The Leucine 130 of the Salmonella effector SifA is crucial for the interaction

L. DIACOVICH, A. DUMONT, D. LAFITTE, E. SOPRANO, C. BIGNON, JP GORVEL*, Y. BOURNE, S. MÉRESSE

Heidelberg, Germany

Workshop; EMBO Fellows Meeting 2009; 2009

Heidelberg, Germany (2009)

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. Besides SifA encloses two domains, a conserved N-terminal translocation signal of 140 residues and a C-terminal domain contained a WxxxE motif, which is propose to interacts with GDP-bound Rho family GTPases. Here we present a molecular dissection and a mutational study of SifA to evaluate the relevance of each domain of this effector on the formation of the SifA-SKIP complex, induction of Sifs, secretion, and virulence. Biochemical and structural analysis confirm that the N-terminal domain of SifA is sufficient to interact with the PH domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant, consistent with a weak transient complex of moderate stability. Mutants in the WxxxE motif are affected in term of stability and secretion only when the tryptophan (W197) residue is modified. While a mutant in the glutamic (E201) residue is still secreted and functional. In contrast a SifA mutant L130D is secreted but it lost the capacity to bind SKIP. In vivo this mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.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. Besides SifA encloses two domains, a conserved N-terminal translocation signal of 140 residues and a C-terminal domain contained a WxxxE motif, which is propose to interacts with GDP-bound Rho family GTPases. Here we present a molecular dissection and a mutational study of SifA to evaluate the relevance of each domain of this effector on the formation of the SifA-SKIP complex, induction of Sifs, secretion, and virulence. Biochemical and structural analysis confirm that the N-terminal domain of SifA is sufficient to interact with the PH domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant, consistent with a weak transient complex of moderate stability. Mutants in the WxxxE motif are affected in term of stability and secretion only when the tryptophan (W197) residue is modified. While a mutant in the glutamic (E201) residue is still secreted and functional. In contrast a SifA mutant L130D is secreted but it lost the capacity to bind SKIP. In vivo this mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.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. Besides SifA encloses two domains, a conserved N-terminal translocation signal of 140 residues and a C-terminal domain contained a WxxxE motif, which is propose to interacts with GDP-bound Rho family GTPases. Here we present a molecular dissection and a mutational study of SifA to evaluate the relevance of each domain of this effector on the formation of the SifA-SKIP complex, induction of Sifs, secretion, and virulence. Biochemical and structural analysis confirm that the N-terminal domain of SifA is sufficient to interact with the PH domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant, consistent with a weak transient complex of moderate stability. Mutants in the WxxxE motif are affected in term of stability and secretion only when the tryptophan (W197) residue is modified. While a mutant in the glutamic (E201) residue is still secreted and functional. In contrast a SifA mutant L130D is secreted but it lost the capacity to bind SKIP. In vivo this mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.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. Besides SifA encloses two domains, a conserved N-terminal translocation signal of 140 residues and a C-terminal domain contained a WxxxE motif, which is propose to interacts with GDP-bound Rho family GTPases. Here we present a molecular dissection and a mutational study of SifA to evaluate the relevance of each domain of this effector on the formation of the SifA-SKIP complex, induction of Sifs, secretion, and virulence. Biochemical and structural analysis confirm that the N-terminal domain of SifA is sufficient to interact with the PH domain of SKIP, forming a 1:1 complex with a micromolar dissociation constant, consistent with a weak transient complex of moderate stability. Mutants in the WxxxE motif are affected in term of stability and secretion only when the tryptophan (W197) residue is modified. While a mutant in the glutamic (E201) residue is still secreted and functional. In contrast a SifA mutant L130D is secreted but it lost the capacity to bind SKIP. In vivo this mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.In vivo this mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.