Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites

Abstract

Protozoan infections are a serious global health problem1,2. Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) eliminate pathogen-infected cells by releasing cytolytic granule contents—granzyme (Gzm) proteases and the pore-forming perforin (PFN)—into the infected cell3. However, these cytotoxic molecules do not kill intracellular parasites. CD8+ CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-γ4,5,6,7,8,9,10. However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterol-poor microbial membranes11,12,13,14, and GNLY, PFN and Gzms rapidly kill intracellular bacteria15. Here we show that GNLY delivers Gzms into three protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii and Leishmania major), in which the Gzms generate superoxide and inactivate oxidative defense enzymes to kill the parasite. PFN delivers GNLY and Gzms into infected cells, and GNLY then delivers Gzms to the intracellular parasites. Killer cell–mediated parasite death, which we term 'microbe-programmed cell death' or 'microptosis', is caspase independent but resembles mammalian apoptosis, causing mitochondrial swelling, transmembrane potential dissipation, membrane blebbing, phosphatidylserine exposure, DNA damage and chromatin condensation. GNLY-transgenic mice are protected against infection by T. cruzi and T. gondii, and survive infections that are lethal to wild-type mice. Thus, GNLY-, PFN- and Gzm-mediated elimination of intracellular protozoan parasites is an unappreciated immune defense mechanism.

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Figure 1: GNLY, PFN and GzmB kill parasites.
Figure 2: GNLY and GzmB cause oxidative damage–mediated parasite programmed cell death.
Figure 3: GNLY+/− mice are more resistant to T. cruzi infection than WT mice.
Figure 4: GNLY+/− mice are more resistant to T. gondii infection than WT mice.

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Acknowledgements

This work was supported by the US National Institutes of Health grant T32HL066987 (F.D.), a Scholar Fellowship from Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (CAPES) and the David Rockefeller Center for Latin American Studies at Harvard University (R.T.G.), a Science Without Borders Scholar Fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (R.B.P.) and the Brazilian National Institute of Science and Technology for Vaccines grant CNPq/Fapemig/MS 573547/2008-4 (R.T.G.). We thank S. Wilkinson (Queen Mary University, London) and J. Kelly (London School of Hygiene and Tropical Medicine) for T. cruzi overexpression plasmids, K. Okuda (University of Massachusetts Medical School) for L. major, K. Engelberg and M.J. Gubbels (Boston College) for T. gondii, and M. Ericsson and R. Bronson (Harvard Medical School) for technical support.

Author information

F.D., S.M., R.T.G. and J.L. designed the experiments, analyzed the data and wrote the manuscript. F.D. and S.M. performed experiments with help from J.A.A., M.W. and R.B.P. B.A.B. provided advice and the use of her laboratory.

Correspondence to Farokh Dotiwala or Ricardo T Gazzinelli or Judy Lieberman.

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Dotiwala, F., Mulik, S., Polidoro, R. et al. Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites. Nat Med 22, 210–216 (2016) doi:10.1038/nm.4023

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