Molecular and Cellular Biology of Salmonella’s Infections: Structural and functional analysis of the SifA:SKIP complex.

DIACOVICH L., SOPRANO E., BIGNON C., LAFITTE D., BOURNE Y., MÉRESSE S.

Villars-sur-Ollon, Switzerland.

Conferencia; EMBO conference series: At the joint edge of cellular microbiology & cell biology.; 2008

European Molecular Biology Organization (EMBO)

SifA is a Salmonella effector that is translocated into the cytoplasm of infected cells by the pathogenicity island-2 encoded type III secretion system. SifA is required for the formation of Salmonella-induced filaments (Sifs) and for the stability of the Salmonella-containing vacuole (SCV). Previous studies demonstrated that, upon translocation, the SifA activity is mediated by the interaction with the eukaryotic host protein SKIP. In turn, the SifA-SKIP complex regulates the recruitment of the molecular motor kinesin-1 on the bacterial vacuole. SKIP is made of two distinct functional domains. The N-terminal domain harboring a RUN motif is involved in the interaction with kinesin-1 while the C-terminal Pleckstrin homology (PH) motif binds to SifA in infected cells. Here we present a functional dissection of SifA domains required for SifA-SKIP complex formation. Complementary co-imunoprecipitation and pull down analysis of the deletion SifA mutants reveals that the N-terminal domain of SifA is sufficient to interact with the PH domain of SKIP. Purified SifA and SKIP(PH) proteins forms a 1:1 complex with a micromolar dissociation constant, consistent with a weak transient complex of moderate stability. Finally, proteins crystals suitable for X-ray diffraction studies were obtained from the native SifA-SKIP(PH) complex and from the Seleno-methionine derivate proteins. The understanding of this kinesin modulation mechanism will provide evidence of how effectors activate a cellular signalization pathway and define its role during the course of the pathogenic process.