Calpain-dependent cytoskeletal rearrangement exploited for anthrax toxin endocytosis

Abstract

The protective antigen component of Bacillus anthracis toxins can interact with at least three distinct proteins on the host cell surface, capillary morphogenesis gene 2 (CMG2), tumor endothelial marker 8, and β 1 -integrin, and, with the assistance of other host proteins, enters targeted cells by receptor-mediated endocytosis. Using an antisense-based phenotypic screen, we discovered the role of calpains in this process. We show that functions of a ubiquitous Ca 2+ -dependent cysteine protease, calpain-2, and of the calpain substrate talin-1 are exploited for association of anthrax toxin and its principal receptor, CMG2, with higher-order actin filaments and consequently for toxin entry into host cells. Downregulated expression of calpain-2 or talin-1, or pharmacological interference with calpain action, did not affect toxin binding but reduced endocytosis and increased the survival of cells exposed to anthrax lethal toxin. Adventitious expression of wild-type talin-1 promoted toxin endocytosis and lethality, whereas expression of a talin-1 mutant (L432G) that is insensitive to calpain cleavage did not. Disruption of talin-1, which links integrin-containing focal adhesion complexes to the actin cytoskeleton, facilitated association of toxin bound to its principal cell-surface receptor, CMG2, with higher-order actin filaments undergoing dynamic disassembly and reassembly during endocytosis. Our results reveal a mechanism by which a bacterial toxin uses constitutively occurring calpain-mediated cytoskeletal rearrangement for internalization.lethal factor | calpastatin | MDL28170

Acquired resistance to innate immune clearance promotes Klebsiella pneumoniae ST258 pulmonary infection

“…Thrombospondin is involved in actin-based cellular dynamics important in inhibiting neutrophil function (30,31). Proteins involved in calpain activation, a Ca 2+ -dependent protease critical for phagocytosis (32), were suppressed to a much greater degree by KP35, consistent with the finding that this strain is resistant to phagocytic killing ( Figure 5D). The proteomic data were confirmed with functional assays.…”

Anthrax protective antigen is a calcium-dependent serine protease

“…Additionally, inhibitors of anthrax lethal toxin formation might work via another mechanism than suggested. For this reason we examined the effect of two compounds, EGA and calpain-inhibitor MDL281700, utilized in studies investigating the inhibition of lethal toxin formation, on the proteolytic activity of PA 83 [18,19]. These compounds however did not affect PA 83 proteolytic activity.…”

Attenuation of Blood-Brain Barrier Breakdown and Hyperpermeability by Calpain Inhibition

“…Calpain-1 (also known as -calpain) and calpain-2 (also known as m-calpain) are predominantly expressed in the central and peripheral nervous systems (14,15). Calpain-1 is activated under low calcium concentrations (3-50 M), whereas calpain-2 is activated only under high concentrations (400 -800 M) of calcium in the cell (23,24).…”

“…Intracellular calcium levels and the endogenous inhibitor of calpains, namely calpastatin, tightly regulate calpain levels endogenously (9,13). Calpains-1 and -2 are the predominant calpains in the central nervous system (14,15). An increased calpain activity was observed following TBI in laboratory rodents (16,17) and human patients (12).…”