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B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection.

Nothelfer K, Arena ET, Pinaud L, Neunlist M, Mozeleski B, Belotserkovsky I, Parsot C, Dinadayala P, Burger-Kentischer A, Raqib R, Sansonetti PJ, Phalipon A - J. Exp. Med. (2014)

Bottom Line: The induction of a type three secretion apparatus (T3SA)-dependent B cell death is observed in the human CL-01 B cell line in vitro, as well as in mouse B lymphocytes in vivo.The presence of bacterial co-signals is required to sensitize B cells to apoptosis and to up-regulate tlr2, thus enhancing IpaD binding.Apoptotic B lymphocytes in contact with Shigella-IpaD are detected in rectal biopsies of infected individuals.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Pasteur, INSERM U786, Unité de Pathogénie Microbienne Moléculaire, 75015 Paris, FranceInstitut Pasteur, INSERM U786, Unité de Pathogénie Microbienne Moléculaire, 75015 Paris, France.

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S. flexneri–induced B cell apoptosis is dependent on the virulence factor IpaD. (A) In vitro infections of the CL-01 B cell line with WT, mxiE, spa15, ipaC, ipaD, and complemented ipaD (ipaD/pIpaD) S. flexneri strains. Fold changes in cell number and percentages of PI+ cells are presented for infection with each strain over infection with the T3SA− strain. Asterisks indicate statistical differences to T3SA− bacteria. (B) CL-01 cell number and percentages of AnnV+PI− (Annexin V+PI−) and PI+ populations at 24 and 48 h p.i. for uninfected cells incubated with 25 µg/ml His-tagged IpaD protein (UI + IpaD), T3SA−-infected cells, cells infected with T3SA− and co-incubated with IpaD (T3SA− + IpaD), and WT infected cells. Data are presented as fold change over the uninfected control. Asterisks indicate statistically significant differences between T3SA− and T3SA− + IpaD. (C) Percentages of apoptotic AnnV+PI− cells after 24 h of co-incubation with T3SA− and different concentrations of IpaD. Asterisks indicate statistical differences to T3SA− bacteria alone. Three independent experiments were performed in triplicate for each condition/bacterial strain and data are presented as mean ± SEM. Statistically significant differences were determined by one-way (C) or two-way (A and B) ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig4: S. flexneri–induced B cell apoptosis is dependent on the virulence factor IpaD. (A) In vitro infections of the CL-01 B cell line with WT, mxiE, spa15, ipaC, ipaD, and complemented ipaD (ipaD/pIpaD) S. flexneri strains. Fold changes in cell number and percentages of PI+ cells are presented for infection with each strain over infection with the T3SA− strain. Asterisks indicate statistical differences to T3SA− bacteria. (B) CL-01 cell number and percentages of AnnV+PI− (Annexin V+PI−) and PI+ populations at 24 and 48 h p.i. for uninfected cells incubated with 25 µg/ml His-tagged IpaD protein (UI + IpaD), T3SA−-infected cells, cells infected with T3SA− and co-incubated with IpaD (T3SA− + IpaD), and WT infected cells. Data are presented as fold change over the uninfected control. Asterisks indicate statistically significant differences between T3SA− and T3SA− + IpaD. (C) Percentages of apoptotic AnnV+PI− cells after 24 h of co-incubation with T3SA− and different concentrations of IpaD. Asterisks indicate statistical differences to T3SA− bacteria alone. Three independent experiments were performed in triplicate for each condition/bacterial strain and data are presented as mean ± SEM. Statistically significant differences were determined by one-way (C) or two-way (A and B) ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: To identify the virulence factors responsible for the induction of human B cell death, CL-01 cells were infected with a panel of S. flexneri mutants. To discriminate between varying types of translocated effectors (Parsot, 2009), we tested the contribution of effectors whose expression is dependent on the transcriptional factor MxiE, using a mxiE mutant, as well as those binding the chaperone Spa15, by using a spa15 mutant. Neither mutant was impaired in its ability to induce CL-01 B cell death, as each exhibited a profile similar to that of WT bacteria both for cell number and percentage of PI+ cells (Fig. 4 A). We thus assessed the contribution of the virulence factors IpaC and IpaD, which are required for pore formation, translocation of T3SA effectors, and invasion of host cells (Ménard et al., 1993; Blocker et al., 1999; Parsot, 2009). Surprisingly, a different outcome was observed with the two noninvasive ipaC and ipaD mutants. CL-01 B cell death was induced by the ipaC mutant, but not by the ipaD mutant, which exhibited cell numbers and percentages of PI+ cells comparable to that of T3SA− bacteria. The WT bacteria-induced cell death phenotype was observed using a complemented ipaD/pIpaD strain (Fig. 4 A). These results indicate that IpaD, which is located at the tip of the T3SA needle (Espina et al., 2006; Sani et al., 2007; Epler et al., 2012), is required to trigger CL-01 B cell death independently of its role in translocation of T3SA effectors and that IpaD-mediated signals may account for the cell death observed in noninvaded CL-01 B lymphocytes.


B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection.

Nothelfer K, Arena ET, Pinaud L, Neunlist M, Mozeleski B, Belotserkovsky I, Parsot C, Dinadayala P, Burger-Kentischer A, Raqib R, Sansonetti PJ, Phalipon A - J. Exp. Med. (2014)

S. flexneri–induced B cell apoptosis is dependent on the virulence factor IpaD. (A) In vitro infections of the CL-01 B cell line with WT, mxiE, spa15, ipaC, ipaD, and complemented ipaD (ipaD/pIpaD) S. flexneri strains. Fold changes in cell number and percentages of PI+ cells are presented for infection with each strain over infection with the T3SA− strain. Asterisks indicate statistical differences to T3SA− bacteria. (B) CL-01 cell number and percentages of AnnV+PI− (Annexin V+PI−) and PI+ populations at 24 and 48 h p.i. for uninfected cells incubated with 25 µg/ml His-tagged IpaD protein (UI + IpaD), T3SA−-infected cells, cells infected with T3SA− and co-incubated with IpaD (T3SA− + IpaD), and WT infected cells. Data are presented as fold change over the uninfected control. Asterisks indicate statistically significant differences between T3SA− and T3SA− + IpaD. (C) Percentages of apoptotic AnnV+PI− cells after 24 h of co-incubation with T3SA− and different concentrations of IpaD. Asterisks indicate statistical differences to T3SA− bacteria alone. Three independent experiments were performed in triplicate for each condition/bacterial strain and data are presented as mean ± SEM. Statistically significant differences were determined by one-way (C) or two-way (A and B) ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig4: S. flexneri–induced B cell apoptosis is dependent on the virulence factor IpaD. (A) In vitro infections of the CL-01 B cell line with WT, mxiE, spa15, ipaC, ipaD, and complemented ipaD (ipaD/pIpaD) S. flexneri strains. Fold changes in cell number and percentages of PI+ cells are presented for infection with each strain over infection with the T3SA− strain. Asterisks indicate statistical differences to T3SA− bacteria. (B) CL-01 cell number and percentages of AnnV+PI− (Annexin V+PI−) and PI+ populations at 24 and 48 h p.i. for uninfected cells incubated with 25 µg/ml His-tagged IpaD protein (UI + IpaD), T3SA−-infected cells, cells infected with T3SA− and co-incubated with IpaD (T3SA− + IpaD), and WT infected cells. Data are presented as fold change over the uninfected control. Asterisks indicate statistically significant differences between T3SA− and T3SA− + IpaD. (C) Percentages of apoptotic AnnV+PI− cells after 24 h of co-incubation with T3SA− and different concentrations of IpaD. Asterisks indicate statistical differences to T3SA− bacteria alone. Three independent experiments were performed in triplicate for each condition/bacterial strain and data are presented as mean ± SEM. Statistically significant differences were determined by one-way (C) or two-way (A and B) ANOVA with Bonferroni post-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: To identify the virulence factors responsible for the induction of human B cell death, CL-01 cells were infected with a panel of S. flexneri mutants. To discriminate between varying types of translocated effectors (Parsot, 2009), we tested the contribution of effectors whose expression is dependent on the transcriptional factor MxiE, using a mxiE mutant, as well as those binding the chaperone Spa15, by using a spa15 mutant. Neither mutant was impaired in its ability to induce CL-01 B cell death, as each exhibited a profile similar to that of WT bacteria both for cell number and percentage of PI+ cells (Fig. 4 A). We thus assessed the contribution of the virulence factors IpaC and IpaD, which are required for pore formation, translocation of T3SA effectors, and invasion of host cells (Ménard et al., 1993; Blocker et al., 1999; Parsot, 2009). Surprisingly, a different outcome was observed with the two noninvasive ipaC and ipaD mutants. CL-01 B cell death was induced by the ipaC mutant, but not by the ipaD mutant, which exhibited cell numbers and percentages of PI+ cells comparable to that of T3SA− bacteria. The WT bacteria-induced cell death phenotype was observed using a complemented ipaD/pIpaD strain (Fig. 4 A). These results indicate that IpaD, which is located at the tip of the T3SA needle (Espina et al., 2006; Sani et al., 2007; Epler et al., 2012), is required to trigger CL-01 B cell death independently of its role in translocation of T3SA effectors and that IpaD-mediated signals may account for the cell death observed in noninvaded CL-01 B lymphocytes.

Bottom Line: The induction of a type three secretion apparatus (T3SA)-dependent B cell death is observed in the human CL-01 B cell line in vitro, as well as in mouse B lymphocytes in vivo.The presence of bacterial co-signals is required to sensitize B cells to apoptosis and to up-regulate tlr2, thus enhancing IpaD binding.Apoptotic B lymphocytes in contact with Shigella-IpaD are detected in rectal biopsies of infected individuals.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Pasteur, INSERM U786, Unité de Pathogénie Microbienne Moléculaire, 75015 Paris, FranceInstitut Pasteur, INSERM U786, Unité de Pathogénie Microbienne Moléculaire, 75015 Paris, France.

Show MeSH
Related in: MedlinePlus