Inhibition of anthrax protective antigen outside and inside the cell.
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, beta-cyclodextrins, has recently been described. A member of this group, per-6-(3-aminopropylthio)-beta-cyclodextrin (AmPrbetaCD), 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 AmPrbetaCD, 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 AmPrbetaCD inhibits receptor-mediated cell uptake but not the binding of LFn-Al to PA/ANTXR complexes, suggesting that AmPrbetaCD works outside the cell. Moreover, AmPrbetaCD and LFn-Al synergistically protect RAW 264.7 cells from PA-mediated LF toxicity, confirming that AmPrbetaCD did not affect the binding of LFn-Al to receptor-associated PA. In contrast, AmPrbetaCD did not inhibit PA-mediated internalization of LFn-Lip, suggesting that multiplexing of LFn on the liposomal surface overcomes the inhibiting effects of AmPrbetaCD. 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 beta-cyclodextrins in combination with LFn-Lip loaded with antianthrax drugs against intracellular targets.
Backer
Marina V
MV
SibTech, Inc., 705 North Mountain Road, Newington, CT 06111, USA.
Patel
Vimal
V
Jehning
Brian T
BT
Claffey
Kevin P
KP
Karginov
Vladimir A
VA
Backer
Joseph M
JM
D000818:Animals; D000942:Antigens, Bacterial / Q000378:metabolism*; D001408:Bacillus anthracis / Q000187:drug effects* / Q000254:growth & development / Q000276:immunology; D001427:Bacterial Toxins / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D008164:Luminescent Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000166:cytology / Q000187:drug effects* / Q000382:microbiology; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D047392:beta-Cyclodextrins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D008164:Luminescent Proteins; D010446:Peptide Fragments; C030325:anthrax toxin; D047392:beta-Cyclodextrins; C505831:per-6-(3-aminopropylthio)-beta-cyclodextrin