title: Metalloprotease NleC suppresses host NF-κB/inflammatory responses by cleaving p65 and interfering with the p65/RPS3 interaction.
abstract: Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p65¹⁻³⁸ fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p65¹⁻³⁸ fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a 'specifier' subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.
authors.affiliation: W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America; Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Andrea
authors.initials: A
authors.lastname: Hodgson
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Wier
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Kai
authors.initials: K
authors.lastname: Fu
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Xin
authors.initials: X
authors.lastname: Sun
authors.affiliation: Division of Gastroenterology, Department of Pediatrics, BC's Children's Hospital and Child and Family Research Institute, Vancouver, British Columbia, Canada.
authors.forename: Hongbing
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Wenxin
authors.initials: W
authors.lastname: Zheng
authors.affiliation: Division of Gastroenterology, Department of Pediatrics, BC's Children's Hospital and Child and Family Research Institute, Vancouver, British Columbia, Canada.
authors.forename: Ho Pan
authors.initials: HP
authors.lastname: Sham
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Kaitlin
authors.initials: K
authors.lastname: Johnson
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Scott
authors.initials: S
authors.lastname: Bailey
authors.affiliation: Division of Gastroenterology, Department of Pediatrics, BC's Children's Hospital and Child and Family Research Institute, Vancouver, British Columbia, Canada.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Vallance
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, United States of America; Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Fengyi
authors.initials: F
authors.lastname: Wan
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000276:immunology* / Q000378:metabolism; D044082:Citrobacter rodentium; D004756:Enterobacteriaceae Infections / Q000276:immunology* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006790:Host-Parasite Interactions / Q000502:physiology*; D015151:Immunoblotting; D047468:Immunoprecipitation; D007249:Inflammation / Q000276:immunology / Q000378:metabolism; D045726:Metalloproteases / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000276:immunology* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D051996:Transcription Factor RelA / Q000276:immunology / Q000378:metabolism; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D016328:NF-kappa B; C498144:Rela protein, mouse; D012269:Ribosomal Proteins; C488930:Rsp3a1 protein, mouse; D051996:Transcription Factor RelA; D045726:Metalloproteases