Purified Bacillus anthracis Lethal Toxin Complex Formed in Vitro and during Infection Exhibits Functional and Biological Activity

Rekha G Panchal,Kelly M Halverson,Wilson Ribot,Douglas Lane,Tara Kenny,Teresa G Abshire,John W Ezzell,Timothy A Hoover,Bradford Powell,Stephen Little,John J Kasianowicz,Sina Bavari

doi:10.1074/jbc.m412210200

Abstract

Anthrax protective antigen (PA, 83 kDa), a pore-forming protein, upon protease activation to 63 kDa (PA(63)), translocates lethal factor (LF) and edema factor (EF) from endosomes into the cytosol of the cell. The relatively small size of the heptameric PA(63) pore (approximately 12 angstroms) raises questions as to how large molecules such as LF and EF can move through the pore. In addition, the reported high binding affinity between PA and EF/LF suggests that EF/LF may not dissociate but remain complexed with activated PA(63). In this study, we found that purified (PA(63))(7)-LF complex exhibited biological and functional activities similar to the free LF. Purified LF complexed with PA(63) heptamer was able to cleave both a synthetic peptide substrate and endogenous mitogen-activated protein kinase kinase substrates and kill susceptible macrophage cells. Electrophysiological studies of the complex showed strong rectification of the ionic current at positive voltages, an effect similar to that observed if LF is added to the channels formed by heptameric PA(63) pore. Complexes of (PA(63))(7)-LF found in the plasma of infected animals showed functional activity. Identifying active complex in the blood of infected animals has important implications for therapeutic design, especially those directed against PA and LF. Our studies suggest that the individual toxin components and the complex must be considered as critical targets for anthrax therapeutics.

Highlights

Lethal and edema toxins play key pathogenesis roles as virulence factors produced by Bacillus anthracis, the etiologic agent of anthrax
There is no direct evidence showing that full-length LF dissociates from the PA63 heptamer complex either during or after translocation through the protective antigen (PA) pore or that LF bound to the PA63 heptamer is capable of cleaving its substrates
SDS-PAGE of purified (PA63)7-LF complex shows the presence of LF, PA63 monomer, and SDS-stable and heat-resistant PA63 heptamer (Fig. 1D, lane 3)

Summary

Introduction

Lethal and edema toxins play key pathogenesis roles as virulence factors produced by Bacillus anthracis, the etiologic agent of anthrax. LF, a Znϩ2-dependent metalloprotease, cleaves several members of the mitogen-activated protein kinase kinase (MAPKK) family [1,2,3,4], and in complex with PA, is responsible for the lethal action of anthrax toxin. Prior studies by Singh et al [17] showed that PA63 in either the monomeric or the oligomeric form can bind LF in a 1:1 ratio. Both the enzymatic moieties EF and LF bind the heptamerized PA63 competitively and with high affinity (Kd ϳ1 nM) [18]. We provide experimental evidence showing that LF bound to the PA63 heptamer is active and exhibits biological and biophysical properties similar to those of free LF

Methods

Results

Conclusion