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. 2013;4:2973.
doi: 10.1038/ncomms3973.

Effectors of Animal and Plant Pathogens Use a Common Domain to Bind Host Phosphoinositides

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Free PMC article

Effectors of Animal and Plant Pathogens Use a Common Domain to Bind Host Phosphoinositides

Dor Salomon et al. Nat Commun. .
Free PMC article

Abstract

Bacterial Type III Secretion Systems deliver effectors into host cells to manipulate cellular processes to the advantage of the pathogen. Many host targets of these effectors are found on membranes. Therefore, to identify their targets, effectors often use specialized membrane-localization domains to localize to appropriate host membranes. However, the molecular mechanisms used by many domains are unknown. Here we identify a conserved bacterial phosphoinositide-binding domain (BPD) that is found in functionally diverse Type III effectors of both plant and animal pathogens. We show that members of the BPD family functionally bind phosphoinositides and mediate localization to host membranes. Moreover, NMR studies reveal that the BPD of the newly identified Vibrio parahaemolyticus Type III effector VopR is unfolded in solution, but folds into a specific structure upon binding its ligand phosphatidylinositol-(4,5)-bisphosphate. Thus, our findings suggest a possible mechanism for promoting refolding of Type III effectors after delivery into host cells.

Figures

Figure 1
Figure 1. VopR localizes to the PM
(a) Localization of galactose-inducible eGFP-fusion proteins in BY4741 yeast cells. Bar = 5 μm. (b) Localization of eGFP-fusion proteins in transfected HeLa cells. Bar = 10 μm.
Figure 2
Figure 2. VopR1–125 specifically binds PIP2
(a) Lipid overlay assay with purified GST-fusion proteins. Membranes were immunoblotted with anti-GST antibodies. (b) Localization of galactose-inducible proteins in MSS4 wild-type or temperature-sensitive mutant yeast at the restrictive temperature (37 °C). Bar = 5 μm. (c) Localization of indicated proteins in transfected HeLa cells 2 h post infection with Vibrio strains expressing (bottom panels), or deleted for (top panels), vpa0450. Bar = 10 μm.
Figure 3
Figure 3. PIP2 induces VopR1–125 folding in solution
(a) 15N/1H HSQC spectra of Gβ1 (black) and His6-Gβ1-VopR1–125 (red). (b) 15N/1H HSQC spectra of Gβ1 (black) and His6-Gβ1-VopR1–125 (blue) in the presence of PI(4,5)P2-diC8 (at 1:1 molar ratio). Dashed line indicates region shown in expansion on right. All spectra were recorded at 25 °C on a 600 MHz NMR with VopR samples at 200 μM protein.
Figure 4
Figure 4. VopS contains a PIP2-binding BPD
(a) HeLa cells 5 h after infection with effector-less Yersinia induced to express the indicated proteins. Actin (red) and DNA (Blue) are shown. Bar = 20 μm. (b) Localization of eGFP-fusion protein in transfected HeLa cells. Bar 10 μm. (c) Localization of galactose-inducible eGFP-fusion proteins in BY4741 yeast cells. Bar = 5 μm. (d) Localization of eGFP-fusion proteins in transfected HeLa cells. Bar = 10 μm. (e) Localization of galactose-inducible VopS1–125–eGFP in MSS4 wild-type or temperature-sensitive mutant yeast at the restrictive temperature (37 °C). Bar = 5 μm. (f) Localization of VopS1–125–eGFP in transfected HeLa cells 2 h after infection with Vibrio strains expressing (bottom panel), or deleted for (top panel), vpa0450. Bar = 10 μm.
Figure 5
Figure 5. BPDs of VPA0450, YpkA, and HopA1 bind phosphoinositides and localize to eukaryotic membranes
(a) The ‘core’ region of the VopR (GI:28898457) BPD (top), and the VopS (GI:28898460), VPA0450 (GI:28900305), YpkA (GI:51593847) and HopA1 (GI:28872465) aligned BPDs (bottom), with corresponding secondary structure prediction shown above: α-helices (red) and β-strands (green); and functional sequence motif indicated by asterisks. Conserved residues are highlighted yellow (mainly hydrophobic), grey (small), polar motif (blue) and functional motif (black). (b) Lipid overlay assay with purified His6-Gβ1-fusion proteins. Membranes were immunoblotted with anti-His antibodies. (c) Localization of eGFP-fusion protein in transfected HeLa cells. Bar = 10 μm. (d) Localization of eGFP-fusion protein in Agrobacterium-infiltrated tomato epidermal cells. White box indicates region shown in expansion on the right. Chloroplast auto-fluorescence (red) is shown in right panel. Bar = 10 μm.
Figure 6
Figure 6. A conserved motif required for membrane localization of BPDs
Localization of galactose-inducible eGFP-fusion proteins in BY4741 yeast cells. WT, wild-type protein. Bar = 5 μm. (a) Localization of VopS mutations. (b) Localization of BPDVPA0450, BPDHopA1 and BPDVopR mutants.
Figure 7
Figure 7. Model for Type III effectors refolding and targeting in host cells
Chaperones (Green ball) bind to otherwise unstructured host-localization domains of Type III effectors (Blue strand) to prevent aggregation while inside the bacterium. To translocate through the T3SS (Red cylinders), effectors are separated from the chaperones and must unfold to allow passage through the ‘needle’ structure. Once inside the host cell, the unfolded effectors can bind a host-specific ligand (Yellow ball; in this case PIP2 on the PM) and promote both refolding of the effector (Blue cylinder) and localization to the appropriate cellular compartment.

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