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

Abstract

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.

Activated cytotoxic T cells within zoonotic cutaneous leishmaniasis lesions

“…It was recently demonstrated that GrB, delivered into infected host cells through the action of PFN and then into intracellular parasites by Grly, is able to kill Trypanosoma cruzi , Toxoplasma gondii and Leishmania major , in a PFN‐, Grly‐ and GrB‐dependent, but caspase‐independent, manner. GrB generate superoxide and inactivate oxidative defense enzymes to kill intracellular parasites . In our study, GrB and Grly mRNA levels were higher in ZCL compared to normal healthy skin indicating the presence of a GrB and Grly‐dependent cytotoxic activity and suggesting the outgoing of active process of parasite killing.…”

Granzyme B Disrupts Central Metabolism and Protein Synthesis in Bacteria to Promote an Immune Cell Death Program

“…More recently GNLY and Gzms were shown to rapidly kill Ec, Lm and Staphylococcus aureus (Dotiwala et al, 2016; Walch et al, 2014). Because earlier studies indicated that MTB was susceptible to high GNLY concentrations (Stenger et al, 1998) and because of the clinical importance of mycobacterial infection, we also examined whether GNLY and GzmB also efficiently kill mycobacteria, using the nonpathogenic M. smegmatis (Ms) strain as a model (Figure S1A).…”

“…The cytotoxic granule pore-forming protein, perforin (PFN), delivers the death-inducing granzyme (Gzm) serine proteases into the target cell, where they cleave multiple substrates to kill the target cell. These cytotoxic cells also kill intracellular bacteria and protozoa (Dotiwala et al, 2016; Walch et al, 2014). Granulysin (GNLY), another pore-forming protein in the cytotoxic granules of killer cells of most mammals, but not rodents, selectively permeabilizes microbial membranes (Stenger et al, 1998) and delivers the Gzms into intracellular microbes to cause rapid microbial death before the host cell is killed.…”

Programmed cell death as a defence against infection

“…Similarly, NK cytotoxicity initiated by inflammasome detection of S. Typhimurium in a colitis model was recently shown to promote inflammation 128 and Prf1 −/− mice were also susceptible to C. rodentium during the innate phase of infection 129 . Interestingly, human, but not mouse, NK cells have additional means to directly kill bacteria, since granulysin delivered upon NK degranulation is directly toxic for both bacteria and parasites 130,131 .…”