SummaryBacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. The precise mechanisms and the bacterial factors allowing B. cereus to circumvent host immune responses remain to be elucidated. We have previously shown that B. cereus induces macrophage cell death by an unknown mechanism. Here we identified the toxic component from the B. cereus supernatant. We report that Haemolysin II (HlyII) provokes macrophage cell death by apoptosis through its pore-forming activity. The HlyII-induced apoptotic pathway is caspase 3 and 8 dependent, thus most likely mediated by the death receptor pathway. Using insects and mice as in vivo models, we show that deletion of hlyII strongly reduces virulence. In addition, we show that after infection of Bombyx mori larvae, the immune cells are apoptotic, demonstrating that HlyII induces apoptosis of phagocytic cells in vivo. Altogether, our results clearly unravel HlyII as a novel virulence protein that induces apoptosis in phagocytic cells in vitro and in vivo.

EBV infects a large proportion of the human population worldwide and is one of the major viruses with human B lymphocyte tropism. It can immortalize human B lymphocytes and controls their resistance to apoptosis. EBV infection is associated with several lymphomas, including Burkitt’s lymphoma. In this report we show that EBV infection leads to the post-transcriptional down-regulation of expression of the proapoptotic protein Bim. This process involves the phosphorylation of BimEL by the constitutive EBV-activated kinase ERK1/2, followed by its degradation through the proteasome pathway. We also show that ectopic expression of BimEL in EBV-positive Burkitt’s lymphoma cells can enhance the sensitivity of these cells to serum deprivation-dependent apoptosis. Thus, EBV-mediated resistance to growth factor deprivation in human B lymphocytes is dependent on BimEL expression. Our data suggest that this regulatory pathway is an important contributor to the oncogenic potential of EBV.

IntroductionHumoral immunity is a highly orchestrated process involving antigen-specific T-B cell interactions leading naive B cells to (1) rapidly become activated, proliferate and differentiate into short-lived plasma cells secreting low affinity antibodies, and (2) generate high-affinity antigen-specific antibody secreting B cells after somatic hypermutations and recombination of immunoglobulin genes in the germinal center. 1 This cellular process allows for the formation of memory B cells and long-lived antibodyforming cells (AFCs). 2 Generation and persistence of these cells are critical for the life-long production of high-affinity antibodies against the immunizing antigen which is an important component of immunologic memory. Apoptosis is indispensable for selection of high-affinity effector cells and for maintenance of self-tolerance. B cells expressing low affinity antibodies are deleted by apoptosis, whereas clones expressing BCR with enhanced affinity for the immunogen are positively selected. 1,3,4 Apoptosis is also crucial for immune system homeostasis by inducing the death of the clonally expanded lymphocytes once the antigen has been eliminated. 5 Proteins of the Bcl-2 family play a critical role in controlling the humoral immune response. Immunized transgenic mice overexpressing antiapoptotic Bcl-2 or Bcl-xL in their lymphocytes exhibit a profound increase in the numbers of antigen-specific B cells and antibody secreting plasma cells compared with wild type mice. 6-9 The Bcl-2 proteins are key regulators of cell survival and are classified into 3 sub-groups. 10 The pro-survival members (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1) are essential for cell survival. Bax, Bak are proapoptotic and required for activation of the downstream phases of apoptosis, including permeabilization of the outer mitochondrial membrane (MOMP) with consequent activation of the caspase cascade that elicits cellular demolition.The so-called BH3-only proteins (Bad, Bid, Bim/Bod, Bik/Blk/ Nbk, Hrk/DP5, Bmf, Noxa, and Puma/Bbc3) share with each other and the wider Bcl-2 family only the BH3 region and are essential for initiation of apoptosis signaling. 11 BH3-only proteins play an important role in the homeostasis of the immune system. 5 For instance, Bim-deficient mice accumulate abnormally increased numbers of B cells and develop hypergammaglobulinemia, which, on a mixed C57BL/6 ϫ 129SV background, progresses to fatal immune complex mediated systemic lupus erythematosus (SLE)-like autoimmune kidney disease. 12 Moreover, immunized Bim Ϫ/Ϫ mice exhibited an abnormal excess of antigen-specific memory B cells and antibody-forming cells. 13 However, the less marked phenotype of the Bim Ϫ/Ϫ mice compared with the Bcl-2 transgenic mice indicates that other BH3-only proteins may also contribute to the apoptosis of activated B cells during humoral immune responses. Analyses of Bid Ϫ/Ϫ , Bad Ϫ/Ϫ , Bik Ϫ/Ϫ single knock-out as well as Bim Ϫ/Ϫ Bid Ϫ/Ϫ , Bim Ϫ/Ϫ Bad Ϫ/Ϫ and Bim Ϫ/Ϫ Bik Ϫ/Ϫ mice have demonstrated that Bid, Bad and Bik are not...

SummaryBacillus cereus is a Gram-positive spore-forming bacterium causing food poisoning and serious opportunistic infections. These infections are characterized by bacterial accumulation despite the recruitment of phagocytic cells. The precise mechanisms and the bacterial factors allowing B. cereus to circumvent host immune responses remain to be elucidated. We have previously shown that B. cereus induces macrophage cell death by an unknown mechanism. Here we identified the toxic component from the B. cereus supernatant. We report that Haemolysin II (HlyII) provokes macrophage cell death by apoptosis through its pore-forming activity. The HlyII-induced apoptotic pathway is caspase 3 and 8 dependent, thus most likely mediated by the death receptor pathway. Using insects and mice as in vivo models, we show that deletion of hlyII strongly reduces virulence. In addition, we show that after infection of Bombyx mori larvae, the immune cells are apoptotic, demonstrating that HlyII induces apoptosis of phagocytic cells in vivo. Altogether, our results clearly unravel HlyII as a novel virulence protein that induces apoptosis in phagocytic cells in vitro and in vivo.

The pro-apoptotic BH3-only protein Bim has a major role in hematopoietic homeostasis, particularly in the lymphocyte compartment, where it strongly affects immune function. The three major Bim isoforms (Bim EL , Bim L and Bim S ) are generated by alternative splicing. Bim EL , the most abundant isoform, contains a unique sequence that has been reported to be the target of phosphorylation by several MAP kinases. In particular, Erk1/2 has been shown to interact with Bim EL through the DEF2 domain of Bim EL and specifically phosphorylate this isoform, thereby targeting it for ubiquitination and proteasomal degradation. To examine the physiological importance of this mechanism of regulation and of the alternative splicing of Bim, we have generated several Bim knock-in mouse strains and analyzed their hematopoietic system. Although mutation in the DEF2 domain reduces Bim EL degradation in some circumstances, this mutation did not significantly increase Bim's pro-apoptotic activity in vivo nor impact on the homeostasis of the hematopoietic system. We also show that Bim EL and Bim L are interchangeable, and that Bim S is dispensable for the function of Bim. Hence, we conclude that physiological regulation of Bim relies on mechanisms independent of its alternative splicing or the Erk-dependent phosphorylation of Bim EL .

The protein Puma (p53-upregulated modulator of apoptosis) belongs to the BH3-only group of the Bcl-2 family and is a major regulator of apoptosis. Although the transcriptional regulation of Puma is clearly established, little is known about the regulation of its expression at the protein levels. We show here that various signals--including the cytokine TGFβ, the death effector TRAIL or chemical drugs such as anisomycin--downregulate Puma protein levels via a novel pathway based on the sequential activation of caspase-3 and a protease inhibited by the serpase inhibitor N-tosyl-L-phenylalanine chloromethyl ketone. This pathway is specific for Puma because (1) the levels of other BH3-only proteins, such as Bim and Noxa were not modified by these stimuli and (2) this caspase-mediated degradation was dependent on both the BH3 and C-terminal domains of Puma. Our data also show that Puma is regulated during the caspase-3-dependent differentiation of murine embryonic stem cells and suggest that this pathway may be relevant and important during caspase-mediated cell differentiation not associated with apoptosis.

The Epstein-Barr virus (EBV) generally latently infects its target cells with expression of genes conferring resistance to apoptosis. However, the modulation of apoptotic signals during lytic cycle remains poorly understood. We show here that resulting from viral reactivation in the EBV-positive Mutu-I and Akata Burkitt's lymphoma cell lines, a two steps proteasome-dependent downregulation of expression of the proapoptotic protein BimEL occurs. The first drop might be EBV-independent, is ERK 1/2 dependent, and BimEL is phosphorylated on Ser69. A second dramatic drop of the BimEL level observed during the lytic cycle is dependent of EBV-late-gene expression, ERK 1/2 independent, and no further phosphorylation of BimEL on Ser69 occurred. These results demonstrate for the first time, that the lytic cycle contributes to downregulation of BimEL and then could add to protection against apoptosis.

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