Binding to Na(+) /H(+) exchanger regulatory factor 2 (NHERF2) affects trafficking and function of the enteropathogenic Escherichia coli type III secretion system effectors Map, EspI and NleH.
Enteropathogenic Escherichia coli (EPEC) strains are diarrhoeal pathogens that use a type III secretion system to translocate effector proteins into host cells in order to colonize and multiply in the human gut. Map, EspI and NleH1 are conserved EPEC effectors that possess a C-terminal class I PSD-95/Disc Large/ZO-1 (PDZ)-binding motif. Using a PDZ array screen we identified Na(+)/H(+) exchanger regulatory factor 2 (NHERF2), a scaffold protein involved in tethering and recycling ion channels in polarized epithelia that contains two PDZ domains, as a common target of Map, EspI and NleH1. Using recombinant proteins and co-immunoprecipitation we confirmed that NHERF2 binds each of the effectors. We generated a HeLa cell line stably expressing HA-tagged NHERF2 and found that Map, EspI and NleH1 colocalize and interact with intracellular NHERF2 via their C-terminal PDZ-binding motif. Overexpression of NHERF2 enhanced the formation and persistence of Map-induced filopodia, accelerated the trafficking of EspI to the Golgi and diminished the anti-apoptotic activity of NleH1. The binding of multiple T3SS effectors to a single scaffold protein is unique. Our data suggest that NHERF2 may act as a plasma membrane sorting site, providing a novel regulatory mechanism to control the intracellular spatial and temporal effector protein activity.
Martinez
Eric
E
Centre for Molecular Microbiology and Infection, Imperial College London, UK.
Schroeder
Gunnar N
GN
Berger
Cedric N
CN
Lee
Sau Fung
SF
Robinson
Keith S
KS
Badea
Luminita
L
Simpson
Nandi
N
Hall
Randy A
RA
Hartland
Elizabeth L
EL
Crepin
Valerie F
VF
Frankel
Gad
G
D054308:Enteropathogenic Escherichia coli / Q000378:metabolism / Q000472:pathogenicity*; D004847:Epithelial Cells / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D037521:Virulence Factors / Q000378:metabolism*
D029968:Escherichia coli Proteins; C556113:EspI protein, E coli; C512346:Map protein, E coli; C556114:NleH protein, E coli; D010750:Phosphoproteins; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; D037521:Virulence Factors; C093376:sodium-hydrogen exchanger regulatory factor