title: The apoptotic response to pneumolysin is Toll-like receptor 4 dependent and protects against pneumococcal disease.
abstract: Pneumolysin, the cholesterol-dependent cytolysin of Streptococcus pneumoniae, induces inflammatory and apoptotic events in mammalian cells. Toll-like receptor 4 (TLR4) confers resistance to pneumococcal infection via its interaction with pneumolysin, but the underlying mechanisms remain to be identified. In the present study, we found that pneumolysin-induced apoptosis is also mediated by TLR4 and confers protection against invasive disease. The interaction between TLR4 and pneumolysin is direct and specific; ligand-binding studies demonstrated that pneumolysin binds to TLR4 but not to TLR2. Involvement of TLR4 in pneumolysin-induced apoptosis was demonstrated in several complementary experiments. First, macrophages from wild-type mice were significantly more prone to pneumolysin-induced apoptosis than cells from TLR4-defective mice. In gain-of-function experiments, we found that epithelial cells expressing TLR4 and stimulated with pneumolysin were more likely to undergo apoptosis than cells expressing TLR2. A specific TLR4 antagonist, B1287, reduced pneumolysin-mediated apoptosis in wild-type cells. This apoptotic response was also partially caspase dependent as preincubation of cells with the pan-caspase inhibitor zVAD-fmk reduced pneumolysin-induced apoptosis. Finally, in a mouse model of pneumococcal infection, pneumolysin-producing pneumococci elicited significantly more upper respiratory tract cell apoptosis in wild-type mice than in TLR4-defective mice, and blocking apoptosis by administration of zVAD-fmk to wild-type mice resulted in a significant increase in mortality following nasopharyngeal pneumococcal exposure. Overall, our results strongly suggest that protection against pneumococcal disease is dependent on the TLR4-mediated enhancement of pneumolysin-induced apoptosis.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Children's Hospital, Boston, MA 02115, USA.
authors.forename: Amit
authors.initials: A
authors.lastname: Srivastava
authors.forename: Philipp
authors.initials: P
authors.lastname: Henneke
authors.forename: Alberto
authors.initials: A
authors.lastname: Visintin
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Morse
authors.forename: Victoria
authors.initials: V
authors.lastname: Martin
authors.forename: Claire
authors.initials: C
authors.lastname: Watkins
authors.forename: James C
authors.initials: JC
authors.lastname: Paton
authors.forename: Michael R
authors.initials: MR
authors.lastname: Wessels
authors.forename: Douglas T
authors.initials: DT
authors.lastname: Golenbock
authors.forename: Richard
authors.initials: R
authors.lastname: Malley
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects; D051379:Mice; D008815:Mice, Inbred Strains; D009305:Nasopharynx / Q000382:microbiology / Q000473:pathology; D011008:Pneumococcal Infections / Q000276:immunology* / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000378:metabolism; D013301:Streptolysins / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D001426:Bacterial Proteins; D061945:Caspase Inhibitors; D008070:Lipopolysaccharides; D013301:Streptolysins; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C032290:plY protein, Streptococcus pneumoniae; D020169:Caspases; C115671:B 1287