Globally, nearly 2 billion people are infected with the intracellular protozoan Toxoplasma gondii1. This persistent infection can cause severe disease in immunocompromised people and is epidemiologically linked to major mental illnesses2 and cognitive impairment3. There are currently no options for curing this infection. The lack of effective therapeutics is due partly to a poor understanding of the essential pathways that maintain long-term infection. Although it is known that Toxoplasma replicates slowly within intracellular cysts demarcated with a cyst wall, precisely how it sustains itself and remodels organelles in this niche is unknown. Here, we identify a key role for proteolysis within the parasite lysosomal organelle (the vacuolar compartment or VAC) in turnover of autophagosomes and persistence during neural infection. We found that disrupting a VAC-localized cysteine protease compromised VAC digestive function and markedly reduced chronic infection. Death of parasites lacking the VAC protease was preceded by accumulation of undigested autophagosomes in the parasite cytoplasm. These findings suggest an unanticipated function for parasite lysosomal degradation in chronic infection, and identify an intrinsic role for autophagy in the T. gondii parasite and its close relatives. This work also identifies a key element of Toxoplasma persistence and suggests that VAC proteolysis is a prospective target for pharmacological development.

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This work was supported by National Institutes of Health grants R01AI120627 (to V.B.C. and M.D.C.), R01GM111703 (to M.B.) and R01AI060767 (to I.C.). Z.D. and A.J.S. received fellowship support from the American Heart Association. B.M.H. and G.K. were supported by National Institutes of Health training grant T32AI007528. O.L.M. was supported by a National Institutes of Health Ruth Kirschstein National Research Service Award F31AI118274. The authors thank S. Meshinchi and J. Whitfield at the University of Michigan for help with electron microscopy and cytokine analysis, respectively, as well as the Johns Hopkins University Microscopy facility. The authors thank L. Weiss, W. Sullivan Jr and L.D. Sibley for the provision of antibodies for this study.

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Author notes

    • Zhicheng Dou
    • , Alyssa J. Miller
    •  & Beth M. Hayes

    Present addresses: Department of Biology, Clemson University, Clemson, South Carolina 29634, USA (Z.D.); Graduate Program in Cellular and Molecular Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA (A.J.M.); Department of Biochemistry and Molecular Biology, University of California at San Francisco, California, 94143 USA (B.M.H.).


  1. Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA

    • Manlio Di Cristina
    • , Zhicheng Dou
    • , Geetha Kannan
    • , My-Hang Huynh
    • , Olivia L. McGovern
    • , Tracey L. Schultz
    • , Aric J. Schultz
    • , Alyssa J. Miller
    • , Beth M. Hayes
    •  & Vern B. Carruthers
  2. Department of Chemistry, Biology and Biotechnology, University of Perugia, Perugia 06122, Italy

    • Manlio Di Cristina
    • , Matteo Lunghi
    •  & Carla Emiliani
  3. Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Wouter van der Linden
    •  & Matthew Bogyo
  4. DIMNP-UMR 5235 CNRS, Université de Montpellier, 34095 Montpellier Cedex 5, France

    • Sébastien Besteiro
  5. Department of Molecular Microbiology and Immunology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205, USA

    • Isabelle Coppens


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M.D.C., Z.D., M.L., O.L.M., G.K., M.-H.H., A.J.M., B.M.H., C.E. and V.B.C. designed the experiments. M.D.C., A.J.S., A.J.M., B.M.H. and V.B.C. wrote the manuscript. W.v.d.L. performed chemical synthesis. M.D.C., Z.D., M.L., O.L.M., G.K., M.H.-H., T.L.S., A.J.S., A.J.M., B.M.H. and I.C. performed the experiments. C.E., M.B. and S.B. provided advice or reagents that were essential for completion of the study. All authors discussed the findings and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Manlio Di Cristina or Vern B. Carruthers.

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