Toxin Transport by A-B Type of Toxins in Eukaryotic Target Cells and Its Inhibition by Positively Charged Heterocyclic Molecules

Abstract

A-B types of toxins are among the most potent bacterial protein toxins produced by gram-positive bacteria. Prominent examples are the tripartite anthrax toxin of Bacillus anthracis and the different A-B type clostridial toxins that are the causative agents of severe human and animal diseases and could serve as biological weapons. The components of all these toxins comprise one binding/transport (B) subunit and one or two separate, non-linked enzymatically active (A) subunits. The A and B subunits are separately produced and secreted by the pathogenic gram-positive bacteria and must assemble on the surface of eukaryotic target cells to form biologically active toxin complexes. The B components are cleaved by proteases to generate the biologically active species that binds to receptors on the surface of the target cells and form there oligomers which bind the A subunits. The AB complexes are internalized by receptor-mediated endocytosis and reach early or late endosomes that become acidified. Subsequently, the B components form channels in endosomal membranes that are indispensable for the transport of the enzymatic subunits across these membranes into the cytosol of target cells via the trans-membrane channels. In addition to the channels formed by the B components, host cell factors including chaperones and further folding helper enzymes are involved in the import of the enzymatic subunits into the cytosol of eukaryotic cells. Positively charged heterocyclic molecules, such as chloroquine and related aminoquinolinium and azolopyridinium salts have been shown in recent years to bind with high affinity to the channels formed by the B components of binary toxins. Since binding to the B components is also a prerequisite for transport of the A components across the endosomal membranes the channel blockers also prevent transport of the A subunits into the host cell cytosol. The inhibition of toxin uptake into cells by such pharmacological compounds should also be of clinically interest because the toxins are the major virulence factors causing anthrax on the one hand and severe enteric disease on the other hand. Therefore, the novel toxin inhibitors should be attractive compounds for an application in combination with antibiotics to prevent or treat the diseases associated with binary toxins. Here the different processes involved in channel block in vitro and inhibition of intoxication of living target cells are reviewed in some detail.

Human peptide α‐defensin‐1 interferes withClostridioides difficiletoxins TcdA, TcdB, and CDT

“…Planar lipid bilayer membranes have widely been used for the study of reconstituted binding components of different AB‐type toxins . Bacterial binding components form heptameric channels that function as protein conducting nanopores for the transport of the enzymatic subunits into the target cells via the early or late endosomal pathway . Small amounts of the binding components added to one side of the membranes allow the formation of many asymmetric transmembrane channels without the need of the native host cell receptors .…”

Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication

“…A binding/transport (B) subunit binds to its receptor on the cell surface, where it assembles with its respective separate enzymatically active (A) subunit to form the AB complexes, which are then internalized by receptor‐mediated endocytosis (for review, see refs. 12 and 13). The B sub‐units of the clostridial actin ADP‐ribosylating toxins C2IIa, ɩ b (Ib), and CDTb share sequence and structure homology with the B subunit of anthrax toxin, the protective antigen (PA) (10–12, 14, 15).…”

“…Later, after endocytosis, they form pores in the membranes of acidified endosomal vesicles, which serve as translocation channels for their A subunits. The A subunits translocate as (at least partially) unfolded proteins through these pores into the cytosol, where they become refolded to exhibit their specific enzyme activities (12, 13). For the clostridial binary toxins, components of the host cell heat shock protein 90 machinery facilitate the transport of the A sub‐units across endosomal membranes into the cytosol (16).…”

“…If the B transmembrane pores are blocked or not formed, the A subunits cannot reach the cytosol, and therefore, cells are protected from intoxication. Thus, the identification and detailed characterization of such compounds is of particular scientific and clinical interest (13).…”

Structure of the cell-binding component of theClostridium difficilebinary toxin reveals a novel macromolecular assembly

“…Once active CDTa is delivered into the host cell cytoplasm, ADPribosylation of G-actin occurs catalytically at Arg-177 (42). ADP-ribosylated G-actin then leads to Factin filament dissociation (43), destruction of the cytoskeleton, increased microtubule protrusions, accelerated bacterial adhesion, and a "death spiral" for host cells (44)(45)(46). In this study, a combination of biophysical and structural biology methods was used to define the molecular structure of the CDTb component of the toxin at the atomic level.…”