INVESTIGADORES
DIACOVICH Lautaro
congresos y reuniones científicas
Título:
The Leucine 130 of the Salmonella effector SifA is crucial for the interaction
Autor/es:
L. DIACOVICH, A. DUMONT, D. LAFITTE, E. SOPRANO, C. BIGNON, JP GORVEL*, Y. BOURNE, S. MÉRESSE
Lugar:
Heidelberg, Germany
Reunión:
Workshop; EMBO Fellows Meeting 2009; 2009
Institución organizadora:
Heidelberg, Germany (2009)
Resumen:
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
mutant accumulate kinesin at the SCV, it is not able to make Sifs, and is attenuate for virulence in mice.
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 re