Anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of mammalian cells, is cleaved by the cell surface protease furin, and then captures either of the two other toxin proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis and LF and EF translocate to the cytosol. EF is a calcium and calmodulin-dependent adenylyl cyclase that causes large and unregulated increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs). Entry of anthrax toxin into cells depends on two related cell surface receptors, tumor endothelium marker 8 (TEM8) and capillary morphogenesis gene product 2(CMG2). TEM8 was initially identified as a protein upregulated in colon cancers. CMG2 has substantial sequence similarity to this candidate tumor marker. The tissue distribution and the relative importance of the two toxin receptors in toxin action have not been well understood. During the current period, we approached this important question by generating mice conditionally deleted for each of the two receptors. Surprisingly, the knockout mice lacking each receptor were viable and showed only subtle phenotypic changes. These mice and cells obtained from them were tested for sensitivity to the two anthrax toxins. The most striking result was that the mice lacking TEM8 were almost indistinguishable from normal mice in response to toxin, arguing that the TEM8 receptor plays a very small role in facilitating toxin action in mice. In contrast, mice lacking CMG2 were highly resistant to challenge with PA and either LF or EF, showing the dominant role played by CMG2. In these mice, death occurred only much later and with higher doses of toxin, again suggesting that the TEM8 receptor (remaining present in these mice) plays a limited and qualitatively different role in toxin action. By breeding the two knockout mice, progeny were obtained lacking both receptors. These mice were completely resistant to doses of toxin that were highly lethal to normal mice. Analysis of the knockout mice was extended to bacterial challenges. The CMG2 mice were highly resistant to infection with the Sterne strain of B. anthracis. This proves that the uptake of anthrax toxin through the CMG2 receptor plays a key role in facilitating anthrax infections, and validates this protein as a therapeutic target. To explain the greater role of CMG2, we isolated and cultured primary cells from each of the knockout mice. These were used in competitive binding studies (e.g., Schild analysis) that showed PA binds with 10-fold higher affinity to CMG2 than to TEM8. This difference may explain at least in part the greater role of CMG2 in toxin action in mice. In this period we also extended use of a fusion of the N-terminal portion of LF (LFn, amino acids 1-254) with beta-lactamase (BLA). The availability of the fluorescent beta-lactase substrate CCF2/AM makes the LFnBLA fusion protein a sensitive reporter on the process of LF internalization into cultured cells and animal tissues. We improved the potency of this reporter by restoring the N-terminus of the recombinant protein to the native AGG.. sequence that was recently shown to enhance the activity of full-size, native LF. In addition, we developed methods for producing this protein in avirulent strains of B. anthracis, which potentially can yield more protein than the previously used E. coli expression system. The LFnBLA protein is being used in high throughput chemical genomic screens to identify chemicals that block steps in the action of anthrax toxin or the cancer therapeutic agents based on this toxin. The results of one such screen were recently published, as is reported under a parallel project.

Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2009
Total Cost
$1,066,759
Indirect Cost
City
State
Country
Zip Code
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 :
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
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
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
Phillips, Damilola D; Fattah, Rasem J; Crown, Devorah et al. (2013) Engineering anthrax toxin variants that exclusively form octamers and their application to targeting tumors. J Biol Chem 288:9058-65

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