine clamp. We found that the on-rates of the blockage were virtually conserved, but the residence times and, correspondingly, the binding constants dropped by more than an order of magnitude, which also reduced the difference between the efficiencies of the two blockers. These data will aid in design of improved toxin channel blocking drugs. The anthrax toxin proteins constitute a highly efficient system for delivering cytotoxic enzymes to the cytosol of tumor cells. However, exogenous proteins delivered to the cytosol of cells are subject to ubiquitination on lysines and proteasomal degradation, which limit their potency. We created fusion proteins containing modified ubiquitins with their C-terminal regions fused to the Pseudomonas exotoxin A catalytic domain (PEIII) in order to achieve delivery and release of PEIII to the cytosol. In analyses reported in 2013, we showed that fusion proteins in which all seven lysines of wild-type ubiquitin were retained while the site cleaved by cytosolic deubiquitinating enzymes (DUBs) was removed were nontoxic, apparently due to rapid ubiquitination and proteasomal degradation. Fusion proteins in which all lysines of wild-type ubiquitin were substituted by arginine had high potency, exceeding that of a simple fusion lacking ubiquitin. This variant was less toxic to nontumor tissues in mice than the fusion protein lacking ubiquitin and was very efficient for tumor treatment in mice. The potency of these proteins was highly dependent on the number of lysines retained in the ubiquitin domain and on retention of the C-terminal ubiquitin sequence cleaved by DUBs. It appears that rapid cytosolic release of a cytotoxic enzyme (e.g., PEIII) that is itself resistant to ubiquitination is an effective strategy for enhancing the potency of tumor-targeting toxins. Diphthamide is a modified histidine residue in eukaryotic translation elongation factor 2 (eEF2) that is the target for irreversible ADP-ribosylation by diphtheria toxin (DT). In Saccharomyces cerevisiae, the initial steps of diphthamide biosynthesis are well characterized and require the DPH1-DPH5 genes. However, the last pathway step, amidation of the intermediate diphthine to diphthamide, is ill-defined. In 2013, with our collaborators, the genetic interaction landscapes of DPH1-DPH5 were mined to identify a candidate gene for the elusive amidase (YLR143w/DPH6) and confirm involvement of a second gene (YBR246w/DPH7) in the amidation step. Like dph1-dph5, dph6 and dph7 mutants have undermodified eEF2 forms that evade inhibition by DT. Moreover, mass spectrometry shows that dph6 and dph7 mutants specifically accumulate diphthine-modified eEF2, demonstrating failure to complete the final amidation step. Dph6 is a candidate for the elusive amidase, while Dph7 apparently couples diphthine synthase (Dph5) to diphthine amidation. The latter conclusion is based on our observation that dph7 mutants show drastically upregulated interaction between Dph5 and eEF2, indicating that their association is kept in check by Dph7. Physiologically, completion of diphthamide synthesis is required for optimal translational accuracy and cell growth, as indicated by shared traits among the dph mutants including increased ribosomal 21 frameshifting and altered responses to translation inhibitors. Through identification of Dph6 and Dph7 as components required for the amidation step of the diphthamide pathway, this work paves the way for a detailed mechanistic understanding of diphthamide formation. The monoclonal antibody S9.6 binds DNARNA hybrids with high affinity, making it useful in research and diagnostic applications, such as in microarrays and in the detection of R-loops. Our group is one of the few sources of the antibody, and we fill dozens of requests for it each year. In 2013 we described a single-chain variable fragment (scFv) of S9.6 and reported its affinities for various synthetic nucleic acid hybrids as measured by surface plasmon resonance (SPR). S9.6 exhibits dissociation constants of approximately 0.6 nM for DNARNA and, surprisingly, 2.7 nM for RNARNA hybrids that are AU-rich. The affinity of the S9.6 scFv did not appear to be strongly influenced by various buffer conditions or by ionic strength below 500 mM NaCl. The smallest epitope that was strongly bound by the S9.6 scFv contained six base pairs of DNARNA hybrid. This work provides information that will increase the value of S9.6 in basic research and as a reagent for diagnostics.

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Liu, Shihui; Ma, Qian; Fattah, Rasem et al. (2017) Anti-tumor activity of anthrax toxin variants that form a functional translocation pore by intermolecular complementation. Oncotarget 8:65123-65131
Bachran, Christopher; Leppla, Stephen H (2016) Tumor Targeting and Drug Delivery by Anthrax Toxin. Toxins (Basel) 8:
Moayeri, Mahtab; Leppla, Stephen H; Vrentas, Catherine et al. (2015) Anthrax Pathogenesis. Annu Rev Microbiol :
Andrade, Bruno B; Pavan Kumar, Nathella; Amaral, Eduardo P et al. (2015) Heme Oxygenase-1 Regulation of Matrix Metalloproteinase-1 Expression Underlies Distinct Disease Profiles in Tuberculosis. J Immunol 195:2763-73
Vrentas, Catherine; Ghirlando, Rodolfo; Keefer, Andrea et al. (2015) Hfqs in Bacillus anthracis: Role of protein sequence variation in the structure and function of proteins in the Hfq family. Protein Sci 24:1808-19
Bachran, Christopher; Gupta, Pradeep K; Bachran, Silke et al. (2014) Reductive methylation and mutation of an anthrax toxin fusion protein modulates its stability and cytotoxicity. Sci Rep 4:4754
Hu, Zonglin; Leppla, Stephen H; Li, Baoguang et al. (2014) Antibodies specific for nucleic acids and applications in genomic detection and clinical diagnostics. Expert Rev Mol Diagn 14:895-916
Bachran, Christopher; Morley, Thomas; Abdelazim, Suzanne et al. (2013) Anthrax toxin-mediated delivery of the Pseudomonas exotoxin A enzymatic domain to the cytosol of tumor cells via cleavable ubiquitin fusions. MBio 4:e00201-13
Sastalla, Inka; Fattah, Rasem; Coppage, Nicole et al. (2013) The Bacillus cereus Hbl and Nhe tripartite enterotoxin components assemble sequentially on the surface of target cells and are not interchangeable. PLoS One 8:e76955
Leysath, Clinton E; Phillips, Damilola D; Crown, Devorah et al. (2013) Anthrax edema factor toxicity is strongly mediated by the N-end rule. PLoS One 8:e74474

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