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Infection and Immunity, October 2009, p. 4679-4687, Vol. 77, No. 10
0019-9567/09/$08.00+0     doi:10.1128/IAI.00264-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Investigation of New Dominant-Negative Inhibitors of Anthrax Protective Antigen Mutants for Use in Therapy and Vaccination {triangledown}

Sha Cao,1 Aizhen Guo,1,2* Ziduo Liu,1 Yadi Tan,1 Gaobing Wu,1 Chengcai Zhang,1,3 Yaxing Zhao,2 and Huanchun Chen1,2*

National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China,1 College of Veterinary Medicine, Provincial Key Laboratory of Preventive Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China,2 Laboratoire de Chimie Bactérienne, IBSM, CNRS-UPR9043, 31 Chemin Joseph Aiguie, 13402 Marseille Cedex 20, France3

Received 7 March 2009/ Returned for modification 17 April 2009/ Accepted 8 July 2009

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 µg/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-1β (IL-1β) 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.

* Corresponding author. Mailing address: Shizishan 1, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, People's Republic of China. Phone: 0086-27-87286861. Fax: 0086-27-87282608. E-mail for Aizhen Guo: aizhen{at}mail.hzau.edu.cn. E-mail for Huanchun Chen: chenhch{at}mail.hzau.edu.cn

{triangledown} Published ahead of print on 20 July 2009.

Editor: S. R. Blanke

Infection and Immunity, October 2009, p. 4679-4687, Vol. 77, No. 10
0019-9567/09/$08.00+0     doi:10.1128/IAI.00264-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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