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Antimicrobial Agents and Chemotherapy, January 2007, p. 245-251, Vol. 51, No. 1
0066-4804/07/$08.00+0     doi:10.1128/AAC.00983-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Inhibition of Anthrax Protective Antigen Outside and Inside the Cell{triangledown} ,{dagger}

Marina V. Backer,1 Vimal Patel,1 Brian T. Jehning,1 Kevin P. Claffey,2 Vladimir A. Karginov,3 and Joseph M. Backer1*

SibTech, Inc., Newington, Connecticut 06111,1 University of Connecticut, Health Center, Farmington, Connecticut 06030,2 Innovative Biologics, Inc., Manassas, Virginia 201103

Received 7 August 2006/ Returned for modification 26 August 2006/ Accepted 16 October 2006

In the course of Bacillus anthracis infection, B. anthracis lethal factor (LF) and edema factor bind to a protective antigen (PA) associated with cellular receptors ANTXR1 (TEM8) or ANTXR2 (CMG2), followed by internalization of the complex via receptor-mediated endocytosis. A new group of potential antianthrax drugs, ß-cyclodextrins, has recently been described. A member of this group, per-6-(3-aminopropylthio)-ß-cyclodextrin (AmPrßCD), was shown to inhibit the toxicity of LF in vitro and in vivo. In order to determine which steps in lethal factor trafficking are inhibited by AmPrßCD, we developed two targeted fluorescent tracers based on LFn, a catalytically inactive fragment of LF: (i) LFn site specifically labeled with the fluorescent dye AlexaFluor-594 (LFn-Al), and (ii) LFn-decorated liposomes loaded with the fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (LFn-Lip). Both tracers retained high affinity to PA/ANTXR complexes and were readily internalized via receptor-mediated endocytosis. Using fluorescent microscopy, we found that AmPrßCD inhibits receptor-mediated cell uptake but not the binding of LFn-Al to PA/ANTXR complexes, suggesting that AmPrßCD works outside the cell. Moreover, AmPrßCD and LFn-Al synergistically protect RAW 264.7 cells from PA-mediated LF toxicity, confirming that AmPrßCD did not affect the binding of LFn-Al to receptor-associated PA. In contrast, AmPrßCD did not inhibit PA-mediated internalization of LFn-Lip, suggesting that multiplexing of LFn on the liposomal surface overcomes the inhibiting effects of AmPrßCD. Notably, internalized LFn-Al and LFn-Lip protected cells that overexpressed anthrax receptor TEM8 from PA-induced, LF-independent toxicity, suggesting an independent mechanism for PA inhibition inside the cell. These data suggest the potential for the use of ß-cyclodextrins in combination with LFn-Lip loaded with antianthrax drugs against intracellular targets.

* Corresponding author. Mailing address: SibTech, Inc., 705 North Mountain Road, Newington, CT 06111. Phone: (860) 953-1753. Fax: (860) 953-1317. E-mail: jbacker{at}sibtech.com.

{dagger} Supplemental material for this article may be found at http://aac.asm.org/.

{triangledown} Published ahead of print on 30 October 2006.

Antimicrobial Agents and Chemotherapy, January 2007, p. 245-251, Vol. 51, No. 1
0066-4804/07/$08.00+0     doi:10.1128/AAC.00983-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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