Toxoplasma gondii-Induced Activation of EGFR Prevents Autophagy Protein-Mediated Killing of the Parasite

Toxoplasma gondii resides in an intracellular compartment (parasitophorous vacuole) that excludes transmembrane molecules required for endosome-lysosome recruitment. Thus, the parasite survives by avoiding lysosomal degradation. However, autophagy can re-route the parasitophorous vacuole to the lysosomes and cause parasite killing. This raises the possibility that T. gondii may deploy a strategy to prevent autophagic targeting to maintain the non-fusogenic nature of the vacuole. We report that T. gondii activated EGFR in endothelial cells, retinal pigment epithelial cells and microglia. Blockade of EGFR or its downstream molecule, Akt, caused targeting of the parasite by LC3(+) structures, vacuole-lysosomal fusion, lysosomal degradation and killing of the parasite that were dependent on the autophagy proteins Atg7 and Beclin 1. Disassembly of GPCR or inhibition of metalloproteinases did not prevent EGFR-Akt activation. T. gondii micronemal proteins (MICs) containing EGF domains (EGF-MICs; MIC3 and MIC6) appeared to promote EGFR activation. Parasites defective in EGF-MICs (MIC1 ko, deficient in MIC1 and secretion of MIC6; MIC3 ko, deficient in MIC3; and MIC1-3 ko, deficient in MIC1, MIC3 and secretion of MIC6) caused impaired EGFR-Akt activation and recombinant EGF-MICs (MIC3 and MIC6) caused EGFR-Akt activation. In cells treated with autophagy stimulators (CD154, rapamycin) EGFR signaling inhibited LC3 accumulation around the parasite. Moreover, increased LC3 accumulation and parasite killing were noted in CD154-activated cells infected with MIC1-3 ko parasites. Finally, recombinant MIC3 and MIC6 inhibited parasite killing triggered by CD154 particularly against MIC1-3 ko parasites. Thus, our findings identified EGFR activation as a strategy used by T. gondii to maintain the non-fusogenic nature of the parasitophorous vacuole and suggest that EGF-MICs have a novel role in affecting signaling in host cells to promote parasite survival

RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye

<p>Macroautophagy/autophagy is an intracellular stress survival and recycling system whereas phagocytosis internalizes material from the extracellular milieu; yet, both pathways utilize lysosomes for cargo degradation. Whereas autophagy occurs in all cells, phagocytosis is performed by cell types such as macrophages and the retinal pigment epithelial (RPE) cells of the eye where it is supported by the noncanonical autophagy process termed LC3-associated phagocytosis (LAP). Autophagy and LAP are distinct pathways that use many of the same mediators and must compete for cellular resources, suggesting that cells may regulate both processes under homeostatic and stress conditions. Our data reveal that RPE cells promote LAP through the expression of RUBCN/Rubicon (RUN domain and cysteine-rich domain containing Beclin 1-interacting protein) and suppress autophagy through the activation of EGFR (epidermal growth factor receptor). In the morning when photoreceptor outer segments (POS) phagocytosis and LAP are highest, RUBCN expression is increased. At the same time, outer segment phagocytosis activates the EGFR resulting in MTOR (mechanistic target of rapamycin [serine/threonine kinase]) stimulation, the accumulation of SQSTM1/p62, and the phosphorylation of BECN1 (Beclin 1, autophagy related) on an inhibitory residue thereby suppressing autophagy. Silencing <i>Rubcn</i>, preventing EGFR activity or directly inducing autophagy in RPE cells by starvation inhibits phagocytic degradation of POS. Thus, RPE cells regulate lysosomal pathways during the critical period of POS phagocytosis to support retinal homeostasis.</p

<i>Toxoplasma gondii</i> induces FAK-Src-STAT3 signaling during infection of host cells that prevents parasite targeting by autophagy

<div><p>Targeting of <i>Toxoplasma gondii</i> by autophagy is an effective mechanism by which host cells kill the protozoan. Thus, the parasite must avoid autophagic targeting to survive. Here we show that the mammalian cytoplasmic molecule Focal Adhesion Kinase (FAK) becomes activated during invasion of host cells. Activated FAK appears to accompany the formation of the moving junction (as assessed by expression the parasite protein RON4). FAK activation was inhibited by approaches that impaired β1 and β3 integrin signaling. FAK caused activation of Src that in turn mediated Epidermal Growth Factor Receptor (EGFR) phosphorylation at the unique Y845 residue. Expression of Src-resistant Y845F EGFR mutant markedly inhibited ROP16-independent activation of STAT3 in host cells. Activation of FAK, Y845 EGFR or STAT3 prevented activation of PKR and eIF2α, key stimulators of autophagy. Genetic or pharmacologic inhibition of FAK, Src, EGFR phosphorylation at Y845, or STAT3 caused accumulation of the autophagy protein LC3 and LAMP-1 around the parasite and parasite killing dependent on autophagy proteins (ULK1 and Beclin 1) and lysosomal enzymes. Parasite killing was inhibited by expression of dominant negative PKR. Thus, <i>T</i>. <i>gondii</i> activates a FAK→Src→Y845-EGFR→STAT3 signaling axis within mammalian cells, thereby enabling the parasite to survive by avoiding autophagic targeting through a mechanism likely dependent on preventing activation of PKR and eIF2α.</p></div

FAK mediates Src activation during <i>T</i>. <i>gondii</i> infection.

<p><i>A</i>, <i>B</i>, RPE cells (<i>A</i>) or CHO cells (<i>B</i>) were challenged with RH <i>T</i>. <i>gondii</i> for the indicated time points. Cell lysates were used to examine expression of total FAK and phospho-Y397 FAK by immunoblot. <i>C</i>, MIC8KOi parasites were cultured in HFF with or without anhydrotetracycline (ATc; 1 μg/ml). Tachyzoites were harvested and incubated with A549 cells for the indicated time points. Expression of total FAK and phospho-Y397 FAK were assessed by immunoblot. Relative densities of phospho-FAK were compared to that of uninfected (control) cells. Relative density of phospho-FAK for uninfected samples was given a value of 1 Densitometry data represent means ± SEM of 3 independent experiments. <i>D</i>, CHO cells were incubated with YFP <i>T</i>. <i>gondii</i> (RH) for 2.5 min followed by assessment of phospho-Y397 FAK and RON4 expression by immunofluorescence. <i>T</i>. <i>gondii</i> was pseudocolored blue. Arrowheads indicate co-localization of RON4 with phospho-Y397FAK or accumulation of phospho-Y397 FAK around an intracellular tachyzoite. Original magnification X630. Quantification of phospho-Y397 FAK and RON4 signaling intensity for the intracellular parasite is shown underneath. <i>E</i>, A549 cells were incubated with vehicle or FAK inhibitor (1 μM) prior to challenge with RH <i>T</i>. <i>gondii</i>. Cell lysates were obtained and used to probe for total Src and phospho-Y416 Src. A vertical line was inserted between densitometry data of lysates from control and FAK inhibitor-treated cells to indicate that relative densities of phospho-FAK from infected cells treated with or without FAK inhibitor were compared to bands from their respective uninfected (control) cells. Relative density of phospho-FAK for uninfected samples was given a value of 1. <i>F</i>, mHEVc cells transduced with lentiviral vectors that express either FAK shRNA or control shRNA were incubated with RH <i>T</i>. <i>gondii</i>. Immunoblots and densitometries were assessed as above. Experiments shown are representative of 3–4 independent experiments.</p

Src signaling induced by <i>T</i>. <i>gondii</i> triggers trans-activation of EGFR that is required to prevent parasite killing within host cells.

<p><i>A</i>, A549 cells were transfected with control siRNA or Src siRNA followed by challenge with RH <i>T</i>. <i>gondii</i>. Expression of total EGFR and phospho-Y845 EGFR was assessed by immunoblot. A vertical line was inserted between densitometry data of lysates from cells transfected with control or Src siRNA to indicate that relative densities of phospho-Y845 EGFR from infected cells were compared to bands from their respective uninfected (control) cells. Relative density of phospho-Y845 EGFR for uninfected samples was given a value of 1. Densitometry data represent means ± SEM of 3 independent experiments. <i>B</i>, NMuMG with stable expression of WT EGFR or EGFR AA mutant were challenged with RH <i>T</i>. <i>gondii</i>. Monolayers were examined at 2 and 24 h to determine the percentages of infected cells and at 24 h to ascertain the numbers of <i>T</i>. <i>gondii</i> tachyzoites per 100 cells. <i>C</i>, A549 cells were transfected with plasmids encoding WT EGFR or Y845F EGFR followed by challenge with RH <i>T</i>. <i>gondii</i>. Monolayers were examined at 2 and 24 h to determine the percentages of infected cells, and at 24 h to ascertain the numbers of <i>T</i>. <i>gondii</i> tachyzoites, <i>T</i>. <i>gondii</i>-containing vacuoles per 100 cells and parasites per vacuole. Results are shown as the mean ± SEM of 3 independent experiments. ** <i>P</i> < 0.01; *** <i>P</i> < 0.001.</p

Src/EGFR-induced STAT3 signaling prevents autophagic killing of <i>T</i>. <i>gondii</i>.

<p><i>A</i>, A549 cells were transfected with control siRNA or STAT3 siRNA and challenged with RH <i>T</i>. <i>gondii</i>. Monolayers were examined microscopically at 2 and 24 h to determine the percentages of infected cells, and at 24 h to ascertain the numbers of <i>T</i>. <i>gondii</i> tachyzoites, <i>T</i>. <i>gondii</i>-containing vacuoles per 100 cells and parasites per vacuole. <i>B</i>, A549 cells transfected with control siRNA or STAT3 siRNA were challenged with <i>T</i>. <i>gondii</i>-RFP (RH). Expression of LC3 was examined by immunofluorescence 5 h post-challenge to determine the percentage of cells with LC3 accumulation around the parasite. Arrowheads indicate accumulation of LC3 around the parasite. Original magnification X630. <i>C</i>, A549 cells transfected with control siRNA or STAT3 siRNA were challenged with <i>T</i>. <i>gondii</i>-YFP (RH). Expression of LAMP-1 was examined by fluorescence microscopy 8 h post-challenge. The percentage of cells with LAMP-1 accumulation around the parasite was determined. Arrowheads indicate accumulation of LAMP-1 around the parasite. <i>D</i>, A549 cells transfected with Beclin 1 siRNA were transfected with STAT3 siRNA followed by challenge with RH <i>T</i>. <i>gondii</i>. Monolayers were examined 24 h post-challenge. <i>E</i>, mHEVc cells transfected with WT STAT3 or Y705F STAT3 were challenged with <i>T</i>. <i>gondii</i>-RFP (RH). Expression of LC3 was examined by immunofluorescence 5 h post-challenge. LC3 accumulation around the parasite was assessed as above. <i>F</i>, mHEVc cells transfected with WT STAT3 or Y705F STAT3 were challenged with RH <i>T</i>. <i>gondii</i> followed by addition of leupeptin/pepstatin (lysosomal inhibitors, LI). Monolayers were examined at 2 and 24 h post-challenge. Results are shown as the mean ± SEM of 3 independent experiments. ** <i>P</i> < 0.01; *** <i>P</i> < 0.001.</p

Blockade of Src induces accumulation of the autophagy protein LC3 around the parasite, vacuole-lysosome fusion and killing of the parasite dependent on autophagy the proteins ULK1 and Beclin 1.

<p><i>A</i>, A549 cells were challenged with RH <i>T</i>. <i>gondii</i> after transfection with either control siRNA or Src siRNA. Monolayers were examined at 2 and 24 h to determine the percentages of infected cells, and at 24 h to ascertain the numbers of <i>T</i>. <i>gondii</i> tachyzoites, <i>T</i>. <i>gondii</i>-containing vacuoles per 100 cells and parasites per vacuole. <i>B</i>, A549 cells transfected with either control siRNA or Src siRNA were challenged with <i>T</i>. <i>gondii</i>-RFP (RH). Expression of LC3 was examined by immunofluorescence 5 h post-challenge. Arrowheads indicate accumulation of LC3 around the parasite. Original magnification X630. <i>C</i>, A549 cells transfected with either control siRNA or Src siRNA were challenged with <i>T</i>. <i>gondii</i>-YFP (RH). Expression of LAMP-1 was examined by immunofluorescence 8 h and 12 h post-challenge. Arrowheads indicate accumulation of LAMP-1 around the parasite. <i>D</i>, mHEVc cells were transfected with control siRNA or ULK1 siRNA followed by treatment with or without PP2 prior to challenge with RH <i>T</i>. <i>gondii</i>. Monolayers were examined by light microscopy 24 h post-infection. <i>E</i>, A549 cells transfected with control siRNA or Src siRNA were transfected with Beclin 1 siRNA. Cells were challenged with RH <i>T</i>. <i>gondii</i> and monolayers were examined by light microscopy 24 h post-infection. <i>F</i>, A549 cells transfected control siRNA or Src siRNA were infected with RH <i>T</i>. <i>gondii</i>. Leupeptin plus pepstatin (Lys Inhibitors) were added post-infection and monolayers were examined microscopically 24 h post-challenge. <i>G</i>-<i>I</i>, mHEVc cells transduced with lentiviral vectors that express either FAK shRNA or control shRNA were transfected with ULK1 siRNA (<i>G</i>), Beclin 1 siRNA (<i>H</i>) or control siRNA followed by incubation with RH <i>T</i>. <i>gondii</i>. mHEVc cells challenged with RH <i>T</i>. <i>gondii</i> were also incubated with leupeptin plus pepstatin (Lys Inhibitors) (<i>I</i>). Monolayers were examined microscopically 24 h post-challenge. Results are shown as the mean ± SEM of 3 independent experiments. ** <i>P</i> < 0.01; *** <i>P</i> < 0.001.</p

<i>T</i>. <i>gondii</i> induces Src activation in mammalian cells.

<p><i>A</i>, Human retinal pigment epithelial cells (RPE), human lung epithelial cells (A549) and mouse endothelial cells (mHEVc) were challenged with tachyzoites of the RH (type I) strain of <i>T</i>. <i>gondii</i>. Cell lysates were obtained at the indicated time points and used to probe for total Src and phospho-Y416 Src. <i>B</i>, Mouse microglia (BV-2) were challenged with RH <i>T</i>. <i>gondii</i> tachyzoites as above. C, RPE were incubated with tachyzoites of the M49 or VAND strains of <i>T</i>. <i>gondii</i> followed by assessment of Src phosphorylation. Densitometry data represent means ± SEM of 3–4 independent experiments. Relative density of phospho-Src signal was obtained by normalization to total Src signal followed by normalization relative to the uninfected control samples (0 min time-point). Relative density of phospho-Src for uninfected samples was given a value of 1.</p

Inhibition of β integrin signaling impairs <i>T</i>. <i>gondii</i>-induced FAK activation.

<p><i>A</i>, RPE were incubated with cRADfV or cRGDfV prior to challenge with RH <i>T</i>. <i>gondii</i>. Total FAK and phospho-Y397 were assessed by immunoblot. A vertical line was inserted between densitometry data of lysates from cRADfV- and cRGDfV-treated cells to indicate that relative densities of phospho-FAK from infected cells were compared to bands from their respective uninfected (control) cells. Relative density of phospho-FAK for uninfected samples was given a value of 1. <i>B</i>, MDA-MB-231 human breast epithelial cells transduced with vector encoding control shRNA or β1 integrin shRNA were incubated with or without a neutralizing anti-α_vβ3 mAb prior to challenge with RH <i>T</i>. <i>gondii</i>. Total FAK and phospho-Y397 were assessed by immunoblot. Densitometries were calculated as above. Densitometry data represent means ± SEM of 3 independent experiments.</p