title: Secretion of the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase by the LEE-encoded type III secretion system in enteropathogenic Escherichia coli.
abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional housekeeping protein secreted by pathogens and involved in adhesion and/or virulence. Previously we reported that enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli secrete GAPDH into the culture medium. This bacterial protein binds human plasminogen and fibrinogen and remains associated with Caco-2 cells upon infection. In these pathogens, GAPDH secretion is not linked to outer membrane vesicles and depends on growth conditions, although the secretion mechanism is still unknown. EPEC is an attaching and effacing pathogen able to secrete and translocate multiple effector proteins into infected cells through a type III secretion system (T3SS). The secretion process is often dependent on a bacterial chaperone. The chaperone CesT displays broad substrate specificity and plays a central role in the recruitment of multiple type III effectors to the T3SS apparatus. Here we provide genetic evidences on GAPDH secretion through T3SS by EPEC grown in DMEM. Secretion of GAPDH is increased in ΔsepD mutants and abolished in mutants defective in the type III ATPase EscN. Complementation with escN gene restores GAPDH secretion. In addition, we prove by means of pull down experiments, overlay immunoblotting and biolayer interferometry a novel interaction between GAPDH and the chaperone CesT. This interaction, which is strong and slow dissociating, may stabilize a population of GAPDH molecules in a secretion competent-state and target them to the type III secretion apparatus. This is the first description of CesT interaction with a housekeeping protein and its export through T3SS.
authors.affiliation: Departament de Bioquímica i Biología Molecular, Institut de Biomedicina de la Universitat de Barcelona (IBUB), Facultat de Farmàcia, Universitat de Barcelona, 08028 Barcelona, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Aguilera
authors.forename: Elaine
authors.initials: E
authors.lastname: Ferreira
authors.forename: Rosa
authors.initials: R
authors.lastname: Giménez
authors.forename: Francisco José
authors.initials: FJ
authors.lastname: Fernández
authors.forename: Marta
authors.initials: M
authors.lastname: Taulés
authors.forename: Juan
authors.initials: J
authors.lastname: Aguilar
authors.forename: M Cristina
authors.initials: MC
authors.lastname: Vega
authors.forename: Josefa
authors.initials: J
authors.lastname: Badia
authors.forename: Laura
authors.initials: L
authors.lastname: Baldomà
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003470:Culture Media; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
chemical_list: D003470:Culture Media; D017931:DNA Primers; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases