title: Flagellin-induced NLRC4 phosphorylation primes the inflammasome for activation by NAIP5.
abstract: The Nlrc4 inflammasome contributes to immunity against intracellular pathogens that express flagellin and type III secretion systems, and activating mutations in NLRC4 cause autoinflammation in patients. Both Naip5 and phosphorylation of Nlrc4 at Ser533 are required for flagellin-induced inflammasome activation, but how these events converge upon inflammasome activation is not known. Here, we showed that Nlrc4 phosphorylation occurs independently of Naip5 detection of flagellin because Naip5 deletion in macrophages abolished caspase-1 activation, interleukin (IL)-1β secretion, and pyroptosis, but not Nlrc4 phosphorylation by cytosolic flagellin of Salmonella Typhimurium and Yersinia enterocolitica. ASC speck formation and caspase-1 expression also were dispensable for Nlrc4 phosphorylation. Interestingly, Helicobacter pylori flagellin triggered robust Nlrc4 phosphorylation, but failed to elicit caspase-1 maturation, IL-1β secretion, and pyroptosis, suggesting that it retained Nlrc4 Ser533 phosphorylating-activity despite escaping Naip5 detection. In agreement, the flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas deletion of the S. Typhimurium flagellin carboxy-terminus prevented caspase-1 maturation only. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation prepares Nlrc4 for subsequent activation by the flagellin sensor Naip5.
authors.affiliation: Department of Medical Protein Research, VIB, Ghent B-9000, Belgium; Department of Biochemistry, Ghent University, Ghent B-9000, Belgium;
authors.forename: Magdalena
authors.initials: M
authors.lastname: Matusiak
authors.affiliation: Department of Medical Protein Research, VIB, Ghent B-9000, Belgium; Department of Biochemistry, Ghent University, Ghent B-9000, Belgium;
authors.forename: Nina
authors.initials: N
authors.lastname: Van Opdenbosch
authors.affiliation: Department of Medical Protein Research, VIB, Ghent B-9000, Belgium; Department of Biochemistry, Ghent University, Ghent B-9000, Belgium;
authors.forename: Lieselotte
authors.initials: L
authors.lastname: Vande Walle
authors.affiliation: Centre d'Infection et d'Immunité de Lille, Institut Pasteur de Lille, Lille 59019, France; and.
authors.forename: Jean-Claude
authors.identifier: http://orcid.org/0000-0001-9854-4652
authors.initials: JC
authors.lastname: Sirard
authors.affiliation: Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105-2794.
authors.forename: Thirumala-Devi
authors.initials: TD
authors.lastname: Kanneganti
authors.affiliation: Department of Medical Protein Research, VIB, Ghent B-9000, Belgium; Department of Biochemistry, Ghent University, Ghent B-9000, Belgium; mohamed.lamkanfi@vib-ugent.be.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Lamkanfi
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D020170:Caspase 1 / Q000378:metabolism; D017931:DNA Primers; D004789:Enzyme Activation; D005408:Flagellin / Q000378:metabolism*; D058847:Inflammasomes / Q000378:metabolism*; D051379:Mice; D051616:Neuronal Apoptosis-Inhibitory Protein / Q000378:metabolism*; D010766:Phosphorylation; D060888:Real-Time Polymerase Chain Reaction; D012486:Salmonella typhimurium / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015008:Yersinia enterocolitica / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D058847:Inflammasomes; C512299:Ipaf protein, mouse; C496806:Naip5 protein, mouse; D051616:Neuronal Apoptosis-Inhibitory Protein; D005408:Flagellin; D012694:Serine; D020170:Caspase 1
keywords: NLRC4; Salmonella; caspase-1; flagellin; inflammasome