Electrochemical detection of the binding of Bacillus anthracis protective antigen (PA) to the membrane receptor on macrophages through release of nitric oxide.

Anthrax is a serious bacterial disease of man and animals whose pathogenesis involves the secretion of lethal toxins in the host. The intracellular delivery of toxic complexes involves a complex structural rearrangement of sub-domains of the exotoxin protective antigen (PA). We have used a biocompatible microelectrode array, coated with J774 mouse macrophages, to detect PA binding and intracellular signaling resulting in nitric oxide (NO) release. We have found that exposure of macrophages to PA in vitro activates the inducible isoform of NO synthase (iNOS), thus increasing the extracellular concentration of NO and nitrite, in a dose- and time-dependent manner. However, the cell-binding domain 4 of PA (PA4) could substitute for full-length PA to achieve equivalent NO release, suggesting that the heptamerisation of PA, ultimately required to deliver toxic complexes into the cell, is not a requirement for the activation of an intracellular cascade through the ERK 1/2 and the PI-3K/ Akt kinase pathways and that these events could be triggered by the binding of PA4 alone to its cell membrane receptor. Further, we have found that pre-incubation of the cells with azidothymidine, a pro-oxidant drug, significantly improves the limit of detection of rPA-induced NO release thus offering a sensitive tool for the analysis of the kinetics of anthrax intoxication and ultimately drug discovery.