title: Flagella from five Cronobacter species induce pro-inflammatory cytokines in macrophage derivatives from human monocytes.
abstract: Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10) in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng) induced the release of IL-8 (3314-6025 pg/ml), TNF-α (39-359 pg/ml), and IL-10 (2-96 pg/ml), in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200) suppressed the secretion of IL-8, TNF-α, and IL-10 between 95-100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria.
authors.affiliation: Laboratorio de Bacteriología Intestinal, Departamento de Infectología, Hospital Infantil de México Federico Gómez, México D.F., México.
authors.forename: Ariadnna
authors.initials: A
authors.lastname: Cruz-Córdova
authors.forename: Luz M
authors.initials: LM
authors.lastname: Rocha-Ramírez
authors.forename: Sara A
authors.initials: SA
authors.lastname: Ochoa
authors.forename: Bertha
authors.initials: B
authors.lastname: González-Pedrajo
authors.forename: Bertha
authors.initials: B
authors.lastname: Gónzalez-Pedrajo
authors.forename: Norma
authors.initials: N
authors.lastname: Espinosa
authors.forename: Carlos
authors.initials: C
authors.lastname: Eslava
authors.forename: Ulises
authors.initials: U
authors.lastname: Hernández-Chiñas
authors.forename: Guillermo
authors.initials: G
authors.lastname: Mendoza-Hernández
authors.forename: Alejandra
authors.initials: A
authors.lastname: Rodríguez-Leviz
authors.forename: Pedro
authors.initials: P
authors.lastname: Valencia-Mayoral
authors.forename: Stanislaw
authors.initials: S
authors.lastname: Sadowinski-Pine
authors.forename: Rigoberto
authors.initials: R
authors.lastname: Hernández-Castro
authors.forename: Iris
authors.initials: I
authors.lastname: Estrada-García
authors.forename: Onofre
authors.initials: O
authors.lastname: Muñoz-Hernández
authors.forename: Irma
authors.initials: I
authors.lastname: Rosas
authors.forename: Juan
authors.initials: J
authors.lastname: Xicohtencatl-Cortes
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D059126:Cronobacter / Q000276:immunology* / Q000648:ultrastructure; D016207:Cytokines / Q000378:metabolism*; D005407:Flagella / Q000737:chemistry / Q000187:drug effects / Q000276:immunology* / Q000648:ultrastructure; D005408:Flagellin / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007070:Immunoglobulin A / Q000276:immunology / Q000494:pharmacology; D018836:Inflammation Mediators / Q000378:metabolism*; D008264:Macrophages / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology; D016415:Sequence Alignment; D051198:Toll-Like Receptor 5 / Q000037:antagonists & inhibitors / Q000276:immunology
chemical_list: D000906:Antibodies; D016207:Cytokines; D007070:Immunoglobulin A; D018836:Inflammation Mediators; D051198:Toll-Like Receptor 5; D005408:Flagellin