title: Investigation of new dominant-negative inhibitors of anthrax protective antigen mutants for use in therapy and vaccination.
abstract: The lethal toxin (LeTx) of Bacillus anthracis plays a key role in the pathogenesis of anthrax. The protective antigen (PA) is a primary part of the anthrax toxin and forms LeTx by combination with lethal factor (LF). Phenylalanine-427 (F427) is crucial for PA function. This study was designed to discover potential novel therapeutic agents and vaccines for anthrax. This was done by screening PA mutants that were mutated at the F427 residue for a dominant-negative inhibitory (DNI) phenotype which was nontoxic but inhibited the toxicity of the wild-type LeTx. For this, PA residue F427 was first mutated to each of the other 19 naturally occurring amino acids. The cytotoxicity and DNI phenotypes of the mutated PA proteins were tested in the presence of 1 microg/ml LF in RAW264.7 cells and were shown to be dependent on the individual amino acid replacements. A total of 16 nontoxic mutants with various levels of DNI activity were identified in vitro. Among them, F427D and F427N mutants had the highest DNI activities in RAW264.7 cells. Both mutants inhibited LeTx intoxication in mice in a dose-dependent way. Furthermore, they induced a Th2-predominant immune response and protected mice against a challenge with five 50% lethal doses of LeTx. The protection was correlated mainly with a low level of interleukin-1 beta (IL-1 beta) and with high levels of PA-specific immunoglobulin G1, IL-6, and tumor necrosis factor alpha. Thus, PA DNI mutants, such as F427D and F427N mutants, may serve in the development of novel therapeutic agents and vaccines to fight B. anthracis infections.
authors.affiliation: National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Sha
authors.initials: S
authors.lastname: Cao
authors.forename: Aizhen
authors.initials: A
authors.lastname: Guo
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
authors.forename: Yadi
authors.initials: Y
authors.lastname: Tan
authors.forename: Gaobing
authors.initials: G
authors.lastname: Wu
authors.forename: Chengcai
authors.initials: C
authors.lastname: Zhang
authors.forename: Yaxing
authors.initials: Y
authors.lastname: Zhao
authors.forename: Huanchun
authors.initials: H
authors.lastname: Chen
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D000881:Anthrax / Q000517:prevention & control* / Q000628:therapy*; D022122:Anthrax Vaccines / Q000235:genetics* / Q000276:immunology*; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology*; D000992:Antitoxins / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology*; D002460:Cell Line; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D050505:Mutant Proteins / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D022122:Anthrax Vaccines; D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D000992:Antitoxins; D001427:Bacterial Toxins; D007074:Immunoglobulin G; D053583:Interleukin-1beta; D015850:Interleukin-6; D050505:Mutant Proteins; D014409:Tumor Necrosis Factor-alpha; C030325:anthrax toxin