Group B Streptococcus Induces Macrophage Apoptosis by Calpain Activation

Abstract

Group B Streptococcus (GBS) has developed several strategies to evade immune defenses. We show that GBS induces macrophage (Mφ) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine Mφ apoptosis. GBS causes a massive intracellular calcium increase, strictly correlated to membrane permeability defects and apoptosis onset. Calcium increase was associated with activation of calcium-dependent protease calpain, demonstrated by casein zymography, α-spectrin cleavage to a calpain-specific fragment, fluorogenic calpain-substrate cleavage, and inhibition of these proteolyses by calpain inhibitors targeting the calcium-binding, 3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, or active site (four different inhibitors), by calpain small-interfering-RNA (siRNA) and EGTA. GBS-induced Mφ apoptosis was inhibited by all micro- and m-calpain inhibitors used and m-calpain siRNA, but not 3-(5-Fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (micro-calpain inhibitor) and micro-calpain siRNA indicating that m-calpain plays a central role in apoptosis. Calpain activation is followed by Bax and Bid cleavage, cytochrome c, apoptosis-inducing factor, and endonuclease G release from mitochondria. In GBS-induced apoptosis, cytochrome c did not induce caspase-3 and -7 activation because they and APAF-1 were degraded by calpains. Therefore, apoptosis-inducing factor and endonuclease G seem the main mediators of the calpain-dependent but caspase-independent pathway of GBS-induced apoptosis. Proapoptotic mediator degradations do not occur with nonhemolytic GBS, not inducing Mφ apoptosis. Apoptosis was reduced by Bax siRNA and Bid siRNA suggesting Bax and Bid degradation is apoptosis correlated. This signaling pathway, different from that of most pathogens, could represent a GBS strategy to evade immune defenses.

Helicobacter pylori Activates Calpain via Toll-Like Receptor 2 To Disrupt Adherens Junctions in Human Gastric Epithelial Cells

“…The following antibodies were used for immunoblotting or immunocytochemistry: mouse monoclonal anti-E-cadherin (DECMA-1 clone), rabbit polyclonal anti-pan-cadherin, and mouse monoclonal anti-␤-catenin (all from Sigma) and mouse monoclonal anti-pan-actin (Thermo Fisher Scientific, Waltham, MA). The selective calpain inhibitor PD150606 (EMD, Darmstadt, Germany) was used at a concentration of 50 M 30 min prior to infection (13). TL2.1, a neutralizing monoclonal antibody against TLR2 (10 g/ml; eBioscience, San Diego, CA) was used 1 h prior to infection.…”

Soft Substrate Up-regulates the Interaction of STIM1 with Store-operated Ca2+Channels That Lead to Normal Epithelial Cell Apoptosis

“…We further studied the mechanisms of -calpain activation in soft substrate by utilizing inhibitors acting at different target sites of calapin. PD150606 and PD151746, two specific inhibitors targeting Ca 2ϩ -binding site of calpain (Fettucciari et al, 2006), inhibited soft substrate-induced calpain activation in a dosedependent manner ( Figure 3C). PD145305, a negative control for PD150606 and PD151746 (Fettucciari et al, 2006), did not show any effect on the activation of calpain.…”

“…PD150606 and PD151746, two specific inhibitors targeting Ca 2ϩ -binding site of calpain (Fettucciari et al, 2006), inhibited soft substrate-induced calpain activation in a dosedependent manner ( Figure 3C). PD145305, a negative control for PD150606 and PD151746 (Fettucciari et al, 2006), did not show any effect on the activation of calpain. In contrast, ALLN, an inhibitor targeting catalytic binding site of calpain (Debiasi et al, 1999), showed no significant effect on soft substrate-induced calpain activity.…”

Calpains promote neutrophil recruitment and bacterial clearance in an acute bacterial peritonitis model

“…Calpain activation downstream of TLRs in macrophages has implicated these proteases in regulating apoptosis and phagocytosis [13,20,21]. To test whether defects in FIP response of calpain KO mice relate to defects in macrophages, we isolated peritoneal macrophages from WT and KO mice, and subjected them to enterobacterial infection ex vivo (using bacterial cultures employed in FIP model).…”

“…In macrophages, calpain activation leads to cleavage of Selenoprotein K (SelK) [13], a transmembrane endoplasmic reticulum protein implicated in regulating Ca ++ flux, ROS production, and cell motility [19]. Calpain activation also allows Group B Streptococcus to evade killing by inducing caspase-independent apoptosis, disruption of the cytoskeleton, and phagosome escape in macrophages [20,21]. Another key calpain substrate relevant to chronic inflammation and immunity is IL-1α, which lacks a signal peptide, undergoes proteolysis in the cytoplasm, and is secreted by both activated and dying cells [22].…”