Abstract
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.
Highlights
Toxoplasma gondii is an obligate intracellular protozoan parasite that infects around a third of the human population worldwide
Toxoplasma gondii resides in a parasitophorous vacuole that excludes transmembrane proteins required for recruitment of endosomes and lysosomes and does not follow the path of classical lysosomal degradation
We report that T. gondii accomplishes this task by causing EGFR activation in host cells
Summary
Toxoplasma gondii is an obligate intracellular protozoan parasite that infects around a third of the human population worldwide. Tachyzoites of T. gondii infect virtually any nucleated cell through active invasion. This process is dependent on the parasite actin-myosin motor and sequential secretion of proteins from micronemes and rhoptries, specialized organelles present in the apical end of the parasite [1]. T. gondii micronemal proteins (MICs) are expressed at the parasite surface membrane and they interact with host cell receptors [2]. The connection between transmembrane MICs to the actin-myosin motor (glideosome) of the parasite together with the binding of host cell receptors by MICs is considered to enable the organism to penetrate host cells [5,6]. The end result is the formation of a parasitophorous vacuole that is devoid of host proteins required for recruitment of endosomes and lysosomes [10]
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