Summary When killer lymphocytes recognize infected cells, perforin delivers cytotoxic proteases (granzymes) into the target cell to trigger apoptosis. What happens to intracellular bacteria during this process is unclear. Human, but not rodent, cytotoxic granules also contain granulysin, an antimicrobial peptide. Here we show that granulysin delivers granzymes into bacteria to kill diverse bacterial strains. In E. coli, granzymes cleave electron transport chain complex I and oxidative stress defense proteins, generating ROS that rapidly kill bacteria. ROS scavengers and bacterial antioxidant protein overexpression inhibit bacterial death. Bacteria overexpressing a GzmB-uncleavable mutant of the complex I subunit nuoF or strains that lack complex I still die, but more slowly, suggesting that granzymes disrupt multiple vital bacterial pathways. Mice expressing transgenic granulysin are better able to clear L. monocytogenes. Thus killer cells play an unexpected role in bacterial defense.

Protozoan infections are a serious global health problem 1,2 . 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 cell 3 . However, these cytotoxic molecules do not kill intracellular parasites. CD8 + CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-g 4-10 . However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterolpoor microbial membranes 11-14 , and GNLY, PFN and Gzms rapidly kill intracellular bacteria 15 . 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.