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. 2019 Apr 19;294(16):6405-6415.
doi: 10.1074/jbc.RA118.007086. Epub 2019 Feb 7.

The Legionella Effector RavD Binds phosphatidylinositol-3-phosphate and Helps Suppress Endolysosomal Maturation of the Legionella-containing Vacuole

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Free PMC article

The Legionella Effector RavD Binds phosphatidylinositol-3-phosphate and Helps Suppress Endolysosomal Maturation of the Legionella-containing Vacuole

Colleen M Pike et al. J Biol Chem. .
Free PMC article

Abstract

Upon phagocytosis into macrophages, the intracellular bacterial pathogen Legionella pneumophila secretes effector proteins that manipulate host cell components, enabling it to evade lysosomal degradation. However, the bacterial proteins involved in this evasion are incompletely characterized. Here we show that the L. pneumophila effector protein RavD targets host membrane compartments and contributes to the molecular mechanism the pathogen uses to prevent encounters with lysosomes. Protein-lipid binding assays revealed that RavD selectively binds phosphatidylinositol-3-phosphate (PI(3)P) in vitro We further determined that a C-terminal RavD region mediates the interaction with PI(3)P and that this interaction requires Arg-292. In transiently transfected mammalian cells, mCherry-RavD colocalized with the early endosome marker EGFP-Rab5 as well as the PI(3)P biosensor EGFP-2×FYVE. However, treatment with the phosphoinositide 3-kinase inhibitor wortmannin did not disrupt localization of mCherry-RavD to endosomal compartments, suggesting that RavD's interaction with PI(3)P is not necessary to anchor RavD to endosomal membranes. Using superresolution and immunogold transmission EM, we observed that, upon translocation into macrophages, RavD was retained onto the Legionella-containing vacuole and was also present on small vesicles adjacent to the vacuole. We also report that despite no detectable effects on intracellular growth of L. pneumophila within macrophages or amebae, the lack of RavD significantly increased the number of vacuoles that accumulate the late endosome/lysosome marker LAMP-1 during macrophage infection. Together, our findings suggest that, although not required for intracellular replication of L. pneumophila, RavD is a part of the molecular mechanism that steers the Legionella-containing vacuole away from endolysosomal maturation pathways.

Keywords: Legionella pneumophila; Legionnaires' disease; bacterial effectors; bacterial pathogenesis; cellular localization; host–pathogen interaction; immune evasion; infection; phosphoinositide; virulence factor.

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

Figures

Figure 1.
Figure 1.
RavD localizes to membrane compartments positive for PI(3)P and binds PI(3)P in vitro. A, RavD is cytosolic and membrane-bound. HEK293T cells producing mCherry-RavD or mCherry alone were homogenized, and the post-nuclear supernatant (PNS) was subjected to cellular fractionation. An anti-mCherry antibody was used to detect the presence of RavD. Antibodies against tubulin and calnexin were used to mark the cytosolic (Cyto) and membrane (Mem) fractions, respectively. B, confocal images of HeLa cells transiently co-transfected with plasmids encoding either EGFP-2×FYVE or EGFP and mCherry-RavD or mCherry. Scale bars = 10 μm (insets, 2 μm). C, protein–lipid overlay assay showing that GST-RavD specifically recognizes PI(3)P. A nitrocellulose membrane prespotted with 100 pmol of each indicated lipid was incubated with purified GST-RavD. RavD retained on the membrane was detected by incubation with anti-GST and an HRP-conjugated secondary anti-rabbit antibody. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PtdIns, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; SIP, sphingosine-1-phosphate; PI, phosphatidylinositol; P, phosphate; P2, biphosphate; P3, triphosphate; PA, phosphatidic acid; PS, phosphatidylserine. D, liposomes containing the indicated lipids were incubated with GST-RavD, GST-SidM, or GST alone and subjected to ultracentrifugation to separate bound from unbound protein. Input and float samples were separated on 4–15% TGX stain–free SDS-PAGE gels. Results are representatives of at least two independent experiments with similar outcomes.
Figure 2.
Figure 2.
The PI(3)P binding region of RavD is positioned within a C-terminal region. A, schematic of the predicted secondary structure of RavD (obtained using the JPred Protein Secondary Structure Prediction Server) (blue, α helix; yellow, β sheet); the position of relevant amino acid (aa) residues is marked. B, alignment of RavD's conserved arginine residues in the indicated Legionella species. C, protein–lipid overlay assays show that the GST-RavD190–325 and GST-RavDR237A variants recognize PI(3)P whereas GST-RavD1–189 and GST-RavDR292A do not. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PtdIns, phosphatidylinositol; PE, phosphatidylethanolamine; PC, phosphatidylcholine; SIP, sphingosine-1-phosphate; PI, phosphatidylinositol; P, phosphate; P2, biphosphate; P3, triphosphate; PA, phosphatidic acid; PS, phosphatidylserine. D, confocal images of HeLa cells transiently co-transfected with plasmids encoding either EGFP-2×FYVE and variants mCherry-RavD190–325, mCherry-RavD1–189, GST-RavDR237A, or RavDR292A. Scale bars = 10 μm (insets, 2 μm). Confocal images and assays are representative of at least two independent experiments with similar outcomes. E, Mander's overlap coefficient for fluorescence signals of EGFP-tagged and mCherry-tagged proteins, as specified. The plot shows the median (black vertical line) and interquartile range (25–75) (gray box) from 15 different cells for each condition. Individual dots represent the Mander's overlap coefficient obtained from a single cell. ***, p ≤ 0.001; ****, p ≤ 0.0001; ns, not significant.
Figure 3.
Figure 3.
RavD localizes to the LCV and surrounding vesicles. A, superresolution structured illumination microscopy image of RAW264.7 macrophages infected at an m.o.i. of 20, fixed 5 h post-infection, and stained with anti-L. pneumophila (green) and anti-HA (red) antibodies. Scale bars = 5 μm (insets, 2 μm). B, representative transmission electron microscopy images showing immunogold localization of HA-RavD in RAW264.7 macrophages. Areas highlighted by rectangles (dashed line) in the top panels are magnified in the bottom panels. HA-RavD localized on the LCV membrane and on vesicles (V) surrounding the LCV. L.p., L. pneumophila. Scale bars = 200 nm in the top panels and 100 nm in the bottom panels. C, the percentage of RavD-positive LCVs gradually increased post-infection. RAW264.7 macrophages were infected with the ΔravD+pHA-RavD or ΔdotA+pHA-RavD for 0.5, 2, 4, and 8 h, and the percentage of HA-RavD-positive vacuoles was determined for each time point. Data are the average and standard deviation from two independent experiments where at least 50 different vacuoles in different cells were assessed.
Figure 4.
Figure 4.
RavD is dispensable for intracellular growth. Monolayers of RAW264.7 macrophages or A. castellanii were infected with WT Lp01, ΔdotA, or ΔravD strains at an m.o.i. of 1 or 0.3, and cells were maintained at 37 °C or 25 °C. The number of intracellular bacteria was determined by recording the number of colony-forming units per milliliter. The mean and standard deviation from three independent experiments is displayed for each time point.
Figure 5.
Figure 5.
Wortmannin does not disrupt colocalization of RavD with endocytic vesicles. A and B, confocal images of HeLa cells transiently co-transfected with plasmids encoding mCherry-RavD or mCherry-RavD190–325 and EGFP-2×FYVE (A) or EGFP-Rab5 (B). 18 h post-transfection, cells were incubated with or without wortmannin. Scale bars = 5 μm.
Figure 6.
Figure 6.
RavD prevents accumulation of LAMP-1 on Legionella-containing vacuoles. A, RAW264.7 macrophages were infected with WT, ΔdotA, ΔravD, or ΔravD+pHA-RavD for 1, 3, 5, and 10 h, and the percentage of LAMP-1–positive vacuoles for each condition was determined. The bar graph displays the average and standard deviation for the percentage of LCVs that were LAMP-1–positive at each time point. Data were obtained from three independent experiments where at least 100 different vacuoles were assessed. Asterisks denote data that showed a significant difference from the control (WT) in an unpaired Student's t test (***, p ≤ 0.001; **, p ≤ 0.01). B, representative confocal microscopy images of LAMP1-stained cells infected with the indicated strains. Scale bars = 2 μm (insets, 0.5 μm).

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