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

et al. (2013) Toxoplasma gondii-Induced Activation of EGFR Prevents Autophagy Protein- Mediated Killing of the Parasite. PLoS Pathog 9(12): e1003809. doi:10.1371/journal.ppat.1003809 Toxoplasma gondii -Induced Activation of EGFR Prevents Autophagy Protein-Mediated Killing of the Parasite Luis Muniz-Feliciano 0 Jennifer Van Grol 0 Jose-Andres C. Portillo 0 Lloyd Liew 0 Bing Liu 0 Cathleen R. Carlin 0 Vern B. Carruthers 0 Stephen Matthews 0 Carlos S. Subauste 0 Eric Y. Denkers, Cornell University, United States of America 0 1 Department of Pathology, Case Western Reserve University , Cleveland , Ohio, United States of America, 2 Division of Infectious Diseases and HIV Medicine, Department of Medicine, Case Western Reserve University School of Medicine , Cleveland , Ohio, United States of America, 3 Division of Molecular Biosciences, Imperial College London , London , United Kingdom , 4 Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine , Cleveland , Ohio, United States of America, 5 Department of Microbiology and Immunology, University of Michigan Medical School , Ann Arbor , Michigan, United States of America, 6 Department of Ophthalmology and Visual Sciences, Case Western Reserve University , Cleveland, Ohio , United States of America 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 CD154activated 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. - Funding: This work was supported by NIH Grants EY018341 (CSS), GM081498 (CRC) and P30 EY11373. LMF and JVG were supported by the Visual Sciences Training Program grant from the National Institutes of Health (T32 EY007157). LMF was also supported by the Immunology Training Program grant from the National Institutes of Health (T32-AI089474). JVG is a recipient of a pre-doctoral fellowship from Prevent Blindness Ohio. JACP is a recipient of a post-doctoral fellowship from the Ohio Lions Eye Research Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Toxoplasma gondii is an obligate intracellular protozoan parasite that infects around a third of the human population worldwide. T. gondii is of clinical importance because it causes encephalitis in immunocompromised individuals and retino-choroiditis in immunocompetent and immunosuppressed patients. T. gondii can also cause congenital infection that may result in cerebral and ocular disease. 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]. Once secreted, T. gondii micronemal proteins (MICs) are expressed at the parasite surface membrane and they interact with host cell receptors [2]. MICs contain adhesive domains such as type I thrombospondin repeats, apple domains, EGF repeats and integrin A domains [3,4]. 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]. Following the release of MICs, rhoptries secrete rhoptry neck proteins (RONs) that are critical for the formation of a structure called the moving junction (MJ) [7,8]. The MJ anchors the parasite to the host cell while the parasite penetrates it. The MJ is also believed to function as a sieve that excludes host type I transmembrane proteins from entering the PV membrane (PVM) [8,9]. The end result is the formation of a parasitophorous vacuole that is devoid of host proteins required for recruitment of endosomes and lysosomes [10]. T. gondii cannot withstand the lysosomal environment. Thus, the non-fusogenic nature of the PV is critical since it allows the parasite to survive and replicate. The immune system can deprive Toxoplasma gondii resides in a parasitophorous vacuole that excludes transmembrane proteins required for recruitment of endosomes and lysosomes and thus, does not follow the path of classical lysosomal degradation. However, the non-fusogenic nature of the vacuole can be reverted when autophagy, a pathway to lysosomal degradation, is upregulated through the immune system or pharmacologically. Maintenance of the non-fusogenic nature of the vacuole is central to parasite survival. Thus, in addition to preventing degradation through a classical lysosomal pathway, T. gondii may also deploy strategies to prevent constitutive levels of autophagy from targeting the pathogen and causing its lysosomal degradation. We report that T. gondii accomplishes this task by causing EGFR activation in host cells. In cells that were not subjected to immune or pharmacologic upregulation of autophagy, blockade of EGFR resulted in parasite encasing by structures that expressed the autophagy protein LC3, vacuole-lysosomal fusion and autophagy protein-dependent killing of the parasite. Moreover, EGF (...truncated)