title: 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 (Mphi) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine Mphi 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, alpha-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 Mphi 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 Mphi 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.
authors.affiliation: Department of Clinical and Experimental Medicine, General Pathology and Immunology Section, S. Maria Hospital, Didactic and Scientific Division of Terni, Perugia University, Italy.
authors.forename: Katia
authors.initials: K
authors.lastname: Fettucciari
authors.forename: Ilaria
authors.initials: I
authors.lastname: Fetriconi
authors.forename: Roberta
authors.initials: R
authors.lastname: Mannucci
authors.forename: Ildo
authors.initials: I
authors.lastname: Nicoletti
authors.forename: Andrea
authors.initials: A
authors.lastname: Bartoli
authors.forename: Stefano
authors.initials: S
authors.lastname: Coaccioli
authors.forename: Pierfrancesco
authors.initials: P
authors.lastname: Marconi
mesh_terms: D000179:Acrylates / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology*; D053477:Apoptotic Protease-Activating Factor 1; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000276:immunology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000276:immunology; D005260:Female; D006868:Hydrolysis; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism / Q000382:microbiology*; D008297:Male; D051379:Mice; D024101:Mitochondrial Proteins / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D013292:Streptococcus agalactiae / Q000276:immunology*; D015854:Up-Regulation / Q000187:drug effects / Q000276:immunology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D000179:Acrylates; C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; D051023:BH3 Interacting Domain Death Agonist Protein; D015853:Cysteine Proteinase Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; C100442:PD 150606; D011506:Proteins; D051028:bcl-2-Associated X Protein; D002154:Calpain; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D002118:Calcium