Abstract
BackgroundSeveral intracellular acting bacterial protein toxins of the AB-type, which are known to enter cells by endocytosis, are shown to produce channels. This holds true for protective antigen (PA), the binding component of the tripartite anthrax-toxin of Bacillus anthracis. Evidence has been presented that translocation of the enzymatic components of anthrax-toxin across the endosomal membrane of target cells and channel formation by the heptameric/octameric PA63 binding/translocation component are related phenomena. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the PA63-channel in a dose dependent way.Methodology/Principal FindingsHere we demonstrate that related positively charged heterocyclic azolopyridinium salts block the PA63-channel in the µM range, when both, inhibitor and PA63 are added to the same side of the membrane, the cis-side, which corresponds to the lumen of acidified endosomal vesicles of target cells. Noise-analysis allowed the study of the kinetics of the plug formation by the heterocycles. In vivo experiments using J774A.1 macrophages demonstrated that the inhibitors of PA63-channel function also efficiently block intoxication of the cells by the combination lethal factor and PA63 in the same concentration range as they block the channels in vitro.Conclusions/SignificanceThese results strongly argue in favor of a transport of lethal factor through the PA63-channel and suggest that the heterocycles used in this study could represent attractive candidates for development of novel therapeutic strategies against anthrax.
Highlights
The main virulence factors of Bacillus anthracis are the poly-Dglutamic acid capsule, which inhibits phagocytosis, and anthrax toxin
We screened a series of heterocyclic azolopyridinium salts and other related azines for their ability to block the trans-membrane channel formed by PA63, the receptorbinding component of B. anthracis
The inhibitory effects of a series of azolopyridinium salts was analyzed in planar lipid bilayer membranes in vitro and the most efficient substances HA1383, HA1495 and HA1568 were subsequently tested for their ability to interfere with PA63-channels in membranes of living cells
Summary
The main virulence factors of Bacillus anthracis are the poly-Dglutamic acid capsule, which inhibits phagocytosis, and anthrax toxin. PA binds to cells, coordinates self-assembly of heptamers and/or octamers on the cell surface, triggers endocytosis of the toxin complexes and delivers EF and LF from endosomal vesicles to the cytosol of the target cell [4,5,6,7,8,9,10]. LF is a highly specific zinc metalloprotease (90 kDa) that removes the N-terminal tail of mitogenactivated protein kinase kinases (MAPKKs) [16,17,18] This cleavage initiates still poorly understood mechanisms leading to subsequent cell death by apoptosis. Several intracellular acting bacterial protein toxins of the AB-type, which are known to enter cells by endocytosis, are shown to produce channels This holds true for protective antigen (PA), the binding component of the tripartite anthrax-toxin of Bacillus anthracis. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the PA63-channel in a dose dependent way
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