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Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.

Brombacher E, Urwyler S, Ragaz C, Weber SS, Kami K, Overduin M, Hilbi H - J. Biol. Chem. (2008)

Bottom Line: Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs.An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole.Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.

ABSTRACT
The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the alpha-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIbeta. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

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C-terminal fragments of SidM localize to LCVs. A, confocal laser scanning micrographs of calnexin-GFP-labeled D. discoideum Ax3 (green), infected at an m.o.i. of 50 for 1 h with L. pneumophila labeled with a serogroup 1-specific antibody (red) and immunostained for M45-SidM, M45-M7, and M45-SidC with an anti-M45 antibody (blue). B, D. discoideum was infected at an m.o.i. of 50 for 1 h with L. pneumophila ΔsidC-sdcA harboring plasmid pCR34 (M45-SidC), pCR52 (M45-SidC-(1–608)), or pEB216 (SidC-(1–586)-M9) and immunostained using antibodies against L. pneumophila serogroup 1 (red) and SidC (green). The experiments were reproduced three (A) or two (B) independent times with similar results.
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fig5: C-terminal fragments of SidM localize to LCVs. A, confocal laser scanning micrographs of calnexin-GFP-labeled D. discoideum Ax3 (green), infected at an m.o.i. of 50 for 1 h with L. pneumophila labeled with a serogroup 1-specific antibody (red) and immunostained for M45-SidM, M45-M7, and M45-SidC with an anti-M45 antibody (blue). B, D. discoideum was infected at an m.o.i. of 50 for 1 h with L. pneumophila ΔsidC-sdcA harboring plasmid pCR34 (M45-SidC), pCR52 (M45-SidC-(1–608)), or pEB216 (SidC-(1–586)-M9) and immunostained using antibodies against L. pneumophila serogroup 1 (red) and SidC (green). The experiments were reproduced three (A) or two (B) independent times with similar results.

Mentions: C-terminal Fragments of SidM Localize to LCVs in Vivo—In vitro, SidM specifically binds to PtdIns(4)P (Fig. 2), a compound that has been identified as a lipid component of LCVs (32). We used D. discoideum to address the question of whether SidM not only localizes to the LCV membrane in infected macrophages (19, 20) but also in amoebae, and whether N-terminal PtdIns(4)P-binding fragments of SidM are still translocated by the Icm/Dot T4SS and bind to the LCV membrane. To this end, we produced M45-tagged SidM and fragments thereof in the wild-type L. pneumophila strain JR32, infected the amoebae, and analyzed the localization by immunofluorescence microscopy (Fig. 5A). Full-length M45-SidM as well as M45-SidC, used as a positive control (33), were translocated and bound to LCV membranes in D. discoideum amoebae, as expected. The SidM fragment M45-M7, including the P4M domain but lacking a 24-kDa N-terminal fragment, was also translocated into D. discoideum and bound to LCV membranes, indicating that SidM possesses a C-terminal translocation signal. Approximately, 66% of calnexin-positive LCVs also stained positive for M45-M7. The translocation of M45-M7 required a functional Icm/Dot T4SS and did not occur in a ΔicmT mutant strain.


Rab1 guanine nucleotide exchange factor SidM is a major phosphatidylinositol 4-phosphate-binding effector protein of Legionella pneumophila.

Brombacher E, Urwyler S, Ragaz C, Weber SS, Kami K, Overduin M, Hilbi H - J. Biol. Chem. (2008)

C-terminal fragments of SidM localize to LCVs. A, confocal laser scanning micrographs of calnexin-GFP-labeled D. discoideum Ax3 (green), infected at an m.o.i. of 50 for 1 h with L. pneumophila labeled with a serogroup 1-specific antibody (red) and immunostained for M45-SidM, M45-M7, and M45-SidC with an anti-M45 antibody (blue). B, D. discoideum was infected at an m.o.i. of 50 for 1 h with L. pneumophila ΔsidC-sdcA harboring plasmid pCR34 (M45-SidC), pCR52 (M45-SidC-(1–608)), or pEB216 (SidC-(1–586)-M9) and immunostained using antibodies against L. pneumophila serogroup 1 (red) and SidC (green). The experiments were reproduced three (A) or two (B) independent times with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2643517&req=5

fig5: C-terminal fragments of SidM localize to LCVs. A, confocal laser scanning micrographs of calnexin-GFP-labeled D. discoideum Ax3 (green), infected at an m.o.i. of 50 for 1 h with L. pneumophila labeled with a serogroup 1-specific antibody (red) and immunostained for M45-SidM, M45-M7, and M45-SidC with an anti-M45 antibody (blue). B, D. discoideum was infected at an m.o.i. of 50 for 1 h with L. pneumophila ΔsidC-sdcA harboring plasmid pCR34 (M45-SidC), pCR52 (M45-SidC-(1–608)), or pEB216 (SidC-(1–586)-M9) and immunostained using antibodies against L. pneumophila serogroup 1 (red) and SidC (green). The experiments were reproduced three (A) or two (B) independent times with similar results.
Mentions: C-terminal Fragments of SidM Localize to LCVs in Vivo—In vitro, SidM specifically binds to PtdIns(4)P (Fig. 2), a compound that has been identified as a lipid component of LCVs (32). We used D. discoideum to address the question of whether SidM not only localizes to the LCV membrane in infected macrophages (19, 20) but also in amoebae, and whether N-terminal PtdIns(4)P-binding fragments of SidM are still translocated by the Icm/Dot T4SS and bind to the LCV membrane. To this end, we produced M45-tagged SidM and fragments thereof in the wild-type L. pneumophila strain JR32, infected the amoebae, and analyzed the localization by immunofluorescence microscopy (Fig. 5A). Full-length M45-SidM as well as M45-SidC, used as a positive control (33), were translocated and bound to LCV membranes in D. discoideum amoebae, as expected. The SidM fragment M45-M7, including the P4M domain but lacking a 24-kDa N-terminal fragment, was also translocated into D. discoideum and bound to LCV membranes, indicating that SidM possesses a C-terminal translocation signal. Approximately, 66% of calnexin-positive LCVs also stained positive for M45-M7. The translocation of M45-M7 required a functional Icm/Dot T4SS and did not occur in a ΔicmT mutant strain.

Bottom Line: Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs.An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole.Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.

ABSTRACT
The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the alpha-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila DeltasidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIbeta. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIbeta to anchor the effectors SidC and SidM to LCVs.

Show MeSH
Related in: MedlinePlus