INFECTION AND IMMUNITY,0019-9567/00/$04.0010

Dec. 2000, p. 6903–6911 Vol. 68, No. 12

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Cellular Internalization of Cytolethal Distending Toxinfrom Haemophilus ducreyi

XIMENA CORTES-BRATTI,1 ESTEBAN CHAVES-OLARTE,1 TERESA LAGERGÅRD,2

AND MONICA THELESTAM1*

Microbiology and Tumorbiology Center, Karolinska Institutet, S-171 77 Stockholm,1 and Department ofMedical Microbiology and Immunology, University of Gothenburg, S-413 46 Gothenburg,2 Sweden

Received 8 June 2000/Returned for modification 28 August 2000/Accepted 9 September 2000

The chancroid bacterium Haemophilus ducreyi produces a toxin (HdCDT) which is a member of the recentlydiscovered family of cytolethal distending toxins (CDTs). These protein toxins prevent the cyclin-dependentkinase cdc2 from being activated, thus blocking the transition of cells from the G2 phase into mitosis, with theconsequent arrest of intoxicated cells in G2. It is not known whether these toxins act by signaling from the cellsurface or intracellularly only. Here we report that HdCDT has to undergo at least internalization before beingable to act. Cellular intoxication was inhibited (i) by removal of clathrin coats via K1 depletion, (ii) by treat-ment with drugs that inhibit receptor clustering into coated pits, and (iii) in cells genetically manipulated tofail in clathrin-dependent endocytosis. Intoxication was also completely inhibited in cells treated with bafilo-mycin A1 or nocodazole and in cells incubated at 18°C, i.e., under conditions known to block the fusion of early en-dosomes with downstream compartments. Moreover, disruption of the Golgi complex by treatment with bre-feldin A or ilimaquinone blocked intoxication. In conclusion, our data indicate that HdCDT enters cells via clathrin-coated pits and has to be transported via the Golgi complex in order to intoxicate cells. This is the first memberof the family of CDTs for which cellular internalization and some details of the pathway have been demonstrated.

Haemophilus ducreyi is a gram-negative organism responsi-ble for chancroid (soft chancre, ulcus molle), a sexually trans-mitted disease characterized by painful, slowly healing genitalulcers and swollen regional lymph nodes (40). Renewed atten-tion has been focused on H. ducreyi, since human immunode-ficiency virus transmission in developing countries has beenassociated with genital ulceration (40). The general pathogenicmechanisms and immunity in chancroid, in particular, the vir-ulence factors responsible for ulceration, remain unclear. Thechancroid bacterium produces a toxin (HdCDT) which belongsto the recently discovered family of cytolethal distending toxins(CDTs) elaborated by various species of gram-negative bacte-ria (24). Three linked genes, cdtABC, encode these toxins,which are composed of the A, B, and C proteins, with molec-ular masses of 26, 30, and 20 kDa, respectively. Expression ofall three genes is needed to create an active toxin, but it is notclear whether the mature (active) holotoxin contains one, two,or all three components. The cdtABC genes from different speciesare related to each other. The highest sequence homology(.95%) was noted between HdCDT and the corresponding Cd-tABC proteins from Actinobacillus actinomycetemcomitans (33),whereas homology to Escherichia coli CDTs is much lower (5).Among the three components, CdtB is the most conserved, with49% identity even between the most distantly related CDTs (24).

All the CDTs known to date induce the same cytotoxic effectin sensitive epithelial target cells: an irreversible arrest in theG2 phase of the cell cycle (4, 6, 45). This effect is accompaniedby a conspicuous enlargement of most cells (24), but T lym-phocytes are an exception (33). The earliest toxin-induced bio-chemical alteration identified so far is tyrosine phosphorylationof the cyclin-dependent kinase cdc2 (4), which was detectablewithin 6 h in cells intoxicated with HdCDT (6). As a result of

tyrosine phosphorylation, this kinase remains inactive and thusis unable to promote the transition of cells from the G2 phaseinto mitosis. The molecular events responsible for this effecthave not yet been clarified for any of the CDTs. This effectresembles the so-called G2 checkpoint response (44), known tooccur after DNA damage induced by certain chemicals orexposure to ionizing radiation. However, no DNA damage wasdetectable in CDT-treated cells prior to tyrosine phosphoryla-tion of cdc2, when measured as DNA synthesis (4, 6) or thepresence of DNA strand breaks (32). The CDTs, particularlyHdCDT, are extremely potent and might enzymatically affectone of the many components involved in the regulation of cdc2activity. However, neither the putative enzymatic activity northe molecular substrate has yet been defined.

Despite these uncertainties, it is reasonable to assume thatthe primary cellular target of HdCDT and the other CDTs islocated intracellularly and that these toxins therefore have toenter cells before being able to exert their effect. Most bacte-rial protein toxins known to act on intracellular targets havebeen shown to enter cells by endocytosis (17). However, forinstance, the heat-stable enterotoxin STa of E. coli induces itsintracellular effect by triggering a transmembrane signal fromoutside the cells (42), and a similar signaling action is a pos-sibility for the CDTs as well. Thus, the aim of the present workwas to determine if cellular internalization of HdCDT is re-quired for its cytotoxic action and, if so, to clarify the majorevents in the internalization pathway. The general experimen-tal approach was to test whether the toxin would be able tointoxicate cells in which the endocytic pathway was blocked atdefined stages.

MATERIALS AND METHODS

Materials. HdCDT was purified from H. ducreyi by immunoaffinity chroma-tography using the neutralizing monoclonal antibody (MAb) M4D4 (26; A. Frisk,M. Lebens, H. Ahmed, C. Johansson, L. Svensson, K. Ahlman, and T. Lagergård,unpublished data). Immunoblotting with specific MAbs (Frisk et al., unpub-lished) showed that the toxin preparation contained both the B and the Ccomponents, while the A component was not detectable. The protein concen-

* Corresponding author. Mailing address: Microbiology and Tumor-biology Center (MTC), Karolinska Institutet, Box 280, S-171 77 Stock-holm, Sweden. Phone: 46-8-728 71 62. Fax: 46-8-33 15 47. E-mail:monica.thelestam@mtc.ki.se.

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tration in the stock solution of this partially purified preparation was 25 mg/ml.A rabbit antiserum reacting with the B and C components was prepared againstthis toxin preparation. L-[3H]leucine (specific activity, 40 to 60 Ci/mmol) wasobtained from NEN Life Science Products (Boston, Mass.). Other reagents wereobtained from Sigma Aldrich Sweden AB unless otherwise stated.

Cell culture and toxin treatment. Human epidermoid larynx carcinoma cells(HEp-2; ATCC no. CCL-23) were cultivated in Eagle’s minimum essential me-dium (MEM) supplemented with 10% fetal bovine serum, 5 mM L-glutamine,penicillin (100 U/ml), and streptomycin (100 mg/ml) in a humid atmospherecontaining 5% CO2. Human cervix carcinoma cells transfected with dominant-negative dynamin (HeLadynK44A) (kindly provided by K. Sandvig, Institute forCancer Research, Montebello, Oslo, Norway) were cultivated in Dulbecco’smodified Eagle’s medium with the same supplements as HEp-2 cells plus 400 mgof Geneticin per ml, 200 ng of puromycin per ml, and no tetracycline or 1 mg oftetracycline per ml. Exponentially growing cells (in 96- or 24-well plates) werecooled on ice for 15 min before the addition of ice-cold toxin (25 ng/ml unlessotherwise stated). After toxin exposure for 15 min at 0°C, the cells were washedthree times with cold Hanks’ balanced salt solution (HBSS). Eagle’s MEM wasadded, and the cells were further incubated at 37°C. Intoxication was scored asthe accumulation of cells in the G2/M phase by flow cytometry or as tyrosinephosphorylation of cdc2 as described below. These two parameters of intoxica-tion were always in agreement.

Cell cycle analysis by flow cytometry. Cells growing in 25-cm2 culture flasks(Costar, Cambridge, Mass.) were treated with 25 ng of HdCDT per ml, incubatedfor various times, rinsed with HBSS, scraped off (Cell Scraper; Nunc, Naperville,Ill.), and processed for flow cytometric analysis of the DNA content as previouslydescribed (6). The data from 104 cells were collected with a FACSort flowcytometer (Becton Dickinson) and analyzed using CellQuest software (BectonDickinson).

Western blotting. Cells were lysed in 300 ml of sodium dodecyl sulfate (SDS)electrophoresis sample buffer, and samples were boiled for 10 min. The proteinswere separated on SDS–10% polyacrylamide gels, transferred to polyvinylidenediflouride membranes (Millipore), and probed with antiphosphotyrosine (Up-state Biotechnology) or anti-cdc2 or anti dynamin (Transduction Laboratories,Lexington, Ky.) antibodies. Blots were developed with an ECL Western blottinganalysis kit (Amersham Pharmacia) using a peroxidase-labeled secondary anti-body. The amount of protein in the lysates was determined by the Bio-Radprotein assay using bovine serum albumin as a standard; 25 to 30 mg was loadedper lane.

Quantification of tyrosine phosphorylation. Western blots were scanned, anddensitometric analysis of tyrosine-phosphorylated cdc2 bands was performedusing Image Quant software (Molecular Dynamics). Each experiment comprisedfour lanes: (i) a control without toxin or drug (C), (ii) a positive control with toxinonly (T), (iii) the actual test with toxin and drug (TD), and (iv) a control withdrug only (D). After densitometer scanning of these four bands (using a PersonalDensitometer from Molecular Dynamics), the value for C was subtracted fromthat for T, and the value for D was subtracted from that for TD. The value forTD 2 D was divided by that for T 2 C, and the resulting ratio was multiplied by100. This final value indicates the cdc2 tyrosine phosphorylation in the test, givenas a percentage of the cdc2 tyrosine phosphorylation in the positive control.

Treatment with proteases. HEp-2 cells were exposed to HdCDT (15 min, 0°C),washed three times with HBSS, and treated with trypsin (2.5 mg/ml) or proteinase K(100 mg/ml) for 9 min at 37°C. The detached cells were seeded in a new well withnormal medium and incubated for 24 h. Control cultures were treated (15 min, 0°C)with toxin (2.5 mg/ml) that had been preincubated for 9 min at 37°C with trypsin orproteinase K and further diluted 1/100 in normal medium to give a final concentra-tion of 25 ng/ml. Samples for Western blotting were taken after 24 h.

Treatment with antibodies. (i) Neutralization before addition of toxin toHEp-2 cells. HdCDT was incubated for 30 min at 37°C with antibody diluted1/100. Cells were treated with the toxin-antibody mixture (15 min, 0°C) andwashed three times with HBSS. Cells were then incubated for 24 h at 37°C infresh medium.

(ii) Neutralization of cell-bound toxin. HEp-2 cells were exposed to HdCDT(15 min, 0°C) and washed three times with HBSS. Cells were then incubated at0°C in medium with antibody diluted 1/100. After 15 min, the temperature wasraised to 37°C; 30 min later, the antibody was removed and incubation at 37°C infresh medium was continued for 24 h.

Potassium depletion. HEp-2 cells were cultivated in six-well plates, washedtwo times with HEPES buffer (pH 7.4) with 100 mM NaCl, and hypotonicallyshocked for 5 min at 37°C with K1 depletion buffer (140 mM NaCl, 2 mM CaCl2,1 mg of glucose per ml, 25 mM HEPES) diluted 1:1 in water. The cells wereincubated in isotonic K1-free medium (50 mM HEPES, 100 mM NaCl, 2 mMCaCl2, 1 mg of glucose per ml) for 1 h and then treated with HdCDT in the samemedium. After the toxin treatment, the cells were incubated without K1 for 1 hand then in normal medium for 24 h.

18°C treatment. Cells were exposed to HdCDT (15 min, 0°C) and incubated for15 min at 37°C to permit endocytic uptake into early endosomes. Thereafter, thecells were incubated at 18°C for 12 h or at 18°C for 12 h followed by 37°C for 12 h.

Treatment with drugs. Cells were preincubated in medium with a drug, ex-posed to HdCDT (15 min, 0°C), and postincubated in the presence of the drug.To exclude the possibility that the drug inhibited the binding of the toxin, thecells in each situation were treated with the toxin plus the drug and postincu-

bated in drug-free medium. Cells were preincubated with various concentrationsof filipin, chlorpromazine, imipramine, and ilimaquinone for 1 h at 37°C; withbrefeldin A (BFA) for 45 min at 37°C; and with nocodazole for 30 min at 4°C.Preincubation with all other drugs was done for 30 min at 37°C. Treatments withilimaquinone and filipin were performed with serum-free medium.

DT treatment. Cells were incubated with 5 mg of diphtheria toxin (DT) per mlfor 30 min at 37°C, washed three times with HBSS, and incubated in normalmedium for 1.5 h. Cells were then incubated with [H3]leucine (1 mCi/ml) inleucine-free medium for 1 h at 37°C. After trypsinization, the cells were treatedfor 30 min at 4°C with trichloroacetic acid (final concentration, 10%). Themacromolecular fraction was precipitated by centrifugation (13,000 rpm in aBiofuge 13 [Heraeus] for 10 min), washed in 10% trichloroacetic acid, anddissolved in 2% Na2CO3–1% SDS–0.1 M NaOH, and 3 ml of scintillation liquid(OptiPhase HiSafe; Fisons Chemicals) was added. Sample counts were deter-mined with a liquid scintillation counter (LKB Wallac).

RESULTS

Cell surface-bound HdCDT is not accessible to activationwith proteases or antibodies. Preincubation of HdCDT withtrypsin or proteinase K before addition to cells inactivated thetoxin. However, when the cell surface-bound toxin was treatedunder the same conditions (9 min at 37°C) with the sameconcentrations of trypsin or proteinase K, the cells were stillstrongly intoxicated, as measured by cdc2 tyrosine phosphory-lation. A similar result was found with the polyclonal antiserumagainst the toxin. Incubation of HdCDT with the antiserum for30 min at 37°C before the binding step neutralized the toxin,whereas antiserum added directly after the binding step wasno longer able to neutralize the toxic effect. These findingssuggested that after binding to the cell surface and upon ele-vation of the temperature, the toxin either is conformationallychanged to become resistant to these treatments or is rapidlyinternalized into a compartment where it is no longer accessi-ble to proteases or antibodies.

HdCDT is internalized by endocytosis. Cells treated withammonium chloride or methylamine were partially protectedagainst intoxication by HdCDT (Fig. 1), cdc2 tyrosine phos-phorylation being reduced to 33.9% 6 13% and 74.0% 6 7%by ammonium chloride and methylamine, respectively. Upontreatment with monensin, the toxic effect was inhibited, as shownby the lack of tyrosine phosphorylation of cdc2 (Fig. 1) andflow cytometric analysis (Fig. 2). There was no evident accu-mulation of cells in the G2 phase 12 h after toxin exposure, incontrast to the results for toxin-treated control cells withoutmonensin. None of these drugs blocked the cell surface bind-ing of the toxin per se, as tested both by preincubating thecells with a drug and by allowing its presence during, but notafter, the toxin-binding step (data not shown). In conclusion,HdCDT appears to require passage via an intracellular low-pHcompartment in order to intoxicate cells with full efficiency,suggesting that the toxin is internalized by endocytosis.

HdCDT is endocytosed via clathrin-coated pits. We testedthe hypothesis that endocytosis of HdCDT takes place viaclathrin-coated pits. Three different experimental approacheswere used: (i) removal of clathrin coats by K1 depletion, (ii)treatment with drugs known to inhibit receptor clustering incoated pits, and (iii) use of a cell line genetically manipulatedto fail in endocytosis via clathrin-coated pits.

(i) Upon removal of clathrin coats by exposure of HEp-2cells to a brief hypotonic shock followed by K1 depletion (20),there was a 40 to 80% reduction of the HdCDT effect (Fig. 3).DT was used as a positive control, since it has been reported toenter cells via clathrin-coated pits (20, 35). The reduction ofthe toxic effect of DT in potassium-depleted cells, measured asinhibition of protein synthesis (Fig. 3, inset), was quantitativelysimilar to the reduction of the HdCDT effect.

(ii) To prevent the assembly of coated pits at the cell surface,HEp-2 cells were exposed to the cationic amphiphilic drug

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chlorpromazine (38) before and after the 15-min HdCDT-treatment. Intoxication in such treated cells was decreased by80% (Fig. 3). A similar decrease resulted after treatment withimipramine, which belongs to the same class of drugs as chlor-promazine (data not shown).

(iii) HeLadynK44A cells were treated with HdCDT. Cells cul-tivated in medium with tetracycline, allowing the expressiononly of endogenous dynamin, were fully sensitive to the toxin,as shown by tyrosine phosphorylation of cdc2 (Fig. 4). In theabsence of tetracycline, these cells overexpress the mutantdynamin, and the clathrin-dependent pathway is inhibited (7).In this situation, the toxic effect of HdCDT was prevented (Fig.4).

The cholesterol-binding drug filipin has been shown to pre-vent the uptake of toxins via caveolae (23). However, treat-

ment of HEp-2 cells with filipin (5 mg/ml) for 1 h before andduring the 15-min toxin-binding step, as well as for 12 h afterbinding, did not affect intoxication, as shown by tyrosine phos-phorylation of cdc2 (data not shown). This observation sug-gests that uptake via caveolae is not an entry mechanismused by HdCDT.

HdCDT requires transport from early endosomes to down-stream vesicular compartments. The next few experimentswere designed to clarify whether the toxin can be translocatedto the cytosol directly from early endosomes or needs to betransported to some downstream compartment(s) before beingable to act. (i) Bafilomycin A1 (BafA1) is a specific inhibitor ofvacuolar proton ATPases and is known to raise the vesicularpH and block protein transport from early to late endosomes(1). Pretreatment of HEp-2 cells with BafA1 completely inhib-

FIG. 1. Effects on HdCDT-induced intoxication of drugs which inhibit endosomal acidification. Cells were preincubated with methylamine (10 mM), NH4Cl (20mM), or monensin (10 mM), treated with the toxin for 15 min, and postincubated for 12 h in the presence of the respective drug. Tyrosine phosphorylation of cdc2in the treated cells was determined by Western blotting as described in Materials and Methods. Blots are representative of two different experiments with each drug.

FIG. 2. Effect of monensin on HdCDT-induced intoxication. Flow cytometry was used to analyze control cells [HdCDT(2)] and toxin-treated cells [HdCDT (1)]in the presence (1) or absence (2) of monensin (10 mM). Samples were prepared 12 h after toxin treatment, and DNA was stained with propidium iodide. The G1and G2/M regions were marked, and the percentages of cells in these phases are shown. One representative experiment of two is shown. FL3-H, relative fluorescence.

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ited the intoxication induced by HdCDT (Fig. 5A). (ii) Themicrotubule-disrupting agent nocodazole is known to block thefusion of endosomal carrier vesicles with downstream compart-ments, such as late endosomes, lysosomes, or the Golgi com-plex (1). We observed that nocodazole treatment of HEp-2cells almost completely inhibited HdCDT-induced intoxica-tion, whereas its addition 1 h after toxin exposure could notprevent the intoxication (Fig. 5B). (iii) Incubation of cells at18°C is another treatment known to block the transport ofproteins from endosomes to lysosomes and/or the Golgi com-plex (28). After the toxin-binding step (15 min, 0°C), followedby a 15-min incubation at 37°C to allow initial endocytic up-take, the cells were incubated for 12 h at 18°C. This treatmentstrongly reduced the intoxication scored as tyrosine phosphor-ylation of cdc2 (Fig. 5C). After incubation at 18°C, a parallelset of similarly treated cells was incubated for another 12 h at37°C. This shift in temperature resulted in complete intoxica-tion compared to the results obtained for control cells incu-bated at 37°C for the full 24 h (Fig. 5D). These results dem-onstrate that HdCDT was unable to act when trapped in earlyendosomes.

An intact Golgi complex is required for intoxication. BFA isa drug well known for its ability to disrupt the Golgi complex(2). When HEp-2 cells were treated with BFA before and aftertoxin exposure, intoxication was completely inhibited, asscored by flow cytometric analysis after 6, 8, and 12 h (Fig. 6Ato C). Since BFA has been reported to interfere with the cellcycle as well (19), an additional experiment was performed toensure that the inhibition by this drug was actually due todisruption of the Golgi complex. BFA was added only 1 h aftertoxin exposure; in this situation, it did not interfere with intox-ication (Fig. 6D), implying that internalization was the stepprevented by BFA, as shown in Fig. 6A to C. The protectiveeffect of BFA was also determined as a lack of tyrosine phos-phorylation of cdc2 (Fig. 7). Similar results were obtained aftertreatment of the cells with ilimaquinone (Fig. 8), another Golgi

complex-disrupting drug (39). Interestingly, after the removalof BFA from toxin-treated cells, intoxication did developwithin 12 h (data not shown). Thus, the endocytosed toxinremained active in some compartment that could fuse withGolgi vesicles only upon restoration of the Golgi complex.Taken together with the finding that HdCDT could not actwhen trapped in endosomes, these observations strongly sug-gest that the toxin is transported to the Golgi complex anddelivered to the cytosol either from there or from the endo-plasmic reticulum (ER).

DISCUSSION

Like all macromolecules having intracellular sites of action,protein toxins acting intracellularly have to cross the plasmamembrane at some stage before being able to attack theirtargets. The known pathways for receptor-mediated uptake ofphysiologically important macromolecules are endocytosis viaclathrin-coated pits or clathrin-independent endocytosis, for

FIG. 3. Effect of K1 depletion and chlorpromazine treatment on tyrosine phosphorylation of cdc2 in HdCDT-treated cells. Cells were depleted of K1 as describedin Materials and Methods and treated with HdCDT; samples were prepared 24 h after toxin exposure. For chlorpromazine treatment, cells were preincubated withchlorpromazine (25 mg/ml, 1 h), treated with HdCDT, and postincubated for 8 h with 10 mg of the drug per ml. Intoxication with DT and measurement of DT activity(inset) were performed as described in Materials and Methods. Error bars indicate standard deviations.

FIG. 4. Effect of HdCDT on HeLadynK44A cells. Tyrosine phosphorylation ofcdc2 after HdCDT treatment was measured in cells expressing only endogenousdynamin (tetracycline 1) and in cells overexpressing dominant-negative dynamin(tetracycline 2). To obtain overexpression, cells were grown in the absence oftetracycline for 2 days before the toxin treatment. Samples were taken 24 h aftertoxin exposure. Blots are representative of four different experiments. P-Tyr,phosphotyrosine.

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example, via caveolae (30). Regardless of the mode of primaryuptake, endocytic vesicles fuse and form endosomes that ma-ture by additional fusion processes. After this stage, endocy-tosed macromolecules may be delivered either to the lysosomesfor degradation or to the Golgi complex for other kinds of pro-cessing. In the Golgi compartment, the endocytic and exocyticpathways meet each other. Mechanisms exist for retrogradetransport from the Golgi compartment to the ER of macromol-ecules containing appropriate signaling sequences (3, 12, 13).

Toxins are masters of exploiting normal cellular physiologicprocesses for their own benefit and can enter cells after binding

to almost any surface structure and using either a clathrin-dependent or a clathrin-independent pathway. Some toxins,for instance, ricin, are able to use both clathrin-dependent andclathrin-independent pathways for initial entry into cells (30).With respect to the subsequent internalization steps, two majorgroups of toxins have been defined: (i) toxins translocateddirectly from endosomes to the cytosol (DT [28] and anthraxtoxin [37]) and (ii) toxins which are transported all the way tothe ER via the Golgi complex before being translocated to thecytosol. Recent data indicate that this latter pathway is used bythe majority of toxins studied to date. Thus, Shiga and Shiga-like toxins (13, 29), pertussis toxin (8, 10), cholera toxin (21), E.coli heat-labile enterotoxin (14), Pseudomonas exotoxin A (46),and the plant toxin ricin (27, 36) are transported in a retro-grade manner to the ER via the Golgi complex. To date, notoxin has been reported to translocate at the level of the Golgicompartment.

We have previously reported that HdCDT, like other CDTs,causes irreversible arrest in the G2 phase of the cell cycle,although the molecular target of the toxin is not yet known (6).In the present study, we provide biochemical data stronglysupporting the notion that HdCDT requires internalization byendocytosis in order to intoxicate HEp-2 and other cells. Wedemonstrate that HdCDT uses a clathrin-dependent pathway.This finding was indirectly suggested by rapid disappearance ofthe toxin from the cell surface, consistent with rapid macro-molecular entry via the clathrin-dependent pathway. Appar-ently, the time needed for warming up the cells enough topermit proteases and antibodies to act sufficed for entry of thetoxin or at least for its clustering into coated pits in a forminaccessible to antibodies or proteases. More direct evidencefor the clathrin-dependent pathway was established using cellsdepleted of potassium or treated with chlorpromazine or imip-ramine, conditions under which clathrin-dependent uptake ofligands into cells is known to cease (38, 43) and which protectcells from intoxication by DT (20).

The clathrin-dependent uptake of HdCDT in HEp-2 cellswas corroborated by the finding that HeLadynK44A cells (over-expressing the dominant-negative dynamin) were resistant tothe toxin. The importance of dynamin in clathrin-mediatedendocytosis is well established (7, 31). It is needed for theconstriction of coated pits and the subsequent budding of thecoated vesicles. Overexpression of dominant-negative dynaminwas shown to specifically block endocytic clathrin-coated ves-icle formation, although fluid-phase uptake continued (7). Thepossibility that active toxin could enter nonspecifically by fluid-phase uptake was thus excluded. The roles of dynamin and itspartners in other intracellular trafficking events are less clear.However, the toxic effect of ricin was recently shown also to beinhibited by the overexpression of dominant-negative dynamin,despite the fact that this toxin can enter cells via clathrin-independent endocytosis (9, 35). In this situation, the deliveryof ricin from endosomes to the Golgi complex was inhibited(16). The complete inhibition of the effect of HdCDT inHeLadynK44A cells might therefore be due to both the inhibi-tion of clathrin-dependent endocytosis and an effect on a laterstep of the endocytic pathway.

The partial inhibition of intoxication by methylamine andammonium chloride, in contrast to the complete inhibition bymonensin or BafA1, suggests that a raised intraendosomal pHper se can only delay HdCDT-induced intoxication. Monensinat the concentration used in this study has been reported toaffect the Golgi complex as well (18), and this effect mightexplain its complete inhibition of intoxication. In addition,monensin was previously shown to inhibit the proliferation ofcells exposed for more than 24 h (15), implying that it might

FIG. 5. Effects of BafA1 (50 mM), nocodazole (30 mM), and 18°C incubationon tyrosine phosphorylation of cdc2 in HdCDT-treated cells. (A) Cells wereexposed to BafA1 before and after toxin treatment. The postincubation time was24 h. (B) Cells were exposed to nocodazole before and directly after toxintreatment (panel 1) or received nocodazole 60 min after toxin treatment (panel2). The postincubation time was 8 h. (C and D) Cells were cultivated in eightindividual petri dishes; four were kept as controls, and four were exposed to thetoxin at the same time. (C) One pair of plates (HdCDT 2 and HdCDT 1) wasincubated (Inc.) at 37°C and the other was incubated at 18°C. Samples wereprepared 12 h after toxin treatment. (D) One pair of plates was incubated at 37°Cfor 24 h. The other was incubated at 18°C for the first 12 h (Inc. 1) and thentransferred to 37°C for another 12 h (Inc. 2). Blots are representative of threedifferent experiments with each treatment.

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interfere with the cell cycle. However, we used a shorter incu-bation time (12 h), and the flow cytometric analysis did notindicate any major change in cell cycle distribution (Fig. 2).

In HEp-2 cells, BafA1 appears not to affect the formationand maturation of multivesicular bodies, which are the endo-somes of this particular cell line. However, delivery from suchmature endosomes to lysosomes was reduced by BafA1 (41),and this drug was also reported to block transport from early tolate endosomes in HeLa cells (1). With this background, thecomplete inhibition of intoxication by BafA1 in HEp-2 cells, incontrast to the partial inhibition by agents that only neutralizethe endosomal pH, suggested that HdCDT might need somefurther transport along the endocytic pathway before beingable to act. This conclusion was also supported by the inhibi-tion obtained with the microtubule-disrupting agent nocoda-zole added before toxin internalization and by the fact thatincubation of toxin-treated cells at 18°C was protective againstthe toxic effect. The complete restoration of intoxicating ability

upon transfer of the cells back to 37°C (Fig. 5C) confirmed thatthe toxin was entrapped in vesicles that were not able to fusewith compartments downstream of the endocytic pathway at18°C. Furthermore, the toxin was obviously protected fromdegradation during the delay in these vesicles.

HdCDT failed to intoxicate cells pretreated with BFA, sug-gesting that the relevant downstream compartment is the Golgicomplex. BFA disrupts the Golgi complex, with redistributionof its proteins to the ER as well as inhibition of vesiculartransport from the ER to the Golgi complex (2). It is wellestablished that toxins internalized via the Golgi complex canbe inhibited by BFA. This finding has been demonstrated forall the above-mentioned toxins, which undergo retrogradetransport to the ER via the Golgi complex. For CDTs, how-ever, the interpretation is complicated by the fact that BFA perse can interfere with the cell cycle, arresting cells to a certainextent in the G1 phase (19). For this reason, incubations withBFA were kept as short as possible (6 to 12 h), thereby avoid-

FIG. 6. Effect of BFA on HdCDT-induced intoxication. Flow cytometry was used to analyze control [HdCDT (2)] and toxin-treated [HdCDT (1)] cells in thepresence (1) or absence (2) of BFA. Cells were pretreated with BFA (2.5 mg/ml) for 45 min, exposed to the toxin, and postincubated in normal medium with BFA.Samples were prepared 6 h (A), 8 h (B), and 12 h (C) after toxin treatment. (D) Sample from cells treated with toxin, postincubated for 45 min at 37°C in fresh medium toallow internalization of the toxin, and exposed for 12 h to BFA. Percentages of cells in G1 and G2/M are shown. One representative experiment of three is shown. FL3-H,relative fluorescence.

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ing pronounced G1 arrest due to BFA. In addition, intoxicationdid occur when BFA was added 1 h after the toxin (Fig. 6D).This result indicates that BFA interfered with a step takingplace during the first hour of intoxication, i.e., the internaliza-tion. Our observations with BFA were corroborated with ili-maquinone, another drug causing the fragmentation of Golgimembranes and their dispersion throughout the cytoplasm (22,39). The actions of BFA and ilimaquinone differ in that thelatter does not induce retrograde transport of Golgi enzymesto the ER (39). Thus, two drugs that disrupt the Golgi complexin different ways inhibited HdCDT-induced intoxication.

In conclusion, all our results support the notion that HdCDTundergoes clathrin-dependent endocytosis and vesicular trans-port at least to the Golgi complex before it can induce arrest inthe G2 phase of the cell cycle. It remains to be determinedexperimentally whether HdCDT is transported in a retrogrademanner from the Golgi complex to the ER and translocated tothe cytosol (or possibly to the nucleus) from there. We havenot yet succeeded in detecting HdCDT in any specific intra-cellular compartment, probably because the toxin is very po-tent and only a few molecules may enter at the final destina-tion. This is a common problem with all toxins transported ina retrograde manner; for ricin, this problem was recently

solved by introducing N-glycosylation sites to demonstrate pas-sage via the ER (27).

All the toxins previously found to enter via the Golgi com-plex have been either implied to or actually demonstrated tocontinue to the ER before being translocated to the cytosol.The mechanism for retrograde transport is not yet clear. TheKDEL retrieval system is exploited by Pseudomonas exotoxinA but not by Shiga-like toxin I (12). Toxins translocated fromthe ER to the cytosol have been suggested to disguise them-selves as misfolded proteins, thereby succeeding in being trans-ported across the ER membrane (11, 17). The fate of proteinsbeing extruded from the ER in this manner is usually ubiquiti-nation and proteasomal degradation (25). How toxins avoidthis fate is not known, but an interesting hypothesis is that theyresist ubiquitination because of a relative lack of lysines (11).Indeed, toxins transported in a retrograde manner have veryfew lysines, which are located only near the N or C termini ofthe active toxin component. In contrast, both DT and anthraxtoxin, which are translocated from early endosomes (28, 37),have several lysines scattered all along their active fragments.Interestingly, the entire CdtB component of HdCDT containsonly 3 lysines, which all are near the N terminus, whereas theCdtC component has 13 lysines scattered along the entire

FIG. 6—Continued.

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molecule. A similar pattern of lysine distribution is found inthe CdtB and CdtC components of all the other CDTs. Recentwork suggested that the A. actinomycetemcomitans CdtB com-ponent alone is capable of inducing G2 arrest (33, 34). Con-sidering the lysine pattern and the internalization data pre-sented here, this suggestion is consistent with the possibilitiesthat the CdtB protein of the CDTs is the active component and

that it is transported to the Golgi complex and from thereprobably to the ER.

In conclusion, this work indicates that HdCDT exerts itsaction intracellularly and not by transmembrane signaling. Thisis the first member of the family of CDTs for which cellularinternalization and some details of the pathway have beendemonstrated. Based on the similarities in sequence and action

FIG. 7. Effect of BFA on the tyrosine phosphorylation of cdc2 in HdCDT-treated cells 8 h (A) and 12 h (B) after toxin treatment. Blots are representative of threedifferent experiments. P-tyr, phosphotyrosine.

FIG. 8. Effect of ilimaquinone (Ilimaq.) on HdCDT-induced intoxication. Shown are Flow cytometric analysis (A) and tyrosine phosphorylation of cdc2 (B) incontrol [HdCDT (2)] and toxin-treated [HdCDT (1)] cells with (1) or without (2) ilimaquinone. One representative experiment of two is shown.

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among all the CDTs, it is likely that they all need to be internal-ized via the Golgi complex before being able to intoxicate cells.

ACKNOWLEDGMENTS

We are grateful to Teresa Frisan for stimulating discussions as wellas helpful comments on the manuscript. We also thank Kirsten Sand-vig for kindly providing HeLadynK44A cells and Peter Low for providingantidynamin antibodies.

This work was supported by the Swedish Medical Research Council(grants 05969 and 12630) and the Swedish Institute and by funds fromKarolinska Institutet.

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