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. 2011;6(11):e27311.
doi: 10.1371/journal.pone.0027311. Epub 2011 Nov 2.

RpkA, a highly conserved GPCR with a lipid kinase domain, has a role in phagocytosis and anti-bacterial defense

Tanja Y Riyahi  1 , Frederike Frese, Michael Steinert, Napoleon N Omosigho, Gernot Glöckner, Ludwig Eichinger, Benoit Orabi, Robin S B Williams, Angelika A Noegel
Affiliations

RpkA, a highly conserved GPCR with a lipid kinase domain, has a role in phagocytosis and anti-bacterial defense

Tanja Y Riyahi et al. PLoS One. 2011.

Abstract

RpkA (Receptor phosphatidylinositol kinase A) is an unusual seven-helix transmembrane protein of Dictyostelium discoideum with a G protein coupled receptor (GPCR) signature and a C-terminal lipid kinase domain (GPCR-PIPK) predicted as a phosphatidylinositol-4-phosphate 5-kinase. RpkA-homologs are present in all so far sequenced Dictyostelidae as well as in several other lower eukaryotes like the oomycete Phytophthora, and in the Legionella host Acanthamoeba castellani. Here we show by immunofluorescence that RpkA localizes to endosomal membranes and is specifically recruited to phagosomes. RpkA interacts with the phagosomal protein complex V-ATPase as proteins of this complex co-precipitate with RpkA-GFP as well as with the GST-tagged PIPK domain of RpkA. Loss of RpkA leads to a defect in phagocytosis as measured by yeast particle uptake. The uptake of the pathogenic bacterium Legionella pneumophila was however unaltered whereas its intra-cellular replication was significantly enhanced in rpkA(-). The difference between wild type and rpkA(-) was even more prominent when L. hackeliae was used. When we investigated the reason for the enhanced susceptibility for L. pneumophila of rpkA(-) we could not detect a difference in endosomal pH but rpkA(-) showed depletion of phosphoinositides (PIP and PIP(2)) when we compared metabolically labeled phosphoinositides from wild type and rpkA(-). Furthermore rpkA(-) exhibited reduced nitrogen starvation tolerance, an indicator for a reduced autophagy rate. Our results indicate that RpkA is a component of the defense system of D. discoideum as well as other lower eukaryotes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Localization of RpkA-GFP.
RpkA-GFP expressing AX2 cells fixed with methanol were labeled with monoclonal mouse antibodies against subunit A of the vacuolar H+-ATPase, VatA (mAb 221-35-2), the putative copper transporter p80 (mAb H161), the common lysosomal antigen CLA (mAb 221-450-6) and the post-lysosomal marker vacuolin (mAb 263-79-2). As secondary antibody a goat-anti-mouse-IgG conjugated with Alexa 568 was used. Presence of RpkA-GFP in living AX2 cells on acidic compartments was identified by LysoTracker-Red. RpkA-GFP expressing cells were incubated for 15 min with TRITC-labeled S. cerevisiae and fixed with methanol. rpkA− cells expressing VatM-GFP and RpkA-RFP. Images were collected using the LSM confocal laser scanning microscope. Scale bar, 5 µm.
Figure 2
Figure 2. Recruitment of RpkA-GFP to the phagosomal membrane.
Live cell analysis of TRITC-labeled S. cerevisiae uptake by AX2. Images were collected every 2.3 sec. Asterisks mark the yeast cell (red) which is taken up, arrowheads indicate RpkA-GFP containing vesicles (green) which are delivered to the maturing phagosome. Bar, 5 µm.
Figure 3
Figure 3. Phagocytosis of yeast cells.
AX2 , rpkA− cells, and rpkA− rescue strains 1D9 and 1E7 expressing RpkA-HA were incubated with TRITC-labeled yeast cells and fixed at the indicated time points. The number of phagocytosed yeast particles per cell was quantified at the indicated time points. Results are provided for three independent experiments ± SE, *P<0.05.
Figure 4
Figure 4. RpkA affects L. pneumophila infection.
(A) RpkA is present on vesicles containing heat-killed L. pneumophila . AX2 cells expressing RpkA-GFP were incubated with heat killed rhodamine-labeled L. pneumophila for 10 min prior to fixation. The arrowhead points to a bacterium surrounded by RpkA labeled membrane. Bar, 5 µm. (B) RpkA and VatA are present on vesicles containing living wild type L. pneumophila . rpkA− cells expressing RpkA-HA (rescue strain 1E7) were incubated with living L. pneumophila and fixed after 1 h. Bacteria were visualized with DAPI, RpkA-HA with mAb 3F10 against the HA-tag and goat-anti-rat-IgG conjugated to Alexa 568 as secondary antibody. VatA was visualized with mAb 221-35-2 and goat-anti-mouse-IgG conjugated with Alexa 488 as secondary antibody. Arrowhead, V-ATPase and RpkA-HA positive vesicle containing L. pneumophila. Bar, 5 µm. (C) Loss of RpkA leads to elevated titers of L. pneumophila . AX2, rpkA- and the rescue strains 1D9 and 1E7 were infected with L. pneumophila for 3 h. Not ingested bacteria were removed and viable internal L. pneumophila were quantified (0 h). The quantification was done also at time points 24 and 48 h post infection. CFU, colony forming units. Results are provided for four experiments done in triplicates ± SD, **P<0.01. (D) Infection with L. hackeliae leads to reduced clearing in rpkA− . AX2 and rpkA− were infected with L. hackeliae for 3 h. Non ingested bacteria were removed and viable internal L. hackeliae were quantified (0 h). The quantification was done also at time points 24, 34, 48 and 72 h post infection. CFU, colony forming units. Results are provided for four experiments done in triplicates ± SD, ***P<0.001.
Figure 5
Figure 5. V-ATPase interacts with RpkA but average endosomal pH is uneffected in rpkA-.
(A) V-ATPase co-immunoprecipitates with RpkA-GFP. Polyclonal GFP-antibodies were used for immunoprecipitation of either RpkA-GFP or GFP as a control. Proteins were separated by SDS-PAGE and stained with Coomassie Blue. VatM and VatC were detected as co-precipitates with RpkA-GFP by mass spectrometry. The positions of VatM, RpkA-GFP and VatC are indicated by white asterisks. (B) VatA and VatM-GFP specifically co-precipitate with GST-PIPK 343 – 805 and GST-PIPK 370 – 828. GST, GST-PIPK343 – 805 and GST-PIPK370 – 828 were incubated with a lysate of rpkA- cells expressing VatM-GFP. Western blots of the pull down were probed using GFP-specific mAb K3-184 to detect VatM-GFP at 130 kDa and VatA specific mAb 223-35-2 to detect VatA at 70 kDa. The western blot shows the lysate of 2×105 cells (L), 10 µl of input (I), the pull downs (GST, GST-PIPK343 – 805 and GST-PIPK370 – 828) and respective flow through fractions (FT). In the lower panel a Coomassie-stained gel of the pull downs is shown. (C) Endosomal pH is unaffected in rpkA− . AX2, rpkA− and rescue strain 1E7 were incubated for 3 h with FITC-dextran as a pH probe. Then the excitation ratio at 495 nm/450 nm was measured and the endosomal pH was determined using a standard curve. Results are provided for quadruplet experiments ± SD.
Figure 6
Figure 6. RpkA influences phosphoinositolphosphate turnover. (A) The PIPK370 – 828 domain binds to phosphoinositolphosphates.
PIP-Strip-membranes were incubated over night with GST-PIPK370 – 828 (1 µg/ml) and with GST (1 µg/ml) for control. Binding was detected by incubation with polyclonal GST-antibodies. (B) Loss of RpkA leads to reduced PIP and PIP2 levels. Phosphoinositide turnover in AX2 and rpkA− cells was monitored by permeabilized cells, and labeling phospholipids with [γ-32P] ATP. Subsequently cells were lysed, phospholipids were extracted and separated by TLC, imaged using Typhoon phosphorimager, quantified with “ImageQuant” and normalized according to total lipids. Results are provided for triplicate experiments with duplicate samples ± SD, *P<0.05.
Figure 7
Figure 7. rpkA− cells show reduced tolerance against nitrogen starvation.
AX2 and rpkA− cells were grown in FM-Medium for at least 5 generations, harvested during the exponential growth phase and resuspended at a density of ∼ 3×106 cells/ml in FM medium lacking amino acids. At the indicated time points viability was determined by analyzing the ability to form colonies (PFU, plaque forming units) on bacterial lawns. Results are provided for duplicate experiments with duplicate samples ± SD.
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