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

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.

A Conserved Apicomplexan Microneme Protein Contributes to Toxoplasma gondii Invasion and Virulence

“…Similar effects on invasion due to deletion of other microneme proteins such as M2AP (9) or MIC3 (7) also cause delayed lethality, presumably due to a slower progression of the infection leading to more effective immune clearance. A recent study showed that EGF-containing MICs, including MIC3, activate Akt signaling via EGF receptors to diminish autophagic killing of the parasite in host cells (34). However, recombinant TgSPATR did not promote epidermal growth factor receptor (EGFR)-Akt activation (L. Muniz-Feliciano, J.…”

Toxoplasma gondii Development of Its Replicative Niche: in Its Host Cell and Beyond

“…Parasite avoidance of the phagolysosome is inhibited by treatment of cells with tyrosine kinase inhibitors, and the epidermal growth factor (EGF) receptor, which is a receptor tyrosine kinase, was identified as at least one target of these inhibitors (44). Significantly, parasite growth was attenuated in cells transfected with small interfering RNAs (siRNAs) targeting the EGF receptor and the PVs in these cells appeared as if they were undergoing autophagic destruction.…”

“…These data support a model where EGF/AKT signaling either prevents the invading parasite from entering the endolysosomal pathway or prevents lysosomal recruitment to the PV. While the parasite ligand(s) that activates the EGF receptor is unknown, the receptor is not activated by parasites with knockout mutations in the MIC1 and MIC3 micronemal proteins (44).…”

“…EGF receptor activation is dependent on the expression of at least 2 micronemal proteins, MIC1 and MIC3, and addition of recombinant MIC1 and MIC3 proteins to cells infected with mic1 mic3 double-knockout parasites restores EGF receptor-dependent inhibition of CD40L-induced autophagy (44). Both of these proteins are shed from the parasite's plasma membrane during invasion (101), and it is therefore possible that the released ectodomains can interact with and activate EGF receptor signaling in noninfected cells and thus initiate an antiautophagic response in a host cell prior to its infection.…”

Nucleotide variation in the Toxoplasma gondii micronemal protein 8 gene

“…Micronemal proteins (MICs) are serially secreted from micronemes during T. gondii infection, and play a significant role in the asexual life cycle of this parasite by mediating parasite invasion, motility, and attachment to host cells (Besteiro et al, 2011;Muniz-Feliciano et al, 2013;Sharma and Chitnis, 2013). As an important member of the MIC family, MIC8 participates in T. gondii invasion through interaction with soluble MIC3 (Cérède et al, 2002;Kessler et al, 2008), and is a potential vaccine candidate against acute and chronic T. gondii infections in a mouse model (Liu et al, 2010;Li et al, 2014).…”