title: Laboratory and clinical Pseudomonas aeruginosa strains do not bind glycosphingolipids in vitro or during type IV pili-mediated initial host cell attachment.
abstract: The glycosphingolipids (GSLs) gangliotriaosylceramide (Gg(3)) and gangliotetraosylceramide (Gg(4)) have been implicated as receptors for type IV pili (T4P)-mediated Pseudomonas aeruginosa epithelial cell attachment. Since P. aeruginosa T4P are divided into five groups, the authors determined whether GSLs in general, and Gg(3) and Gg(4) in particular, are specifically bound and required for host epithelial cell attachment of clinical and laboratory strains within these groups. An enterohaemorrhagic Escherichia coli strain, CL56, known to bind to both Gg(3) and Gg(4), provided a positive control. TLC overlay showed no binding of more than 12 P. aeruginosa strains to either Gg(3) or Gg(4) (or other GSLs), while CL56 Gg(3)/Gg(4) binding was readily detectable. GSL ELISA similarly demonstrated no significant P. aeruginosa binding to Gg(3) or Gg(4), compared with CL56. Using a selective chemical inhibitor, epithelial cell GSL synthesis was abrogated, and Gg(3) and Gg(4) expression deleted, but P. aeruginosa attachment was not impaired. Target cell attachment was mediated by T4P, since non-piliated, but flagellated, mutants were unable to bind to the target cells. CFTR (cystic fibrosis transmembrane conductance regulator) has also been implicated as a receptor; however, in this work, overexpression of CFTR had no effect on P. aeruginosa binding. It is concluded that neither Gg(3) nor Gg(4) are specifically recognized by P. aeruginosa, and that endogenous GSLs do not have a role in the attachment of live intact P. aeruginosa to cultured lung epithelial cells. In contrast to whole piliated P. aeruginosa, T4P sheared from such bacteria showed significant Gg(3) and Gg(4) binding, which may explain the results of other studies.
authors.affiliation: Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.|Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Aufaugh
authors.initials: A
authors.lastname: Emam
authors.affiliation: Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Analyn R
authors.initials: AR
authors.lastname: Yu
authors.affiliation: Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Hyun-Joo
authors.initials: HJ
authors.lastname: Park
authors.affiliation: Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Radhia
authors.initials: R
authors.lastname: Mahfoud
authors.affiliation: Department of Surgery, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Julianne
authors.initials: J
authors.lastname: Kus
authors.affiliation: Department of Surgery, University of Toronto, Toronto, Ontario, Canada.|Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Lori L
authors.initials: LL
authors.lastname: Burrows
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.|Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.|Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Clifford A
authors.initials: CA
authors.lastname: Lingwood
mesh_terms: D001422:Bacterial Adhesion; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000382:microbiology; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D010861:Fimbriae, Bacterial / Q000502:physiology; D005732:Gangliosides / Q000378:metabolism; D006028:Glycosphingolipids / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008168:Lung / Q000166:cytology; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D033081:Shear Strength
chemical_list: C011258:G(A1) ganglioside; D005732:Gangliosides; D006028:Glycosphingolipids; C038244:ganglio-N-triaosylceramide