title: YgfZ contributes to secretion of cytotoxic necrotizing factor 1 into outer-membrane vesicles in Escherichia coli.
abstract: Cytotoxic necrotizing factor 1 (CNF1), a Rho GTPase-activating bacterial toxin, has been shown to contribute to invasion by meningitis-causing Escherichia coli K1 of human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. However, CNF1 is a cytosolic protein and it remains unclear how its secretion occurs, contributing to E. coli invasion of HBMEC. To investigate the genetic requirement for CNF1 secretion in E. coli K1 strain RS218, we performed mini-Tn5 in vitro mutagenesis and constructed a transposon mutant library of strain NBC, in which β-lactamase was fused to the C-terminus of CNF1 in the chromosome of strain RS218. We identified a transposon mutant (NBC-1E6) that exhibited reduced β-lactamase activity in its culture supernatant and had the transposon inserted into the ygfZ gene. When ygfZ was deleted from the genome of strain RS218 (ΔygfZ), the translocation of CNF1 into HBMEC was impaired. Subcellular localization analysis of CNF1 demonstrated that YgfZ, a periplasmic protein, contributes to secretion of CNF1 into outer-membrane vesicles (OMVs). The ΔygfZ mutant was significantly defective in invasion of HBMEC compared to the parent E. coli K1 strain. The defects of the ΔygfZ mutant in CNF1 secretion into OMVs and translocation into HBMEC as well as invasion of HBMEC were abrogated by complementation with ygfZ. Taken together, our findings demonstrate that YgfZ contributes to CNF1 secretion into OMVs in meningitis-causing E. coli K1.
authors.affiliation: Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, 200 North Wolfe Street, Room 3157, Baltimore, MD 21287, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Yu
authors.affiliation: Division of Pediatric Infectious Diseases, Johns Hopkins University School of Medicine, 200 North Wolfe Street, Room 3157, Baltimore, MD 21287, USA.
authors.forename: Kwang Sik
authors.initials: KS
authors.lastname: Kim
mesh_terms: D001427:Bacterial Toxins; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004251:DNA Transposable Elements; D042783:Endothelial Cells / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000378:metabolism*; D055354:Exosomes / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D016254:Mutagenesis, Insertional; D037521:Virulence Factors / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D037521:Virulence Factors; C509203:YgfZ protein, E coli; C060296:cytotoxic necrotizing factor type 1