Bordetella type III secretion induces caspase 1-independent necrosis

The Bordetella bronchiseptica type III (TIII) secretion system induces cytotoxicity in infected macrophages and epithelial cells. In this report we characterize the cell death phenotype and compare it to the TIII-dependent cytotoxicity induced by Yersinia enterocolitica and Shigella flexneri. Bordetella bronchiseptica strain RB58 was able to induce cell death in J774A.1 macrophages with the same efficiency as Shigella and Yersinia, but only B. bronchiseptica was able to kill epithelial cells in a TIII-dependent manner. Primary macrophages from caspase 1-/- mice were susceptible to RB58-mediated killing, suggesting that unlike Shigella and Salmonella, caspase 1 does not mediate cell death. RB58-induced cytotoxicity was not inhibited by addition of the pan-caspase inhibitor zVAD, and Western blot analyses of RB58-infected HeLa cells indicated that neither caspase 3 nor 7 was cleaved and PARP remained in its full-length active form. Morphologically the RB58-infected HeLa cells resembled necrotic rather than apoptotic cells, exhibiting cytoplasmic swelling and extensive membrane blebbing in the absence of nuclear changes. The addition of exogenous glycine, which has been shown to prevent necrotic cell death by blocking non-specific ion fluxes across the plasma membrane, blocked RB58-induced cytotoxicity. Addition of cyclosporin A which prevents the opening of the mitochondrial permeability pore, had no effect on RB58-infected cells. We conclude that the B. bronchiseptica TIII secretion system induces a mode of cell death consistent with necrosis that is distinct from that of Yersinia and Shigella.