Uptake of Granulysin via Lipid Rafts Leads to Lysis of Intracellular Listeria innocua Michael Walch, Elisabeth Eppler, Claudia Dumrese, Hanna Barman, Peter Groscurth, and Urs Ziegler 1 The bacteriolytic activity of CTL is mediated by granulysin, which has been reported to kill intracellular Mycobacterium tuber- culosis in dendritic cells (DC) with high efficiency. Despite that crucial effector function, the killing mechanism and uptake of granulysin into target cells have not been well investigated. To this end we analyzed granulysin binding, uptake, and the subse- quent lysis of intracellular Listeria innocua in human DC. Recombinant granulysin was found to be actively taken up by DC into early endosomal Ag 1-labeled endosomes, as detected by immunofluorescence. Further transfer to L. innocua-containing phago- somes was indicated by colocalization of bacterial DNA with granulysin. After uptake of granulysin by DC, lysis of L. innocua was found in a dose-dependent manner. Uptake as well as lysis of Listeria were inhibited after blocking endocytosis by lowering the temperature and by cholesterol depletion of DC. Colocalization of granulysin with cholera toxin during uptake showed binding to and internalization via lipid rafts. In contrast to cholera toxin, which was targeted to the perinuclear compartment, granulysin was found exclusively in endosomal-phagosomal vesicles. Lipid raft microdomains, enriched in the immunological synapse, may thus enhance uptake and transfer of granulysin into bacterial infected host cells. The Journal of Immunology, 2005, 174: 4220 – 4227. C ytotoxic T lymphocytes and NK cells play an essential role in the host defense against intracellular pathogens such as Chlamydia, Listeria, and Mycobacteria (1). The mechanisms of CTL and NK cells involved in clearance of intra- cellular bacteria are release of cytokines (2), especially of IFN- and TNF-, induction of target cell apoptosis (3), and direct me- diation of antibacterial activity (4). The bacteriolytic activity of CTL is mediated by granulysin, a 9-kDa protein stored in cytolytic granules together with perforin and granzyme B (5). It was dis- covered by a subtractive hybridization procedure of late activated T cells (6, 7) and exhibited a vast spectrum of antimicrobial ac- tivity against bacteria, fungi, and parasites (8), either as free mi- croorganisms or located in host cells. The killing of intracellular Mycobacterium tuberculosis by V9/V2 T lymphocytes was shown to be dependent on granulysin (9). Furthermore, it was re- ported that V9/V2 T cells from children with tuberculosis have strongly reduced effector functions, indicated by decreased IFN- production and granulysin expression, which was recovered upon chemotherapy (10). Other groups found granulysin responsible for the antimycobacterial activity of NKT cells (11) or CTL (8). Com- parable results were obtained when investigating Mycobacterium leprae, which also survives within phagosomes of host cells (12, 13). Ochoa et al. (12) showed, by phenotyping of cells in dermal granulomas of leprosy lesions, that there are masses of granulysin- containing cells. These cells were identified as CD4 + T cells. Moreover, the frequency of T cells containing granulysin in lesions reflected the capacity of the patients to restrict the disease. A recent study revealed granulysin-containing CD4 + T cells infiltrating af- fected follicles and perilesional dermis in superficial microbial fol- liculitis (13). Together, these results indicate that there is little doubt that granulysin is crucial for the mediation of antibacterial activity of CTL and NK cells. Granulysin belongs to the saposin-like protein family (SAP- LIP). 2 These proteins share a particular polypeptide motive and affinity to a variety of lipids, especially sphingolipids (14), as well as to cholesterol (15). The interaction of saposins with sphingo- lipids has been extensively investigated. Dependent on the pH value, all saposins were reported to bind negatively charged gan- gliosides (16, 17). Positive charges at neutral pH are crucial for lytic activity of granulysin against bacteria and negatively charged liposomes (18). After binding and clustering of granulysin at the bacterial membrane, deformation of the membrane might lead to bacteriolysis (19). Although the lytic activity of granulysin against a wide spec- trum of microorganisms has been well studied (8, 20, 21), few data are available about the interaction of granulysin with the host cell itself, in particular on binding, uptake, and intracellular trafficking. Binding of granulysin may be mediated by lipid rafts, which are specialized membrane microdomains composed of sphingolipids and cholesterol in the outer exoplasmic leaflet as well as phospho- lipids and cholesterol in the inner cytoplasmic leaflet (22). There is abundant evidence that rafts are involved in a variety of cellular functions, including endocytosis of pathogens (23–25) and bacte- rial endotoxins (26, 27), as well as in protein sorting and ligand- induced signal transduction (28). With respect to granulysin uptake in infected host cells, it is under debate whether other lytic proteins secreted by CTL and NK cells, such as perforin, assist in internalization of granulysin in Division of Cell Biology, Institute of Anatomy, University of Zurich, Zurich, Swit- zerland Received for publication July 15, 2004. Accepted for publication January 26, 2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Urs Ziegler, Division of Cell Biology, Institute of Anatomy, University of Zurich, Winterhurerstrasse 190, CH- 8057 Zurich, Switzerland. E-mail address: ziegler@anatom.unizh.ch 2 Abbreviations used in this paper; SAPLIP, saposin-like protein family; BAD, 2,3- butanedione; CAH, citraconic anhydride; CLSM, confocal laser scanning micros- copy; DAPI, 4,6-diamidine-2-phenyl-indol-dihydrochloride; DC, dendritic cell; EEA-1, early endosomal Ag 1; His-tag, hexahistidine tag; LAMP-1, lysosomal-asso- ciated membrane protein 1; MCD, -methyl-cyclodextrin; TSB, tryptic soy broth. The Journal of Immunology Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00