Supporting Information Figure S1 TNF-a signaling during bacterially induced macrophage apoptosis. The RAW 264.7 mouse macrophage cell line was infected with the N. meningitidis serogroup C strain FAM20 at MOI = 100 for 20 hours. In addition, cells were incubated with an inhibitory TNF-a antibody or treated with an irrelevant isotype-matched antibody. Apoptotic cells were visual- ized using the APOPercentage kit and the relative numbers of apoptotic cells were determined by light microscopy. Values indicate mean6SD of three independent experiments. (TIF) Figure S2 Release of the cytokine HMGB1 after bacte- rially induced macrophage apoptosis. PMA-differentiated References 1. Peak IR, Srikhanta Y, Dieckelmann M, Moxon ER, Jennings MP (2000) Identification and characterisation of a novel conserved outer membrane protein from Neisseria meningitidis. FEMS Immunol Med Microbiol 28: 329–334. 2. Weynants VE, Feron CM, Goraj KK, Bos MP, Denoel PA, et al. (2007) Additive and synergistic bactericidal activity of antibodies directed against minor outer membrane proteins of Neisseria meningitidis. Infect Immun 75: 5434–5442. 3. Pizza M, Scarlato V, Masignani V, Giuliani MM, Arico B, et al. (2000) Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing. Science 287: 1816–1820. 4. Scarselli M, Serruto D, Montanari P, Capecchi B, Adu-Bobie J, et al. (2006) Neisseria meningitidis NhhA is a multifunctional trimeric autotransporter adhesin. Mol Microbiol 61: 631–644. 5. Echenique-Rivera H, Brunelli B, Scarselli M, Taddei AR, Pizza M, et al. (2011) A Naturally Occurring Single-Residue Mutation in the Translocator Domain of Neisseria meningitidis NhhA Affects Trimerization, Surface Localization, and Adhesive Capabilities. Infect Immun 79: 4308–4321. 6. Sjolinder H, Eriksson J, Maudsdotter L, Aro H, Jonsson AB (2008) Meningococcal outer membrane protein NhhA is essential for colonization and disease by preventing phagocytosis and complement attack. Infect Immun 76: 5412–5420. 7. Jendrossek V, Fillon S, Belka C, Muller I, Puttkammer B, et al. (2003) Apoptotic response of Chang cells to infection with Pseudomonas aeruginosa strains PAK and PAO-I: molecular ordering of the apoptosis signaling cascade and role of type IV pili. Infect Immun 71: 2665–2673. 8. Abul-Milh M, Wu Y, Lau B, Lingwood CA, Barnett Foster D (2001) Induction of epithelial cell death including apoptosis by enteropathogenic Escherichia coli expressing bundle-forming pili. Infect Immun 69: 7356–7364. 9. Deghmane AE, Veckerle C, Giorgini D, Hong E, Ruckly C, et al. (2009) Differential modulation of TNF-alpha-induced apoptosis by Neisseria meningi- tidis. PLoS Pathog 5: e1000405. 10. Xaus J, Comalada M, Valledor AF, Lloberas J, Lopez-Soriano F, et al. (2000) LPS induces apoptosis in macrophages mostly through the autocrine production of TNF-alpha. Blood 95: 3823–3831. 11. Sprong T, van der Ley P, Steeghs L, Taw WJ, Verver-Janssen TJ, et al. (2002) Neisseria meningitidis can induce pro-inflammatory cytokine production via pathways independent from CD14 and toll-like receptor 4. Eur Cytokine Netw 13: 411–417. 12. Massari P, Ho Y, Wetzler LM (2000) Neisseria meningitidis porin PorB interacts with mitochondria and protects cells from apoptosis. Proc Natl Acad Sci U S A 97: 9070–9075. 13. Muller A, Gunther D, Dux F, Naumann M, Meyer TF, et al. (1999) Neisserial porin (PorB) causes rapid calcium influx in target cells and induces apoptosis by the activation of cysteine proteases. EMBO J 18: 339–352. N. meningitidis NhhA Induces Macrophage Apoptosis THP-1 cells were infected with the N. meningitidis serogroup C strain FAM20 or NhhA-deficient meningococci at MOI = 100 for 20 hours. Release of the cytokine HMGB1 was examined by indirect ELISA. Values indicate mean6SD of three independent experiments. *, p,0.05. (TIF) Author Contributions Conceived and designed the experiments: HS MS. Performed the experiments: MS GA MH WS. Analyzed the data: MS GA HS. Contributed reagents/materials/analysis tools: HS SSW. Wrote the paper: MS. 14. Muller A, Gunther D, Brinkmann V, Hurwitz R, Meyer TF, et al. (2000) Targeting of the pro-apoptotic VDAC-like porin (PorB) of Neisseria gonor- rhoeae to mitochondria of infected cells. EMBO J 19: 5332–5343. 15. Tunbridge AJ, Stevanin TM, Lee M, Marriott HM, Moir JW, et al. (2006) Inhibition of macrophage apoptosis by Neisseria meningitidis requires nitric oxide detoxification mechanisms. Infect Immun 74: 729–733. 16. Marshall JC, Jia SH, Parodo J, Watson RW (2008) Interleukin-1beta mediates LPS-induced inhibition of apoptosis in retinoic acid-differentiated HL-60 cells. Biochem Biophys Res Commun 369: 532–538. 17. Majde JA (1993) Microbial cell-wall contaminants in peptides: a potential source of physiological artifacts. Peptides 14: 629–632. 18. Marriott HM, Ali F, Read RC, Mitchell TJ, Whyte MK, et al. (2004) Nitric oxide levels regulate macrophage commitment to apoptosis or necrosis during pneumococcal infection. FASEB J 18: 1126–1128. 19. Qin S, Wang H, Yuan R, Li H, Ochani M, et al. (2006) Role of HMGB1 in apoptosis-mediated sepsis lethality. J Exp Med 203: 1637–1642. 20. Griffiths NJ, Hill DJ, Borodina E, Sessions RB, Devos NI, et al. (2011) Meningococcal surface fibril (Msf) binds to activated vitronectin and inhibits the terminal complement pathway to increase serum resistance. Mol Microbiol. 21. Xu G, Shi Y (2007) Apoptosis signaling pathways and lymphocyte homeostasis. Cell Res 17: 759–771. 22. Padron J, Bebelagua Y, Lastre M, Lapinet J, Zayas C, et al. (1999) Nitric oxide participates in the immune response against Neisseria meningitidis serogroup B. FEMS Immunol Med Microbiol 25: 385–389. 23. Hotchkiss RS, Chang KC, Swanson PE, Tinsley KW, Hui JJ, et al. (2000) Caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte. Nat Immunol 1: 496–501. 24. Wesche DE, Lomas-Neira JL, Perl M, Chung CS, Ayala A (2005) Leukocyte apoptosis and its significance in sepsis and shock. J Leukoc Biol 78: 325–337. 25. Wang H, Bloom O, Zhang M, Vishnubhakat JM, Ombrellino M, et al. (1999) HMG-1 as a late mediator of endotoxin lethality in mice. Science 285: 248–251. 26. Sappington PL, Yang R, Yang H, Tracey KJ, Delude RL, et al. (2002) HMGB1 B box increases the permeability of Caco-2 enterocytic monolayers and impairs intestinal barrier function in mice. Gastroenterology 123: 790–802. 27. Yang H, Ochani M, Li J, Qiang X, Tanovic M, et al. (2004) Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc Natl Acad Sci U S A 101: 296–301. 28. Meyer M, Essack M, Kanyanda S, Rees J (2008) A low-cost flow cytometric assay for the detection and quantification of apoptosis using an anionic halogenated fluorescein dye. Biotechniques 45: 317–320. PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e29586