Detailed structural analyses of the anthrax toxin proteins by our group and others continues to offer opportunities to engineer the proteins as tools for cell biology and as the basis of vaccines and therapeutics. Our prior work has provided robust methods for production and purification of the anthrax toxin proteins: protective antigen, lethal factor, and edema factor. A large number of substitution and fusion proteins have been constructed to facilitate analysis and exploitation of these proteins. We previously arranged through the NIH technology transfer office for many of these proteins to be distributed by Kerafast. Their website lists more than 10 of our protein variants that are available to researchers wishing to evaluate their utility. Kerafast also continues to distribute the unique S9.6 monoclonal antibody that recognizes DNA/RNA hybrids. In the current year of 2016, this antibody remained extremely popular, being sold to hundreds of researchers. It is typically used for studying the normal and pathological roles of DNA/RNA hybrids. Transcriptional start sites typically have a region in which nascent mRNA is bound to one DNA strand, displacing the opposite DNA strand, producing what is termed an R-loop. Determining the location and abundance of R-loops provides important information regarding translational processes. During 2016, we supported the NIH technology transfer office in licensing this antibody to EMB/Millipore as an additional distributor. For future work with S9.6, we are developing methods for production of a single chain (scFv) version of the antibody. This protein will also be used by others to obtain crystal structures of the antibody bound to a DNA/RNA hybrid. Our studies on protein expression and characterization have been extended during 2016 to additional proteins produced by various pathogens. A number of expression systems have been developed for production of the mycobacterial proteins ESAT6 and Ag85b. These are being engineered with modifications that will allow them to be used as vaccine candidates and immune modulators. During 2016 we studied the structural properties and biological roles of the small Bacillus anthracis Hfq proteins. Hfq proteins in Gram-negative bacteria play important roles in bacterial physiology and virulence, mediated by binding of the Hfq hexamer to small RNAs and/or mRNAs to post-transcriptionally regulate gene expression. However, the physiological role of Hfqs in Gram-positive bacteria is less clear. B. anthracis, the causative agent of anthrax, uniquely expresses three distinct Hfq proteins, two from the chromosome (Hfq1, Hfq2) and one from its pXO1 virulence plasmid (Hfq3). The protein sequences of Hfq1 and 3 are evolutionarily distinct from those of Hfq2 and of Hfqs found in other Bacilli. We produced each of these three proteins in Escherichia coli. While Hfq2 adopts the expected hexamer structure, Hfq1 does not form similarly stable hexamers in vitro. The impact on the monomer-hexamer equilibrium of varying Hfq C-terminal tail length and other sequence differences among the Hfqs was examined, and a sequence region of the Hfq proteins that was involved in hexamer formation was identified. It was found that, in addition to the distinct higher-order structures of the Hfq homologs, they give rise to different phenotypes. Hfq1 has a disruptive effect on the function of E. coli Hfq in vivo, while Hfq3 expression at high levels is toxic to E. coli but also partially complements Hfq function in E. coli. These results set the stage for future studies of the roles of these proteins in B. anthracis physiology and for the identification of sequence determinants of phenotypic complementation.

Project Start
Project End
Budget Start
Budget End
Support Year
9
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
Zip Code
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:
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
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
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
Phillips, Damilola D; Garboczi, David N; Singh, Kavita et al. (2013) The sub-nanomolar binding of DNA-RNA hybrids by the single-chain Fv fragment of antibody S9.6. J Mol Recognit 26:376-81
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

Showing the most recent 10 out of 31 publications