Rab1 Guanine Nucleotide Exchange Factor SidM Is a Major Phosphatidylinositol 4-Phosphate-binding Effector Protein of Legionella pneumophila

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 ␣-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 ⌬sidM 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 III␤. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase III␤ to anchor the effectors SidC and SidM to LCVs.

Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development

“…However, PI(3)P at late endosomes/lysosomes is expected to be further phosphorylated to PI(3,5)P 2 , by the activity of the PI 5-kinase PIKfyve. Similarly to Legionella effectors, CvpB uses PIs for membrane anchoring, apparently using a nonconventional lipid-binding domain as previously reported for the Legionella effectors SidC and SidM (42,43). Similarly to the Salmonella effector SopB, Coxiella CvpB triggers an increase in PI(3)P levels of infected cells to favor the biogenesis of a spacious replicative compartment (40).…”

Structural basis for substrate recognition by a unique Legionella phosphoinositide phosphatase

“…Intriguingly, PI(4,5)P 2 may be further converted to PI(4)P by the host PI-5-phosphatase OCRL (20). Thus, one plausible hypothesis is that SidF plays a role in the establishment of LCVs with a lipid composition enriched in PI(4)P. It has been suggested that PI(4)P provides specific anchors on the LCV for Legionella effectors, such as SidC/SdcA (18) and SidM(DrrA) (31). Through binding to PI(4)P, these effectors presumably facilitate the fusion of ERderived vesicles with the LCV.…”

Characterization of Enzymes from Legionella pneumophila Involved in Reversible Adenylylation of Rab1 Protein

“…Later it was demonstrated that it contains only GEF activity in a central domain containing residues 340 -533, but that no active displacement (GDI displacement factor activity) occurs (15). The C-terminal domain of the protein was reported to bind phosphatidylinositol-4-phosphate with high affinity (16,17), and the N-terminal domain was shown to be an adenylyltransferase catalyzing the covalent attachment of an AMP moiety to Tyr 77 in the switch II region of Rab1 (18). It was shown that the modifications has only small effects on DrrA-mediated nucleotide exchange, whereas catalysis by the GAPs LepB and TBC1D20 is strongly inhibited.…”