Uptake of Granulysin via Lipid Rafts Leads to Lysis of Intracellular Listeria innocua
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免疫学杂志 2005年第7期
Abstract
The bacteriolytic activity of CTL is mediated by granulysin, which has been reported to kill intracellular Mycobacterium tuberculosis 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 subsequent 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 phagosomes 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.
Introduction
Cytotoxic 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 intracellular bacteria are release of cytokines (2), especially of IFN- and TNF-, induction of target cell apoptosis (3), and direct mediation 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 discovered by a subtractive hybridization procedure of late activated T cells (6, 7) and exhibited a vast spectrum of antimicrobial activity against bacteria, fungi, and parasites (8), either as free microorganisms or located in host cells. The killing of intracellular Mycobacterium tuberculosis by V9/V2 T lymphocytes was shown to be dependent on granulysin (9). Furthermore, it was reported that V9/V2 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). Comparable 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 affected follicles and perilesional dermis in superficial microbial folliculitis (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 (SAPLIP).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 sphingolipids has been extensively investigated. Dependent on the pH value, all saposins were reported to bind negatively charged gangliosides (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 spectrum 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 phospholipids 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, 24, 25) and bacterial 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 infected host cells. Recombinant granulysin killed intracellular located M. tuberculosis only if cells were incubated simultaneously with perforin (8). In contrast, the killing of intracellular M. tuberculosis by CD4+ T cells and CD8+ CTLs was independent of perforin (29). CD4+ T cells that occurred in leprosy lesions were negative for perforin (12). Furthermore, it is known from studies with perforin knockout mice that perforin is not required for the early control of mycobacterial infection in mice (30). Perforin-independent granulysin uptake has also been shown to some extent in Jurkat cells, where homogeneous distribution of granulysin in the cytoplasm of cells was found after incubation with high concentrations of up to 50 μM granulysin (31).
In our study we analyzed binding, uptake, and intracellular trafficking of granulysin using human monocyte-derived dendritic cells (DC) as hosts harboring Listeria innocua. DC are professional APCs that are crucial for the induction of a cellular immune response. They possess several mechanisms to internalize macromolecules and pathogens for Ag processing and presentation to T cells (32). The mechanisms involved in uptake are receptor-mediated endocytosis via clathrin-coated pits, phagocytosis, macropinocytosis, and lipid rafts (25, 33). L. innocua is a Gram-positive, apathogenic bacterium ubiquitously distributed in our environment (34). Upon phagocytosis, it is transferred via endosomes to phagosomes, where it resides until transfer to and lysis in phagolysosomes (35). Uptake and trafficking of granulysin were correlated to established markers of the endocytic pathway as well as to cholera toxin to evaluate whether lipid rafts are involved in a perforin-independent mechanism of granulysin uptake and killing of intracellular L. innocua.
Materials and Methods
Production of recombinant granulysin and anti-granulysin Abs
Due to the postulated C-terminal posttranslational processing of native granulysin in CTLs (5), two recombinant granulysins of different lengths were cloned from cDNA that was reverse transcribed from total RNA extracted from human lymphokine-activated killer cells. Both constructs corresponding to NKG5 started with G 63 and ended with D 132, or L 145, respectively, for granulysin132 or granulysin145. A fragment of human -actin identical in length with granulysin132 was additionally reverse transcribed for use as a control protein, referred to as actinfrag. These inserts were cloned in pEt28a (Novagen), followed by a factor Xa cleavage site (IEGR/G) and a C-terminal hexahistidine tag (His-tag). Proteins were expressed in Escherichia coli BL21 (DE3) additionally transformed with the chloramphenicol-resistant plasmid, pRARE (Novagen) in Luria-Bertoni medium containing 50 μg/ml kanamycin, 34 μg/ml chloramphenicol, and 2% glucose (all from Sigma-Aldrich). Expression was induced with 1 mM isopropyl--D-thiogalactoside (Qbiogene). After lysis of bacteria by adding 1 mg/ml lysozyme, 1% Triton X-100, 50 μg/ml DNase, and 5 μg/ml RNase (all from Sigma-Aldrich), granulysin was purified via nickel affinity chromatography and further renatured according the protocol of Ernst et al. (20). Renatured recombinant granulysin was additionally purified using Sep-Pak Vac 6cc (1 g) C18 cartridges (Waters) and was eluted with 100% acetonitrile containing 0.1% trifluoroacetic acid. After lyophilization, the protein concentration was determined using the Bio-Rad protein assay. Protein purity was estimated by SDS-PAGE and N-terminal sequencing (University of Zurich), revealing the correct first four amino acids as GRDY.
For Ab production and cleavage of the C-terminal His-tag, the dialysate containing granulysin145 was concentrated by lyophilization, and after rehydration, it was treated with factor Xa (Amersham Biosciences) for 2 h at 4°C to remove the His-tag. The efficiency of His-tag removal was assessed by Western blotting and was routinely >99%. After His-Tag cleavage, granulysin145 was reverse phase purified as described above. Polyclonal anti-granulysin Abs were raised in guinea pig (Pineda).
Viability determination of L. innocua
Serial dilutions of L. innocua-loaded cells lysed by adding ice-cold water or suspension-treated L. innocua were spread on tryptic soy broth (TSB; Difco) agar plates. CFU were determined by counting colonies after overnight culture at 37°C, and specific lysis was calculated using the formula ((CFUs in buffer control – CFUs in test incubation)/CFUs in buffer control) x 100.
Alternatively, turbidimetry was used to study specific lysis of bacteria (36). Serial dilutions of cell lysates or treated L. innocua were incubated in 96-well plates (Nunc). Bacterial growth curves were monitored in a microplate reader (Spectra MAX 340; Molecular Devices) at OD600 with discontinuous shaking for 16 h at 37°C. Specific lysis was calculated by determining the time when the maximum population was reached in buffer controls (ODTmax-control). At this point, the OD value (ODTmax-Test) of a shifted growth curve was evaluated, and specific lysis was calculated using the formula ((ODTmax-control – ODmin) – (ODTmax-test – ODmin)/ODTmax-control – ODmin) x 100. All OD values were corrected by subtraction of the baseline OD (ODmin).
Bacteriolytic activity of granulysin
Granulysin was incubated at various concentrations for 3 h at 4 or 37°C with 105/ml L. innocua in 0.01 M Trisma base (pH 8). Actinfrag and buffer alone served as controls. After incubation, the viability of L. innocua was determined as described above. For some experiments, granulysin132 was pretreated with 2,3-butanedione (BAD) or with citraconic anhydride (CAH; both from Sigma-Aldrich) in 10 mM sodium borate buffer for 2 h at room temperature. During incubation, the pH was controlled and adjusted between 8 and 9. Granulysin132 incubated in borate buffer alone served as a control. Binding of BAD and CAH to granulysin was analyzed using electrospray mass spectrometry (University of Zurich; not shown).
To study binding of granulysin to L. innocua, 106/ml bacteria were incubated with granulysin or actinfrag in a concentration of 2.5 μM for 15 min at 37 or 4°C, respectively. Bacteria were washed subsequently three times with ice-cold PBS and fixed with 1.5% paraformaldehyde in PBS containing 1% sucrose.
Isolation and culture of DC
Human DC were generated in vitro from blood-derived precursors as previously described (37). Briefly, human PBMC obtained from venous blood of healthy donors (Blood Bank SRK) were isolated by Ficoll-Paque (Pharmacia Biotech) density centrifugation. The PBMC were cultured in RPMI 1640 supplemented with penicillin/streptomycin (all from Invitrogen Life Technologies) and 10% heat-inactivated pooled human A serum (Blood Bank SRK) for 2 h. The adherent cells were cultured for 6 days in RPMI 1640 supplemented with penicillin/streptomycin, 5% heat-inactivated pooled human A serum (DC culture medium) with rhGM-CSF (50 ng/ml; Novartis), and human rIL-4 (100 U/ml; R&D Systems).
Challenge of DC with Listeria
L. innocua were propagated in TSB at 37°C overnight, diluted 10-fold, and further expanded to an OD600 of 0.5 corresponding to 5 x 107/ml viable bacteria. Bacteria were harvested by centrifugation and washed twice with PBS before opsonization in RPMI 1640 with 50% pooled heat-inactivated human A serum for 30 min at 37°C. Opsonized Listeria were washed in PBS and resuspended in RPMI 1640. DC were challenged for 1 h with a multiplicity of infection of 5. Subsequently, cultures were washed with PBS and incubated for 3 h in DC culture medium containing 25 μg/ml gentamicin (Sigma-Aldrich) to kill extracellular L. innocua. Cholesterol depletion of DC was achieved by addition of -methyl-cyclodextrin (MCD; Sigma-Aldrich) in RPMI 1640 without human serum for 1 h at 37°C.
Granulysin treatment of DC
L. innocua-challenged or unchallenged DC were incubated with various concentrations of granulysin, actinfrag, or culture medium alone for the indicated times at 37 or at 4°C, subsequently washed twice with PBS, and either fixed with 1.5% paraformaldehyde in PBS containing 1% sucrose for immunofluorescence labeling or lysed by adding ice-cold sterile water for 30 min on ice for assessment of viability of L. innocua as described above. DC were also incubated with granulysin132 that was pretreated with BAD or CAH at the indicated concentrations in 10 mM sodium borate buffer or sodium borate buffer alone as a control. After incubation with modified granulysin for 1 h at 4°C, the cells were washed twice with ice-cold PBS and lysed with PBS containing 0.5% Triton. The content of granulysin bound to DC membranes was determined by Western blot analysis. Samples were run on a 15% SDS-PAGE gel and blotted on transfer membranes (Immobilon-P; Millipore). Granulysin was detected using anti-granulysin Ab. As a reference, cellular actin was detected using an anti-actin mAb (AC15; Sigma-Aldrich). The granulysin content bound to DC was measured and calculated relative to cellular actin using Image-J software (National Institutes of Health).
Confocal laser scanning microscopy (CLSM)
Fixed DC were cytospun onto glass slides and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 1 min at room temperature. Unspecific binding was blocked with 0.1% BSA (Fluka) in PBS for 1 h room temperature. Recombinant His-tagged granulysin132, granulysin145 after His-tag removal, or His-tagged actinfrag were detected with an anti-His mAb (1/1,000; Invitrogen Life Technologies) or with guinea pig anti-granulysin antiserum (1/10,000). Lysosomes were labeled with a lysosomal-associated membrane protein 1 (LAMP-1) mAb (1/50), early endosomes were labeled with a rabbit early endosomal Ag 1 (EEA-1) Ab (1/200; Affinity BioReagents), and CD55 was labeled with an anti-CD55 mAb (Accurate Chemical). Omitting the first Abs served as a control for specificity. For detection, the following Abs were used: FITC-conjugated goat anti-mouse or a goat anti-rabbit Ab, Cy3-labeled goat anti-guinea pig Ab (all from Kirkegaard & Perry Laboratories), or Texas Red-conjugated donkey anti-mouse Ab (Jackson ImmunoResearch Laboratories). All Abs were diluted in 0.1% BSA in PBS. DNA was labeled with 1 μg/ml 4,6-diamidine-2-phenyl-indol-dihydrochloride (DAPI; Roche) in PBS for 15 min at room temperature.
For co-uptake experiments L. innocua-challenged DC were simultaneously treated with granulysin or actinfrag in combination with cholera toxin-FITC (10 μg/ml; Molecular Probes), dextran-FITC (1 mg/ml; m.w. = 40,000; Sigma-Aldrich), or transferrin-FITC (25 μg/ml; Molecular Probes) for various time periods. Alternatively, L. innocua-challenged DC were pulsed with granulysin and cholera toxin-FITC for 10 min on ice. Subsequently, cells were washed twice with ice-cold PBS and transferred to 37°C for the indicated chase periods before fixation and processing as described above.
Fluorescent-labeled specimens were examined using a confocal laser scanning microscope (CLSM SP1; Leica). Images were analyzed using the Imaris software package (Bitplane), and threshold levels for calculation of colocalization micrographs were selected above background signals. Images representing single sections through three-dimensional volume stacks are shown.
Results
Bacteriolytic activity of recombinant granulysin
The antibacterial activity of granulysin132 and granulysin145, respectively, was monitored by assessing the viability of L. innocua in suspension. His-tagged granulysin132-treated L. innocua were found to have a lowered viability depending on the granulysin132 concentration used for incubation (Fig. 1A). This activity was identical with the antibacterial activity of granulysin145 without a C-terminal His-tag. At a concentration of 2.5 μM, >90% of the bacteria were killed. Significant bacteriolysis could still be measured at a concentration of 0.15 μM. Actinfrag, a His-tagged fragment of human -actin, identical in length, expression, and purification, used as a control to granulysin, did not affect L. innocua viability.
FIGURE 1. Bacteriolytic activity of recombinant granulysin against L. innocua in suspension. A, L. innocua were incubated for 3 h at 37°C with granulysin132, granulysin145, or actinfrag as a control in various concentrations. Specific lysis was determined in CFU assays. The mean ± SE of three independent experiments are presented. B, L. innocua were treated with 2.5 μM granulysin145, granulysin132, or actinfrag for various time periods at 37 and 4°C. Specific lysis was calculated from bacterial growth curves obtained by turbidimetry. The mean ± SE of three independent experiments are presented (, granulysin132, 37°C; , granulysin145, 37°C; ?, actin132, 37°C; , granulysin132, 4°C; , granulysin145, 4°C). Granulysin132-treated (C) or actinfrag-treated (D) L. innocua were stained with an anti-His mAb for CLSM. Representative phase contrast (left panels) and immunofluorescence images (right panels) are shown. Bar = 4 μm.
To investigate the kinetics of granulysin-induced bacteriolysis, L. innocua in suspension were incubated with 2.5 μM granulysin132 or granulysin145 without His-tag, and lysis was stopped by adding TSB at the indicated time points. Growth inhibition by granulysin was monitored and calculated from Listeria growth curves. The onset of bacteriolysis occurred very rapidly, with >70% of the bacteria killed after 5 min of granulysin treatment at 37°C (Fig. 1B). Fifteen minutes of granulysin incubation at 37°C was sufficient to kill 90% of the bacteria. Lowering the temperature to 4°C during incubation decreased specific bacteriolysis to 55% after 15 min and to 70% after 45 min, but did not abolish lysis (Fig. 1B).
After incubation with 2.5 μM granulysin132 for 15 min and staining with an anti-His mAb, L. innocua were coated by granulysin132 (Fig. 1C). Granulysin132 bound to bacteria was also detected after treatment at 4°C (data not shown). No binding of actinfrag was detected after treating the Listeria with actinfrag (Fig. 1D). According to these findings, granulysin binds and kills L. innocua highly efficiently within minutes at 37 or 4°C.
Granulysin is actively taken up by DC
To lyse intracellular bacteria, granulysin has to enter the cell either by a passive or an active uptake mechanism. Active protein uptake is a temperature-dependent process; therefore, granulysin uptake was investigated at 37 and 4°C. For this purpose, DC were treated for 45 min at 37°C with granulysin132 or actinfrag, and the localization of the proteins was determined by immunofluorescent staining using an anti-His mAb. CLSM revealed a spot-like pattern of immunolabeled granulysin132 within the DC (Fig. 2A). Granulysin132 distribution was identical in L. innocua-challenged DC (data not shown). After incubation for 45 min at 4°C, granulysin132 was found exclusively at the cell membrane, and no significant transfer to an intracellular compartment occurred (Fig. 2B), indicating an active uptake mechanism. Actinfrag was neither bound nor taken up by DC at detectable levels (Fig. 2C).
FIGURE 2. Granulysin132 is actively taken up by human DC. Unchallenged DC were incubated for 45 min with 2.5 μM granulysin132 at 37°C (A) or 4°C (B) or with 2.5 μM actinfrag at 37°C (C). After the incubation, the cells were fixed and stained with the anti-His Ab for CLSM. Representative phase contrast (right panels) and immunofluorescence images (left panels) are shown. Bar = 8 μm. D, Granulysin132 was pretreated with CAH, BAD, or sodium borate buffer before incubation with DC for 60 min at 4°C. The level of bound granulysin was assessed by Western blotting. Granulysin was detected with the anti-granulysin Ab. As a reference band, cellular actin was detected using an anti-actin mAb. E, The activity of modified granulysin132 against L. innocua in suspension was tested with turbidimetry and calculated from bacterial growth curves.
Ernst et al. (20) demonstrated that modification of arginine residues with BAD reduced the binding as well as the lytic capacity of granulysin against E. coli, whereas neither binding nor lytic capacity of granulysin pretreated with CAH to modify lysine residues was affected. Consistent with these results, pretreatment of granulysin132 with BAD reduced the lytic activity against L. innocua in suspension, whereas CAH or borate buffer alone had no effect (Fig. 2E). Preincubation of granulysin with BAD, but not with CAH, significantly decreased binding of granulysin132 to DC cell membranes, as assessed by Western blotting (Fig. 2D). Similar to binding, uptake of granulysin132 at 37°C was not affected by modification of lysine residues (not shown).
Granulysin binding and uptake in DC are associated with lipid rafts
The spot-like staining pattern of granulysin132 within DC resembled vesicles of the endocytic pathway. Because granulysin belongs to the SAPLIP, which has known affinity to sphingolipids (5), a binding and initial uptake mechanism associated with lipid rafts seemed likely. Lipid rafts are highly organized microdomains in the plasma membrane with elevated cholesterol and glycosphingolipid contents (22). The subunit of cholera toxin binds the Gm1 ganglioside in lipid rafts and is a well-established marker to detect such microdomains (38). To examine possible association of granulysin binding and uptake in DC via lipid rafts, co-uptake experiments with fluorescently labeled cholera toxin and granulysin were performed. After incubation of granulysin132 with cholera toxin for 30 min in steady state at 37°C, both proteins were found colocalized in DC (Fig. 3A, see colocalization panel). The colocalization of cholera toxin and granulysin132 was most significant in the peripheral part of the cells. Cholera toxin was concentrated over time in the perinuclear area of the DC, whereas granulysin132 remained in the peripheral compartment. No uptake of the proteins occurred at 4°C (Fig. 3B). Under these conditions granulysin132 and cholera toxin were colocalized and remained in a patch-like pattern at the cell membrane. To exclude an interaction of the His-tag of granulysin132 with the cell membrane, especially with lipid rafts, DC were coincubated with granulysin145 after His-tag removal and cholera toxin at 37 and 4°C (Fig. 3, C and D). After fixation, the samples were stained with the anti-granulysin Ab. We found a staining pattern identical with that achieved with granulysin132, proving that the His-tag did not influence granulysin binding or uptake in DC. The assumption of initial co-uptake and later separation of granulysin132 and cholera toxin was confirmed in pulse-chase experiments. Granulysin132 and cholera toxin were allowed to bind to DC on ice for 10 min, and subsequent to medium replacement, cells were incubated at 37°C for 5–90 min (Fig. 3E). After 5 min, both proteins were colocalized, either still bound to the cell membrane or in the peripheral compartments of the cells. Thirty minutes after binding to the cell membrane, granulysin132 and cholera toxin had separated into different compartments. After 90 min, cholera toxin was found highly concentrated near the nucleus and was totally separated from granulysin132, which remained in the peripheral compartment of the cells (see colocalization panels in Fig. 3F). The association of granulysin with lipid rafts in DC was confirmed by staining of granulysin132-treated DC with an anti-CD55 Ab and an anti-granulysin Ab (Fig. 3G). To test possible involvement of macropinocytosis or clathrin-dependent endocytosis in granulysin132 uptake, dextran-FITC and transferrin-FITC, respectively, were incubated with granulysin132. In neither the initial uptake nor in the later stages was dextran (Fig. 4C) or transferrin (data not shown) colocalized with granulysin132.
FIGURE 3. Granulysin binding and uptake in DC are associated with lipid rafts. L. innocua-challenged DC were coincubated with granulysin (2.5 μM; granulysin132 (A and B) or granulysin145 without His-tag (C and D)) and FITC-labeled cholera toxin (CT; 10 μg/ml) for 30 min at 37°C (A and C) or 4°C (B and D). E and F, For pulse-chase experiments, DC were pulsed with granulysin132 and cholera toxin for 10 min at 4°C and subsequently cultivated at 37°C for the indicated chase periods. Granulysin132 is marked in red, cholera toxin is shown in green, and nuclear and bacterial DNA are stained with DAPI (blue; merged image (E), calculated colocalization (F)). G, Double labeling of granulysin-treated DC with an anti-CD55 Ab and the anti-granulysin Ab. Granulysin132 is marked in red, and CD55 is shown in green. Colocalization images of granulysin132 and cholera toxin or CD55 were calculated using Imaris software. Bars = 8 μm.
FIGURE 4. Cholesterol depletion prevents granulysin132 uptake, but not binding. DC were pretreated with 20 mM MCD for 45 min before incubation with granulysin132 (2.5 μM) and dextran-FITC (1 mg/ml) for 45 min at 37°C (A) or 4°C (B). C, Control cells were pretreated with serum-free medium for 45 min before incubation with granulysin132 and dextran-FITC for 45 min at 37°C. Bars = 8 μm.
Preincubation of DC with MCD, which is known to disrupt lipid raft-mediated uptake by binding cholesterol (39), inhibited the uptake, but not the binding, of granulysin132 in DC. After incubation of DC with MCD for 45 min at 37°C, granulysin132 was detected in patches at the cell membrane (Fig. 4A). In contrast, simultaneously applied dextran-FITC was endocytosed by cholesterol-depleted cells at 37°C, indicating that the fluid phase endocytosis was still functional. After incubation of cholesterol-depleted cells at 4°C, both granulysin132 and dextran-FITC were found at the cell membrane, but no intracellular staining was detected (Fig. 4B). In control cells pretreated with serum-free medium, uptake of both granulysin132 and dextran-FITC was detected at 37°C (Fig. 4C). Similar results were obtained by pretreatment of DC with filipin (1 μg/ml), another lipid raft-disrupting drug (38), before granulysin132 incubation (not shown). Overall, these findings revealed lipid rafts to be critically involved in initial uptake, but not binding of granulysin to DC.
Granulysin was found in early endosomes, but not in lysosomes
To follow trafficking of granulysin132 from lipid rafts, double labeling of granulysin132 with markers specific for the endocytic compartment was performed. In a first step, early endosomes were stained with a polyclonal Ab recognizing the EEA-1. Granulysin132 was colocalized with the EEA-1 (Fig. 5A) in L. innocua-challenged DC after 10 min of incubation. After 60 min, the longest time analyzed for EEA-1 colocalization, granulysin132 still resided in endosomes.
FIGURE 5. Granulysin132 is localized in early endosomes and Listeria-containing phagosomes, but not in lysosomes. A, L. innocua-challenged DC were incubated with 2.5 μM granulysin132 for 30 min at 37°C for colocalization with EEA-1. B, For double staining of granulysin132 and LAMP-1, cells were treated for 90 min with 2.5 μM granulysin132 at 37°C, then stained with a polyclonal anti-granulysin Ab and a polyclonal Ab recognizing EEA-1 or an anti-LAMP-1 mAb, respectively. Nuclear and bacterial DNA was labeled with DAPI (blue); early endosomes and lysosomes, respectively, are marked in green; and granulysin132 is depicted in red. The yellow spots in the merge image (A) indicate colocalization of granulysin132 with EEA-1. Colocalization of the green and red channels was additionally calculated using Imaris software. Arrowheads in B indicate listerial DNA spots that colocalize with LAMP-1, whereas arrows mark DNA spots that coincide with granulysin132. Bar = 8 μm.
Immunostaining of L. innocua-challenged and granulysin132-treated DC with an Ab recognizing LAMP-1 revealed that granulysin132 clearly separated from the LAMP-1-positive compartment (Fig. 5B). DAPI-stained listerial DNA was found in the lysosomal, LAMP-1-positive compartment (arrowheads in Fig. 5B) or in phagosomes, which showed a distinct labeling for granulysin132 (arrows in Fig. 5B). Positive staining of L. innocua for granulysin132 remained detectable even when bacteria were isolated from granulysin132-treated DC (data not shown). Overall, these results indicate that after uptake, granulysin is present in early sorting endosomes of DC and then transferred to phagosomes, where granulysin is able to bind Listeria.
Granulysin mediates lysis of L. innocua in DC
L. innocua-challenged DC were incubated with various concentrations of granulysin132 or granulysin145 without His-tag for 3 h at 37°C. The viability of the intracellular bacteria was tested in CFU assays (Fig. 6A). Granulysin132 (5 μM) killed >41%, and 5 μM granulysin145 killed >46% of the intracellular L. innocua after 3 h. Granulysin132 (2.5 μM) was sufficient to lyse 29% of the intracellular Listeria, very similar to 2.5 μM granulysin145, which killed 26%. Viability reduction of bacteria was strictly dependent on granulysin132 dosage and did not occur with actinfrag treatment. Incubation of DC with granulysin132 at 4°C for 3 h did not impair the viability of intracellular L. innocua (data not shown). To exclude bacteria loss due to detachment of perishing host cells, the viability of DC after granulysin132 incubation was assessed. Granulysin132 incubated in a concentration up to 5 μM had no effect on the viability of L. innocua-challenged DC monitored in lactate dehydrogenase release and thiazolyl blue (MTT) assays (data not shown).
FIGURE 6. Granulysin132 mediates lysis of L. innocua in DC. L. innocua-challenged DCs were incubated for 3 h with granulysin132 in varying concentrations (A) or with 2.5 μM granulysin132 for the indicated time periods at 37°C (B). After granulysin132 incubation, cells were lysed in ice-cold water, and Listeria viability was assessed in CFU assays. The mean ± SD of four independent experiments are presented. C, Deconvoluted CLSM image of intracellular bacteria found in granulysin-treated DC. Bacterial DNA (red) is stained with DAPI, and granulysin132 (green) is labeled with an anti-His Ab. Bar = 2 μm. D, L. innocua-challenged DC were cholesterol-depleted for 45 min with the indicated concentrations of MCD. Subsequently, DC were incubated with 2.5 μM granulysin132 for 3 h at 37°C. After the incubation, cells were lysed, and bacterial viability was assessed in CFU assays. The mean ± SE of three independent experiments are presented. Statistical significance was calculated with Student’s t test for paired samples.
Compared with the fast lysis of extracellular L. innocua, intracellular bacterial killing was delayed. Forty-five minutes of incubation at 37°C with 2.5 μM granulysin132 was required to kill 12% of the intracellular bacteria (Fig. 6B). Intracellular lysis increased to 25% after 90 min of treatment, but never reached the same level as in suspension (see Fig. 1B). The maximal reduction of 31% of the intracellular bacteria was achieved after 3 h of incubation with 2.5 μM granulysin132. Deconvoluted high resolution CLSM images of L. innocua within granulysin132-treated DC demonstrated bacterial DNA that was clearly surrounded by granulysin132 labeling (Fig. 6C). Granulysin132-mediated lysis of intracellular L. innocua was reduced after cholesterol depletion of DC by MCD in a dose-dependent manner (Fig. 6D). Preincubation of DC with 10 mM MCD diminished lysis of intracellular L. innocua to 13%, and only 4% of specific lysis was achieved after pretreatment with 20 mM MCD.
Discussion
In this study we could clearly demonstrate that recombinant granulysin was binding and dose-dependently killing L. innocua as free bacteria grown in suspension. Granulysin-induced lysis of Listeria is in agreement with data obtained by other groups using a variety of microbial pathogens, including L. monocytogenes (8, 20). To date, the binding of granulysin to bacteria has never been directly shown, but can be explained by properties of the SAPLIP family, of which granulysin is a member. SAPLIPs interact with a variety of lipids (14). Bacterial membranes contain mainly acidic phospholipids, such as phosphatidylglycerol and cardiolipin. Positive charges of granulysin (net charge, +11) can be assumed to be the driving force for binding to negatively charged bacterial membranes. Charges in granulysin are not homogeneously distributed, but are slightly polarized toward a region of the molecule corresponding to helix 3 (19). This region was proposed as the site of the initial contact of granulysin to bacterial membranes. After binding, granulysin is suggested to cluster at the bacterial membrane, leading to deformation and, subsequently, lysis by friction of adjacent granulysin molecules.
To reach intracellular bacteria, granulysin has to bind and enter mammalian cells that have apparent differences in the composition of membranes to prokaryotes. The outer leaflet of mammalian cells is mainly composed of zwitterionic phospholipids, such as phosphatidylcholine and sphingomyelin. Cholesterol is abundant in mammalian cells, but is absent in bacterial cell membranes. A third difference is the higher transmembrane potential of prokaryotic cells (40, 41, 42, 43).
Binding of granulysin to mammalian cell membranes can again be explained by properties of the SAPLIP family. The interaction of saposins with sphingolipids has been extensively investigated. Dependent on the pH value, all saposins were reported to bind the negatively charged gangliosides, which are found enriched together with cholesterol in lipid rafts. In our study we could show by modifying arginine residues of granulysin that positive charges of arginine residues contribute crucially to the lytic activity against L. innocua as well as to the binding capacity to DC membranes. Lysine residues seem not to play an important role in lysis of bacteria and binding to eukaryotic cells. This is consistent with the finding that covering the granulysin arginine residues by butanedione reduced binding and lysis of E. coli (20). Granulysin was found bound to lipid rafts, as indicated by its colocalization with cholera toxin and CD55, both known markers for lipid rafts (25, 38, 44). Preincubation of cells with MCD or filipin, which are both known to deplete cholesterol (45), did not abolish binding of granulysin to lipid rafts. Mechanisms of granulysin binding to lipid rafts or even binding partners are still an enigma, but it is known to be cholesterol independent. Clustering of granulysin at ganglioside-rich regions of the plasma membrane could apply local shear stress that might trigger budding and subsequently endocytosis of granulysin-containing membrane vesicles. There is some evidence that local deformation and membrane tension contribute to the regulation of endocytosis (46, 47).
In contrast to binding, uptake of granulysin could be inhibited by cholesterol depletion, but also by incubation at 4°C. Inhibition of uptake at 4°C is a strong indication for an active process (48). Endocytosis of granulysin via the lipid raft/caveolae pathway is distinguished from both the clathrin-dependent and constitutive pinocytic pathways by its sensitivity to cholesterol depletion. In line with this observation is that dextran or transferrin as tracers for constitutive pinocytosis and clathrin-dependent pathway, respectively, were never found colocalized with simultaneously applied granulysin. After endocytosis, granulysin was found to be sorted differently from cholera toxin (49). Additional intracellular trafficking of granulysin from early endosomes may result in fusion of endosomes with phagosomes where granulysin was found to be colocalized with bacteria. Fusion of granulysin-containing early endosomes with phagosomes may be regulated by the small rab5 GTPase, which has been shown to be directly correlated with an accelerated maturation of Listeria-containing phagosomes (50).
In our model of L. innocua-challenged human DC binding, uptake and trafficking of granulysin to phagosomes as well as binding to and lysis of L. innocua were shown to occur in the absence of perforin. In contrast to these results, it was shown that M. tuberculosis was efficiently lysed in vitro in human DC by granulysin only in combination with perforin (8, 20). This combination of perforin and granulysin to achieve antibacterial activity against intracellular pathogens seems necessary at least occasionally. NKT cells and CTL could control mycobacterial infection by relying on granulysin, but not on perforin or Fas/Fas ligand interaction (11, 29). In vivo, it was found that perforin-negative, but granulysin-positive, CD4+ cells are present at sites of bacterial infections (12, 13). This is in line with our in vitro data for granulysin activity. However, data are largely missing about the exact role of perforin in lysis of intracellular bacteria, e.g., M. tuberculosis. Data on perforin-dependent as well as independent uptake and activity of granulysin are not conclusive, and mechanisms of entry into host cells are poorly characterized. Perforin might well have a role in granulysin trafficking to compartments harboring intracellular bacteria. In contrast to apathogenic L. innocua pathogenic species such as M. tuberculosis, Chlamydia spp. and Coxiella spp. have evolved strategies to modify their intracellular compartments (51, 52) or are known to escape from vesicular compartments to the cytosol-like L. monocytogenes (53). Mycobactera, for example, can stop phagosome maturation by inhibiting acquisition of the rab5 effector EEA-1. By excluding this important regulator for vesicular trafficking, fusion processes within the endocytic pathway are impaired (54).
Overall, we propose a model of granulysin-mediated activity against intravacuolar bacteria. According to this model, binding due to electrostatic interactions and clustering of granulysin at membrane microdomains with elevated acidic sphingolipid content would lead to endocytosis as a first step toward lysis of intracellular bacteria. Such lipid raft microdomains were indeed found concentrated in the regions of the immunological synapse (55). After cholesterol-dependent uptake, granulysin is delivered to early sorting endosomes, fusing later with bacteria-harboring phagosomes, where the lysis of the bacteria is induced. Simultaneously secreted IFN- at sites of infection by the CTL would enhance granulysin targeting. There is evidence that activated macrophages up-regulate rab5 in response to IFN- (56). An elevated level of rab5 accelerates not only maturation of Listeria-containing phagosomes (50), but also the likelihood for phagosome-endosome fusion. Thus, the antimicrobial activity mediated by granulysin would be enhanced.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We are very grateful to Marina Balzer and Gery Barmettler for excellent technical assistance, and to Rene Moser, Sonja Latinovic, and Caroline Maake for helpful support. We thank Markus Schneemann and Gabriele Schoedon (Medical Clinic B Research Unit, Department of Medicine, University Hospital of Zurich) for providing L. innocua. We also thank Jack Rohrer (Institute of Physiology, University of Zurich) for kindly providing the EEA-1 as well as the LAMP-1 Abs.
Footnotes
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 microscopy; DAPI, 4,6-diamidine-2-phenyl-indol-dihydrochloride; DC, dendritic cell; EEA-1, early endosomal Ag 1; His-tag, hexahistidine tag; LAMP-1, lysosomal-associated membrane protein 1; MCD, -methyl-cyclodextrin; TSB, tryptic soy broth.
Received for publication July 15, 2004. Accepted for publication January 26, 2005.
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The bacteriolytic activity of CTL is mediated by granulysin, which has been reported to kill intracellular Mycobacterium tuberculosis 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 subsequent 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 phagosomes 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.
Introduction
Cytotoxic 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 intracellular bacteria are release of cytokines (2), especially of IFN- and TNF-, induction of target cell apoptosis (3), and direct mediation 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 discovered by a subtractive hybridization procedure of late activated T cells (6, 7) and exhibited a vast spectrum of antimicrobial activity against bacteria, fungi, and parasites (8), either as free microorganisms or located in host cells. The killing of intracellular Mycobacterium tuberculosis by V9/V2 T lymphocytes was shown to be dependent on granulysin (9). Furthermore, it was reported that V9/V2 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). Comparable 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 affected follicles and perilesional dermis in superficial microbial folliculitis (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 (SAPLIP).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 sphingolipids has been extensively investigated. Dependent on the pH value, all saposins were reported to bind negatively charged gangliosides (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 spectrum 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 phospholipids 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, 24, 25) and bacterial 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 infected host cells. Recombinant granulysin killed intracellular located M. tuberculosis only if cells were incubated simultaneously with perforin (8). In contrast, the killing of intracellular M. tuberculosis by CD4+ T cells and CD8+ CTLs was independent of perforin (29). CD4+ T cells that occurred in leprosy lesions were negative for perforin (12). Furthermore, it is known from studies with perforin knockout mice that perforin is not required for the early control of mycobacterial infection in mice (30). Perforin-independent granulysin uptake has also been shown to some extent in Jurkat cells, where homogeneous distribution of granulysin in the cytoplasm of cells was found after incubation with high concentrations of up to 50 μM granulysin (31).
In our study we analyzed binding, uptake, and intracellular trafficking of granulysin using human monocyte-derived dendritic cells (DC) as hosts harboring Listeria innocua. DC are professional APCs that are crucial for the induction of a cellular immune response. They possess several mechanisms to internalize macromolecules and pathogens for Ag processing and presentation to T cells (32). The mechanisms involved in uptake are receptor-mediated endocytosis via clathrin-coated pits, phagocytosis, macropinocytosis, and lipid rafts (25, 33). L. innocua is a Gram-positive, apathogenic bacterium ubiquitously distributed in our environment (34). Upon phagocytosis, it is transferred via endosomes to phagosomes, where it resides until transfer to and lysis in phagolysosomes (35). Uptake and trafficking of granulysin were correlated to established markers of the endocytic pathway as well as to cholera toxin to evaluate whether lipid rafts are involved in a perforin-independent mechanism of granulysin uptake and killing of intracellular L. innocua.
Materials and Methods
Production of recombinant granulysin and anti-granulysin Abs
Due to the postulated C-terminal posttranslational processing of native granulysin in CTLs (5), two recombinant granulysins of different lengths were cloned from cDNA that was reverse transcribed from total RNA extracted from human lymphokine-activated killer cells. Both constructs corresponding to NKG5 started with G 63 and ended with D 132, or L 145, respectively, for granulysin132 or granulysin145. A fragment of human -actin identical in length with granulysin132 was additionally reverse transcribed for use as a control protein, referred to as actinfrag. These inserts were cloned in pEt28a (Novagen), followed by a factor Xa cleavage site (IEGR/G) and a C-terminal hexahistidine tag (His-tag). Proteins were expressed in Escherichia coli BL21 (DE3) additionally transformed with the chloramphenicol-resistant plasmid, pRARE (Novagen) in Luria-Bertoni medium containing 50 μg/ml kanamycin, 34 μg/ml chloramphenicol, and 2% glucose (all from Sigma-Aldrich). Expression was induced with 1 mM isopropyl--D-thiogalactoside (Qbiogene). After lysis of bacteria by adding 1 mg/ml lysozyme, 1% Triton X-100, 50 μg/ml DNase, and 5 μg/ml RNase (all from Sigma-Aldrich), granulysin was purified via nickel affinity chromatography and further renatured according the protocol of Ernst et al. (20). Renatured recombinant granulysin was additionally purified using Sep-Pak Vac 6cc (1 g) C18 cartridges (Waters) and was eluted with 100% acetonitrile containing 0.1% trifluoroacetic acid. After lyophilization, the protein concentration was determined using the Bio-Rad protein assay. Protein purity was estimated by SDS-PAGE and N-terminal sequencing (University of Zurich), revealing the correct first four amino acids as GRDY.
For Ab production and cleavage of the C-terminal His-tag, the dialysate containing granulysin145 was concentrated by lyophilization, and after rehydration, it was treated with factor Xa (Amersham Biosciences) for 2 h at 4°C to remove the His-tag. The efficiency of His-tag removal was assessed by Western blotting and was routinely >99%. After His-Tag cleavage, granulysin145 was reverse phase purified as described above. Polyclonal anti-granulysin Abs were raised in guinea pig (Pineda).
Viability determination of L. innocua
Serial dilutions of L. innocua-loaded cells lysed by adding ice-cold water or suspension-treated L. innocua were spread on tryptic soy broth (TSB; Difco) agar plates. CFU were determined by counting colonies after overnight culture at 37°C, and specific lysis was calculated using the formula ((CFUs in buffer control – CFUs in test incubation)/CFUs in buffer control) x 100.
Alternatively, turbidimetry was used to study specific lysis of bacteria (36). Serial dilutions of cell lysates or treated L. innocua were incubated in 96-well plates (Nunc). Bacterial growth curves were monitored in a microplate reader (Spectra MAX 340; Molecular Devices) at OD600 with discontinuous shaking for 16 h at 37°C. Specific lysis was calculated by determining the time when the maximum population was reached in buffer controls (ODTmax-control). At this point, the OD value (ODTmax-Test) of a shifted growth curve was evaluated, and specific lysis was calculated using the formula ((ODTmax-control – ODmin) – (ODTmax-test – ODmin)/ODTmax-control – ODmin) x 100. All OD values were corrected by subtraction of the baseline OD (ODmin).
Bacteriolytic activity of granulysin
Granulysin was incubated at various concentrations for 3 h at 4 or 37°C with 105/ml L. innocua in 0.01 M Trisma base (pH 8). Actinfrag and buffer alone served as controls. After incubation, the viability of L. innocua was determined as described above. For some experiments, granulysin132 was pretreated with 2,3-butanedione (BAD) or with citraconic anhydride (CAH; both from Sigma-Aldrich) in 10 mM sodium borate buffer for 2 h at room temperature. During incubation, the pH was controlled and adjusted between 8 and 9. Granulysin132 incubated in borate buffer alone served as a control. Binding of BAD and CAH to granulysin was analyzed using electrospray mass spectrometry (University of Zurich; not shown).
To study binding of granulysin to L. innocua, 106/ml bacteria were incubated with granulysin or actinfrag in a concentration of 2.5 μM for 15 min at 37 or 4°C, respectively. Bacteria were washed subsequently three times with ice-cold PBS and fixed with 1.5% paraformaldehyde in PBS containing 1% sucrose.
Isolation and culture of DC
Human DC were generated in vitro from blood-derived precursors as previously described (37). Briefly, human PBMC obtained from venous blood of healthy donors (Blood Bank SRK) were isolated by Ficoll-Paque (Pharmacia Biotech) density centrifugation. The PBMC were cultured in RPMI 1640 supplemented with penicillin/streptomycin (all from Invitrogen Life Technologies) and 10% heat-inactivated pooled human A serum (Blood Bank SRK) for 2 h. The adherent cells were cultured for 6 days in RPMI 1640 supplemented with penicillin/streptomycin, 5% heat-inactivated pooled human A serum (DC culture medium) with rhGM-CSF (50 ng/ml; Novartis), and human rIL-4 (100 U/ml; R&D Systems).
Challenge of DC with Listeria
L. innocua were propagated in TSB at 37°C overnight, diluted 10-fold, and further expanded to an OD600 of 0.5 corresponding to 5 x 107/ml viable bacteria. Bacteria were harvested by centrifugation and washed twice with PBS before opsonization in RPMI 1640 with 50% pooled heat-inactivated human A serum for 30 min at 37°C. Opsonized Listeria were washed in PBS and resuspended in RPMI 1640. DC were challenged for 1 h with a multiplicity of infection of 5. Subsequently, cultures were washed with PBS and incubated for 3 h in DC culture medium containing 25 μg/ml gentamicin (Sigma-Aldrich) to kill extracellular L. innocua. Cholesterol depletion of DC was achieved by addition of -methyl-cyclodextrin (MCD; Sigma-Aldrich) in RPMI 1640 without human serum for 1 h at 37°C.
Granulysin treatment of DC
L. innocua-challenged or unchallenged DC were incubated with various concentrations of granulysin, actinfrag, or culture medium alone for the indicated times at 37 or at 4°C, subsequently washed twice with PBS, and either fixed with 1.5% paraformaldehyde in PBS containing 1% sucrose for immunofluorescence labeling or lysed by adding ice-cold sterile water for 30 min on ice for assessment of viability of L. innocua as described above. DC were also incubated with granulysin132 that was pretreated with BAD or CAH at the indicated concentrations in 10 mM sodium borate buffer or sodium borate buffer alone as a control. After incubation with modified granulysin for 1 h at 4°C, the cells were washed twice with ice-cold PBS and lysed with PBS containing 0.5% Triton. The content of granulysin bound to DC membranes was determined by Western blot analysis. Samples were run on a 15% SDS-PAGE gel and blotted on transfer membranes (Immobilon-P; Millipore). Granulysin was detected using anti-granulysin Ab. As a reference, cellular actin was detected using an anti-actin mAb (AC15; Sigma-Aldrich). The granulysin content bound to DC was measured and calculated relative to cellular actin using Image-J software (National Institutes of Health).
Confocal laser scanning microscopy (CLSM)
Fixed DC were cytospun onto glass slides and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 1 min at room temperature. Unspecific binding was blocked with 0.1% BSA (Fluka) in PBS for 1 h room temperature. Recombinant His-tagged granulysin132, granulysin145 after His-tag removal, or His-tagged actinfrag were detected with an anti-His mAb (1/1,000; Invitrogen Life Technologies) or with guinea pig anti-granulysin antiserum (1/10,000). Lysosomes were labeled with a lysosomal-associated membrane protein 1 (LAMP-1) mAb (1/50), early endosomes were labeled with a rabbit early endosomal Ag 1 (EEA-1) Ab (1/200; Affinity BioReagents), and CD55 was labeled with an anti-CD55 mAb (Accurate Chemical). Omitting the first Abs served as a control for specificity. For detection, the following Abs were used: FITC-conjugated goat anti-mouse or a goat anti-rabbit Ab, Cy3-labeled goat anti-guinea pig Ab (all from Kirkegaard & Perry Laboratories), or Texas Red-conjugated donkey anti-mouse Ab (Jackson ImmunoResearch Laboratories). All Abs were diluted in 0.1% BSA in PBS. DNA was labeled with 1 μg/ml 4,6-diamidine-2-phenyl-indol-dihydrochloride (DAPI; Roche) in PBS for 15 min at room temperature.
For co-uptake experiments L. innocua-challenged DC were simultaneously treated with granulysin or actinfrag in combination with cholera toxin-FITC (10 μg/ml; Molecular Probes), dextran-FITC (1 mg/ml; m.w. = 40,000; Sigma-Aldrich), or transferrin-FITC (25 μg/ml; Molecular Probes) for various time periods. Alternatively, L. innocua-challenged DC were pulsed with granulysin and cholera toxin-FITC for 10 min on ice. Subsequently, cells were washed twice with ice-cold PBS and transferred to 37°C for the indicated chase periods before fixation and processing as described above.
Fluorescent-labeled specimens were examined using a confocal laser scanning microscope (CLSM SP1; Leica). Images were analyzed using the Imaris software package (Bitplane), and threshold levels for calculation of colocalization micrographs were selected above background signals. Images representing single sections through three-dimensional volume stacks are shown.
Results
Bacteriolytic activity of recombinant granulysin
The antibacterial activity of granulysin132 and granulysin145, respectively, was monitored by assessing the viability of L. innocua in suspension. His-tagged granulysin132-treated L. innocua were found to have a lowered viability depending on the granulysin132 concentration used for incubation (Fig. 1A). This activity was identical with the antibacterial activity of granulysin145 without a C-terminal His-tag. At a concentration of 2.5 μM, >90% of the bacteria were killed. Significant bacteriolysis could still be measured at a concentration of 0.15 μM. Actinfrag, a His-tagged fragment of human -actin, identical in length, expression, and purification, used as a control to granulysin, did not affect L. innocua viability.
FIGURE 1. Bacteriolytic activity of recombinant granulysin against L. innocua in suspension. A, L. innocua were incubated for 3 h at 37°C with granulysin132, granulysin145, or actinfrag as a control in various concentrations. Specific lysis was determined in CFU assays. The mean ± SE of three independent experiments are presented. B, L. innocua were treated with 2.5 μM granulysin145, granulysin132, or actinfrag for various time periods at 37 and 4°C. Specific lysis was calculated from bacterial growth curves obtained by turbidimetry. The mean ± SE of three independent experiments are presented (, granulysin132, 37°C; , granulysin145, 37°C; ?, actin132, 37°C; , granulysin132, 4°C; , granulysin145, 4°C). Granulysin132-treated (C) or actinfrag-treated (D) L. innocua were stained with an anti-His mAb for CLSM. Representative phase contrast (left panels) and immunofluorescence images (right panels) are shown. Bar = 4 μm.
To investigate the kinetics of granulysin-induced bacteriolysis, L. innocua in suspension were incubated with 2.5 μM granulysin132 or granulysin145 without His-tag, and lysis was stopped by adding TSB at the indicated time points. Growth inhibition by granulysin was monitored and calculated from Listeria growth curves. The onset of bacteriolysis occurred very rapidly, with >70% of the bacteria killed after 5 min of granulysin treatment at 37°C (Fig. 1B). Fifteen minutes of granulysin incubation at 37°C was sufficient to kill 90% of the bacteria. Lowering the temperature to 4°C during incubation decreased specific bacteriolysis to 55% after 15 min and to 70% after 45 min, but did not abolish lysis (Fig. 1B).
After incubation with 2.5 μM granulysin132 for 15 min and staining with an anti-His mAb, L. innocua were coated by granulysin132 (Fig. 1C). Granulysin132 bound to bacteria was also detected after treatment at 4°C (data not shown). No binding of actinfrag was detected after treating the Listeria with actinfrag (Fig. 1D). According to these findings, granulysin binds and kills L. innocua highly efficiently within minutes at 37 or 4°C.
Granulysin is actively taken up by DC
To lyse intracellular bacteria, granulysin has to enter the cell either by a passive or an active uptake mechanism. Active protein uptake is a temperature-dependent process; therefore, granulysin uptake was investigated at 37 and 4°C. For this purpose, DC were treated for 45 min at 37°C with granulysin132 or actinfrag, and the localization of the proteins was determined by immunofluorescent staining using an anti-His mAb. CLSM revealed a spot-like pattern of immunolabeled granulysin132 within the DC (Fig. 2A). Granulysin132 distribution was identical in L. innocua-challenged DC (data not shown). After incubation for 45 min at 4°C, granulysin132 was found exclusively at the cell membrane, and no significant transfer to an intracellular compartment occurred (Fig. 2B), indicating an active uptake mechanism. Actinfrag was neither bound nor taken up by DC at detectable levels (Fig. 2C).
FIGURE 2. Granulysin132 is actively taken up by human DC. Unchallenged DC were incubated for 45 min with 2.5 μM granulysin132 at 37°C (A) or 4°C (B) or with 2.5 μM actinfrag at 37°C (C). After the incubation, the cells were fixed and stained with the anti-His Ab for CLSM. Representative phase contrast (right panels) and immunofluorescence images (left panels) are shown. Bar = 8 μm. D, Granulysin132 was pretreated with CAH, BAD, or sodium borate buffer before incubation with DC for 60 min at 4°C. The level of bound granulysin was assessed by Western blotting. Granulysin was detected with the anti-granulysin Ab. As a reference band, cellular actin was detected using an anti-actin mAb. E, The activity of modified granulysin132 against L. innocua in suspension was tested with turbidimetry and calculated from bacterial growth curves.
Ernst et al. (20) demonstrated that modification of arginine residues with BAD reduced the binding as well as the lytic capacity of granulysin against E. coli, whereas neither binding nor lytic capacity of granulysin pretreated with CAH to modify lysine residues was affected. Consistent with these results, pretreatment of granulysin132 with BAD reduced the lytic activity against L. innocua in suspension, whereas CAH or borate buffer alone had no effect (Fig. 2E). Preincubation of granulysin with BAD, but not with CAH, significantly decreased binding of granulysin132 to DC cell membranes, as assessed by Western blotting (Fig. 2D). Similar to binding, uptake of granulysin132 at 37°C was not affected by modification of lysine residues (not shown).
Granulysin binding and uptake in DC are associated with lipid rafts
The spot-like staining pattern of granulysin132 within DC resembled vesicles of the endocytic pathway. Because granulysin belongs to the SAPLIP, which has known affinity to sphingolipids (5), a binding and initial uptake mechanism associated with lipid rafts seemed likely. Lipid rafts are highly organized microdomains in the plasma membrane with elevated cholesterol and glycosphingolipid contents (22). The subunit of cholera toxin binds the Gm1 ganglioside in lipid rafts and is a well-established marker to detect such microdomains (38). To examine possible association of granulysin binding and uptake in DC via lipid rafts, co-uptake experiments with fluorescently labeled cholera toxin and granulysin were performed. After incubation of granulysin132 with cholera toxin for 30 min in steady state at 37°C, both proteins were found colocalized in DC (Fig. 3A, see colocalization panel). The colocalization of cholera toxin and granulysin132 was most significant in the peripheral part of the cells. Cholera toxin was concentrated over time in the perinuclear area of the DC, whereas granulysin132 remained in the peripheral compartment. No uptake of the proteins occurred at 4°C (Fig. 3B). Under these conditions granulysin132 and cholera toxin were colocalized and remained in a patch-like pattern at the cell membrane. To exclude an interaction of the His-tag of granulysin132 with the cell membrane, especially with lipid rafts, DC were coincubated with granulysin145 after His-tag removal and cholera toxin at 37 and 4°C (Fig. 3, C and D). After fixation, the samples were stained with the anti-granulysin Ab. We found a staining pattern identical with that achieved with granulysin132, proving that the His-tag did not influence granulysin binding or uptake in DC. The assumption of initial co-uptake and later separation of granulysin132 and cholera toxin was confirmed in pulse-chase experiments. Granulysin132 and cholera toxin were allowed to bind to DC on ice for 10 min, and subsequent to medium replacement, cells were incubated at 37°C for 5–90 min (Fig. 3E). After 5 min, both proteins were colocalized, either still bound to the cell membrane or in the peripheral compartments of the cells. Thirty minutes after binding to the cell membrane, granulysin132 and cholera toxin had separated into different compartments. After 90 min, cholera toxin was found highly concentrated near the nucleus and was totally separated from granulysin132, which remained in the peripheral compartment of the cells (see colocalization panels in Fig. 3F). The association of granulysin with lipid rafts in DC was confirmed by staining of granulysin132-treated DC with an anti-CD55 Ab and an anti-granulysin Ab (Fig. 3G). To test possible involvement of macropinocytosis or clathrin-dependent endocytosis in granulysin132 uptake, dextran-FITC and transferrin-FITC, respectively, were incubated with granulysin132. In neither the initial uptake nor in the later stages was dextran (Fig. 4C) or transferrin (data not shown) colocalized with granulysin132.
FIGURE 3. Granulysin binding and uptake in DC are associated with lipid rafts. L. innocua-challenged DC were coincubated with granulysin (2.5 μM; granulysin132 (A and B) or granulysin145 without His-tag (C and D)) and FITC-labeled cholera toxin (CT; 10 μg/ml) for 30 min at 37°C (A and C) or 4°C (B and D). E and F, For pulse-chase experiments, DC were pulsed with granulysin132 and cholera toxin for 10 min at 4°C and subsequently cultivated at 37°C for the indicated chase periods. Granulysin132 is marked in red, cholera toxin is shown in green, and nuclear and bacterial DNA are stained with DAPI (blue; merged image (E), calculated colocalization (F)). G, Double labeling of granulysin-treated DC with an anti-CD55 Ab and the anti-granulysin Ab. Granulysin132 is marked in red, and CD55 is shown in green. Colocalization images of granulysin132 and cholera toxin or CD55 were calculated using Imaris software. Bars = 8 μm.
FIGURE 4. Cholesterol depletion prevents granulysin132 uptake, but not binding. DC were pretreated with 20 mM MCD for 45 min before incubation with granulysin132 (2.5 μM) and dextran-FITC (1 mg/ml) for 45 min at 37°C (A) or 4°C (B). C, Control cells were pretreated with serum-free medium for 45 min before incubation with granulysin132 and dextran-FITC for 45 min at 37°C. Bars = 8 μm.
Preincubation of DC with MCD, which is known to disrupt lipid raft-mediated uptake by binding cholesterol (39), inhibited the uptake, but not the binding, of granulysin132 in DC. After incubation of DC with MCD for 45 min at 37°C, granulysin132 was detected in patches at the cell membrane (Fig. 4A). In contrast, simultaneously applied dextran-FITC was endocytosed by cholesterol-depleted cells at 37°C, indicating that the fluid phase endocytosis was still functional. After incubation of cholesterol-depleted cells at 4°C, both granulysin132 and dextran-FITC were found at the cell membrane, but no intracellular staining was detected (Fig. 4B). In control cells pretreated with serum-free medium, uptake of both granulysin132 and dextran-FITC was detected at 37°C (Fig. 4C). Similar results were obtained by pretreatment of DC with filipin (1 μg/ml), another lipid raft-disrupting drug (38), before granulysin132 incubation (not shown). Overall, these findings revealed lipid rafts to be critically involved in initial uptake, but not binding of granulysin to DC.
Granulysin was found in early endosomes, but not in lysosomes
To follow trafficking of granulysin132 from lipid rafts, double labeling of granulysin132 with markers specific for the endocytic compartment was performed. In a first step, early endosomes were stained with a polyclonal Ab recognizing the EEA-1. Granulysin132 was colocalized with the EEA-1 (Fig. 5A) in L. innocua-challenged DC after 10 min of incubation. After 60 min, the longest time analyzed for EEA-1 colocalization, granulysin132 still resided in endosomes.
FIGURE 5. Granulysin132 is localized in early endosomes and Listeria-containing phagosomes, but not in lysosomes. A, L. innocua-challenged DC were incubated with 2.5 μM granulysin132 for 30 min at 37°C for colocalization with EEA-1. B, For double staining of granulysin132 and LAMP-1, cells were treated for 90 min with 2.5 μM granulysin132 at 37°C, then stained with a polyclonal anti-granulysin Ab and a polyclonal Ab recognizing EEA-1 or an anti-LAMP-1 mAb, respectively. Nuclear and bacterial DNA was labeled with DAPI (blue); early endosomes and lysosomes, respectively, are marked in green; and granulysin132 is depicted in red. The yellow spots in the merge image (A) indicate colocalization of granulysin132 with EEA-1. Colocalization of the green and red channels was additionally calculated using Imaris software. Arrowheads in B indicate listerial DNA spots that colocalize with LAMP-1, whereas arrows mark DNA spots that coincide with granulysin132. Bar = 8 μm.
Immunostaining of L. innocua-challenged and granulysin132-treated DC with an Ab recognizing LAMP-1 revealed that granulysin132 clearly separated from the LAMP-1-positive compartment (Fig. 5B). DAPI-stained listerial DNA was found in the lysosomal, LAMP-1-positive compartment (arrowheads in Fig. 5B) or in phagosomes, which showed a distinct labeling for granulysin132 (arrows in Fig. 5B). Positive staining of L. innocua for granulysin132 remained detectable even when bacteria were isolated from granulysin132-treated DC (data not shown). Overall, these results indicate that after uptake, granulysin is present in early sorting endosomes of DC and then transferred to phagosomes, where granulysin is able to bind Listeria.
Granulysin mediates lysis of L. innocua in DC
L. innocua-challenged DC were incubated with various concentrations of granulysin132 or granulysin145 without His-tag for 3 h at 37°C. The viability of the intracellular bacteria was tested in CFU assays (Fig. 6A). Granulysin132 (5 μM) killed >41%, and 5 μM granulysin145 killed >46% of the intracellular L. innocua after 3 h. Granulysin132 (2.5 μM) was sufficient to lyse 29% of the intracellular Listeria, very similar to 2.5 μM granulysin145, which killed 26%. Viability reduction of bacteria was strictly dependent on granulysin132 dosage and did not occur with actinfrag treatment. Incubation of DC with granulysin132 at 4°C for 3 h did not impair the viability of intracellular L. innocua (data not shown). To exclude bacteria loss due to detachment of perishing host cells, the viability of DC after granulysin132 incubation was assessed. Granulysin132 incubated in a concentration up to 5 μM had no effect on the viability of L. innocua-challenged DC monitored in lactate dehydrogenase release and thiazolyl blue (MTT) assays (data not shown).
FIGURE 6. Granulysin132 mediates lysis of L. innocua in DC. L. innocua-challenged DCs were incubated for 3 h with granulysin132 in varying concentrations (A) or with 2.5 μM granulysin132 for the indicated time periods at 37°C (B). After granulysin132 incubation, cells were lysed in ice-cold water, and Listeria viability was assessed in CFU assays. The mean ± SD of four independent experiments are presented. C, Deconvoluted CLSM image of intracellular bacteria found in granulysin-treated DC. Bacterial DNA (red) is stained with DAPI, and granulysin132 (green) is labeled with an anti-His Ab. Bar = 2 μm. D, L. innocua-challenged DC were cholesterol-depleted for 45 min with the indicated concentrations of MCD. Subsequently, DC were incubated with 2.5 μM granulysin132 for 3 h at 37°C. After the incubation, cells were lysed, and bacterial viability was assessed in CFU assays. The mean ± SE of three independent experiments are presented. Statistical significance was calculated with Student’s t test for paired samples.
Compared with the fast lysis of extracellular L. innocua, intracellular bacterial killing was delayed. Forty-five minutes of incubation at 37°C with 2.5 μM granulysin132 was required to kill 12% of the intracellular bacteria (Fig. 6B). Intracellular lysis increased to 25% after 90 min of treatment, but never reached the same level as in suspension (see Fig. 1B). The maximal reduction of 31% of the intracellular bacteria was achieved after 3 h of incubation with 2.5 μM granulysin132. Deconvoluted high resolution CLSM images of L. innocua within granulysin132-treated DC demonstrated bacterial DNA that was clearly surrounded by granulysin132 labeling (Fig. 6C). Granulysin132-mediated lysis of intracellular L. innocua was reduced after cholesterol depletion of DC by MCD in a dose-dependent manner (Fig. 6D). Preincubation of DC with 10 mM MCD diminished lysis of intracellular L. innocua to 13%, and only 4% of specific lysis was achieved after pretreatment with 20 mM MCD.
Discussion
In this study we could clearly demonstrate that recombinant granulysin was binding and dose-dependently killing L. innocua as free bacteria grown in suspension. Granulysin-induced lysis of Listeria is in agreement with data obtained by other groups using a variety of microbial pathogens, including L. monocytogenes (8, 20). To date, the binding of granulysin to bacteria has never been directly shown, but can be explained by properties of the SAPLIP family, of which granulysin is a member. SAPLIPs interact with a variety of lipids (14). Bacterial membranes contain mainly acidic phospholipids, such as phosphatidylglycerol and cardiolipin. Positive charges of granulysin (net charge, +11) can be assumed to be the driving force for binding to negatively charged bacterial membranes. Charges in granulysin are not homogeneously distributed, but are slightly polarized toward a region of the molecule corresponding to helix 3 (19). This region was proposed as the site of the initial contact of granulysin to bacterial membranes. After binding, granulysin is suggested to cluster at the bacterial membrane, leading to deformation and, subsequently, lysis by friction of adjacent granulysin molecules.
To reach intracellular bacteria, granulysin has to bind and enter mammalian cells that have apparent differences in the composition of membranes to prokaryotes. The outer leaflet of mammalian cells is mainly composed of zwitterionic phospholipids, such as phosphatidylcholine and sphingomyelin. Cholesterol is abundant in mammalian cells, but is absent in bacterial cell membranes. A third difference is the higher transmembrane potential of prokaryotic cells (40, 41, 42, 43).
Binding of granulysin to mammalian cell membranes can again be explained by properties of the SAPLIP family. The interaction of saposins with sphingolipids has been extensively investigated. Dependent on the pH value, all saposins were reported to bind the negatively charged gangliosides, which are found enriched together with cholesterol in lipid rafts. In our study we could show by modifying arginine residues of granulysin that positive charges of arginine residues contribute crucially to the lytic activity against L. innocua as well as to the binding capacity to DC membranes. Lysine residues seem not to play an important role in lysis of bacteria and binding to eukaryotic cells. This is consistent with the finding that covering the granulysin arginine residues by butanedione reduced binding and lysis of E. coli (20). Granulysin was found bound to lipid rafts, as indicated by its colocalization with cholera toxin and CD55, both known markers for lipid rafts (25, 38, 44). Preincubation of cells with MCD or filipin, which are both known to deplete cholesterol (45), did not abolish binding of granulysin to lipid rafts. Mechanisms of granulysin binding to lipid rafts or even binding partners are still an enigma, but it is known to be cholesterol independent. Clustering of granulysin at ganglioside-rich regions of the plasma membrane could apply local shear stress that might trigger budding and subsequently endocytosis of granulysin-containing membrane vesicles. There is some evidence that local deformation and membrane tension contribute to the regulation of endocytosis (46, 47).
In contrast to binding, uptake of granulysin could be inhibited by cholesterol depletion, but also by incubation at 4°C. Inhibition of uptake at 4°C is a strong indication for an active process (48). Endocytosis of granulysin via the lipid raft/caveolae pathway is distinguished from both the clathrin-dependent and constitutive pinocytic pathways by its sensitivity to cholesterol depletion. In line with this observation is that dextran or transferrin as tracers for constitutive pinocytosis and clathrin-dependent pathway, respectively, were never found colocalized with simultaneously applied granulysin. After endocytosis, granulysin was found to be sorted differently from cholera toxin (49). Additional intracellular trafficking of granulysin from early endosomes may result in fusion of endosomes with phagosomes where granulysin was found to be colocalized with bacteria. Fusion of granulysin-containing early endosomes with phagosomes may be regulated by the small rab5 GTPase, which has been shown to be directly correlated with an accelerated maturation of Listeria-containing phagosomes (50).
In our model of L. innocua-challenged human DC binding, uptake and trafficking of granulysin to phagosomes as well as binding to and lysis of L. innocua were shown to occur in the absence of perforin. In contrast to these results, it was shown that M. tuberculosis was efficiently lysed in vitro in human DC by granulysin only in combination with perforin (8, 20). This combination of perforin and granulysin to achieve antibacterial activity against intracellular pathogens seems necessary at least occasionally. NKT cells and CTL could control mycobacterial infection by relying on granulysin, but not on perforin or Fas/Fas ligand interaction (11, 29). In vivo, it was found that perforin-negative, but granulysin-positive, CD4+ cells are present at sites of bacterial infections (12, 13). This is in line with our in vitro data for granulysin activity. However, data are largely missing about the exact role of perforin in lysis of intracellular bacteria, e.g., M. tuberculosis. Data on perforin-dependent as well as independent uptake and activity of granulysin are not conclusive, and mechanisms of entry into host cells are poorly characterized. Perforin might well have a role in granulysin trafficking to compartments harboring intracellular bacteria. In contrast to apathogenic L. innocua pathogenic species such as M. tuberculosis, Chlamydia spp. and Coxiella spp. have evolved strategies to modify their intracellular compartments (51, 52) or are known to escape from vesicular compartments to the cytosol-like L. monocytogenes (53). Mycobactera, for example, can stop phagosome maturation by inhibiting acquisition of the rab5 effector EEA-1. By excluding this important regulator for vesicular trafficking, fusion processes within the endocytic pathway are impaired (54).
Overall, we propose a model of granulysin-mediated activity against intravacuolar bacteria. According to this model, binding due to electrostatic interactions and clustering of granulysin at membrane microdomains with elevated acidic sphingolipid content would lead to endocytosis as a first step toward lysis of intracellular bacteria. Such lipid raft microdomains were indeed found concentrated in the regions of the immunological synapse (55). After cholesterol-dependent uptake, granulysin is delivered to early sorting endosomes, fusing later with bacteria-harboring phagosomes, where the lysis of the bacteria is induced. Simultaneously secreted IFN- at sites of infection by the CTL would enhance granulysin targeting. There is evidence that activated macrophages up-regulate rab5 in response to IFN- (56). An elevated level of rab5 accelerates not only maturation of Listeria-containing phagosomes (50), but also the likelihood for phagosome-endosome fusion. Thus, the antimicrobial activity mediated by granulysin would be enhanced.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We are very grateful to Marina Balzer and Gery Barmettler for excellent technical assistance, and to Rene Moser, Sonja Latinovic, and Caroline Maake for helpful support. We thank Markus Schneemann and Gabriele Schoedon (Medical Clinic B Research Unit, Department of Medicine, University Hospital of Zurich) for providing L. innocua. We also thank Jack Rohrer (Institute of Physiology, University of Zurich) for kindly providing the EEA-1 as well as the LAMP-1 Abs.
Footnotes
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 microscopy; DAPI, 4,6-diamidine-2-phenyl-indol-dihydrochloride; DC, dendritic cell; EEA-1, early endosomal Ag 1; His-tag, hexahistidine tag; LAMP-1, lysosomal-associated membrane protein 1; MCD, -methyl-cyclodextrin; TSB, tryptic soy broth.
Received for publication July 15, 2004. Accepted for publication January 26, 2005.
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