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- anthrax (1)
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Background
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. 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 \(PA_{63}\) binding/translocation component are related phenomena. Chloroquine and some 4-aminoquinolones, known as potent drugs against Plasmodium falciparium infection of humans, block efficiently the \(PA_{63}\)-channel in a dose dependent way.
Methodology/Principal Findings
Here we demonstrate that related positively charged heterocyclic azolopyridinium salts block the \(PA_{63}\)-channel in the µM range, when both, inhibitor and \(PA_{63}\) 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 \(PA_{63}\)-channel function also efficiently block intoxication of the cells by the combination lethal factor and \(PA_{63}\) in the same concentration range as they block the channels in vitro.
Conclusions/Significance
These results strongly argue in favor of a transport of lethal factor through the \(PA_{63}\)-channel and suggest that the heterocycles used in this study could represent attractive candidates for development of novel therapeutic strategies against anthrax.
The membrane-bound proton-pumping pyrophosphatase (V-PPase), together with the V-type H+-ATPase, generates the proton motive force that drives vacuolar membrane solute transport. Transgenic plants constitutively overexpressing V-PPases were shown to have improved salinity tolerance, but the relative impact of increasing PPi hydrolysis and proton-pumping functions has yet to be dissected.
For a better understanding of the molecular processes underlying V-PPase-dependent salt tolerance, we transiently overexpressed the pyrophosphate-driven proton pump (NbVHP) in Nicotiana benthamiana leaves and studied its functional properties in relation to salt treatment by primarily using patch-clamp, impalement electrodes and pH imaging.
NbVHP overexpression led to higher vacuolar proton currents and vacuolar acidification. After 3 d in salt-untreated conditions, V-PPase-overexpressing leaves showed a drop in photosynthetic capacity, plasma membrane depolarization and eventual leaf necrosis. Salt, however, rescued NbVHP-hyperactive cells from cell death. Furthermore, a salt-induced rise in V-PPase but not of V-ATPase pump currents was detected in nontransformed plants.
The results indicate that under normal growth conditions, plants need to regulate the V-PPase pump activity to avoid hyperactivity and its negative feedback on cell viability. Nonetheless, V-PPase proton pump function becomes increasingly important under salt stress for generating the pH gradient necessary for vacuolar proton-coupled Na+ sequestration.