Interaction between the SifA virulence factor and its host target SKIP is essential for Salmonella pathogenesis.
SifA is a Salmonella effector that is translocated into infected cells by the pathogenicity island 2-encoded type 3 secretion system. SifA is a critical virulence factor. Previous studies demonstrated that, upon translocation, SifA binds the pleckstrin homology motif of the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the mobilization of the molecular motor kinesin-1 on the bacterial vacuole. SifA exhibits multiple domains containing functional motifs. Here we performed a molecular dissection and a mutational study of SifA to evaluate the relative contribution of the different domains to SifA functions. Biochemical and crystallographic analysis confirmed that the N-terminal domain of SifA is sufficient to interact with the pleckstrin homology domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant. Mutation of the tryptophan residue in the WXXXE motif, which has been proposed to mimic active form of GTPase, deeply affected the stability and the translocation of SifA while mutations of the glutamic residue had no functional impact. A SifA L130D mutant that does not bind SKIP showed a DeltasifA-like phenotype both in infected cells and in the mouse model of infection. We concluded that the WXXXE motif is essential for maintaining the tertiary structure of SifA, the functions of which require the interaction with the eukaryotic protein SKIP.
Diacovich
Lautaro
L
Centre d'Immunologie de Marseille-Luminy, CNRS UMR 6102, INSERM U631, Université de la Méditerranée, Parc Scientifique de Luminy, Case 906, 13288 Marseille Cedex 9.
Dumont
Audrey
A
Lafitte
Daniel
D
Soprano
Elodie
E
Guilhon
Aude-Agnès
AA
Bignon
Christophe
C
Gorvel
Jean-Pierre
JP
Bourne
Yves
Y
Méresse
Stéphane
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054884:Host-Pathogen Interactions; D016547:Kinesins / Q000378:metabolism; D007700:Kinetics; D008264:Macrophages / Q000166:cytology / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009154:Mutation; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012475:Salmonella / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology; D012481:Salmonella Infections, Animal / Q000378:metabolism / Q000382:microbiology; D014774:Virulence / Q000235:genetics; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D006023:Glycoproteins; C421862:SifA protein, Salmonella; D037521:Virulence Factors; D049452:Green Fluorescent Proteins; C531212:Pps protein, mouse; D010744:Phosphoric Monoester Hydrolases; D016547:Kinesins