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Infection and Immunity, January 2003, p. 541-545, Vol. 71, No. 1
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.1.541-545.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Campylobacter jejuni Cytolethal Distending Toxin Promotes DNA Repair Responses in Normal Human Cells

Duane C. Hassane, Robert B. Lee, and Carol L. Pickett*

Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, Kentucky 40536

Received 2 May 2002/ Returned for modification 17 June 2002/ Accepted 10 October 2002


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ABSTRACT
 
Cytolethal distending toxin (CDT) is a multisubunit protein found in various gram-negative bacterial pathogens of humans which is thought to cause cell death by direct DNA damage of host cells. We sought to determine if a cellular response to DNA damage could be detected by exogenous addition of the holotoxin. Exogenous addition of the Campylobacter jejuni 81-176 CDT to primary human fibroblasts resulted in formation of Rad50 foci, which are formed around double-stranded-DNA breaks. Moreover, such foci are formed in both proliferating and nonproliferating cells that are treated with C. jejuni CDT. Fibroblasts that were intoxicated and later stimulated to proliferate failed to divide and remained arrested in the G1 phase of the cell cycle.


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TEXT
 
Campylobacter jejuni is the leading cause of gastrointestinal disease in United States and is found on the majority of uncooked chicken available to consumers from grocers (13, 14, 40). As such, this bacterium's potential virulence determinants merit close investigation. Among these potential determinants is the cytolethal distending toxin (CDT), which is also found in association with a variety of other gram-negative human pathogens that cause diseases affecting mucosal surfaces such as chancroid, periodontitis, and gastroenteritis (2, 6, 38). CDT consists of three polypeptide subunits encoded by the closely linked cdtA, cdtB, and cdtC genes (30, 33). CDT holotoxin possesses maximal activity when all three subunits are present (1, 21). Cultured cells exposed to CDT exhibit a number of effects including cell cycle arrest, enlargement, Cdc2 and ataxia-telangiectasia-mutated protein (ATM) phosphorylation, and cell death (5, 6, 7, 19, 29, 43). All human cell types examined to date by our laboratory appear susceptible to C. jejuni CDT (C. Pickett, unpublished data). C. jejuni CDT may thus participate in diverse functions such as epithelial effacement and immunomodulation. The elucidation of subunit functions has only begun recently. CdtA, with its similarity to the ricin B chain (17, 21), and CdtC appear important for holotoxin binding and transgression through the host cell plasma membrane. CdtB, with its similarity to DNase I, is apparently able to degrade plasmid DNA in vitro (10, 12, 22). Reports concerning mammalian cells and yeast have demonstrated that CdtB, when ectopically expressed or microinjected, can degrade chromatin in vivo (11, 20). Additionally, early transcriptional upregulation of a genotoxin-inducible gene has been shown to occur in yeast (17). Thus, as an intracellular DNase, CdtB apparently kills cells by fatally damaging their DNA. Despite this evidence, previous authors were unable to detect DNA damage as a result of exogenous addition of CDT to cultured cells (34). We thus sought to determine whether DNA repair responses could result from exogenous addition of C. jejuni CDT to cultured cells.

The formation of repair complexes in mammalian cells in response to exposure to DNA-damaging agents such as gamma irradiation is well characterized (3, 15, 24-27). The repair of double-stranded-DNA breaks (DSBs) is typically accomplished by either homologous recombination or nonhomologous end joining (NHEJ) (16, 39). Both of these processes involve Rad50 (4, 18, 37, 41, 42). The predominant repair mechanism in mammalian cells is, however, NHEJ, where fragmented DNA is religated end to end. NHEJ is partially mediated by a complex containing the Rad50 protein (Rad50/Mre11/Nbs1) that is involved in the processing of DSBs (16, 39). DNA damage produces nuclear foci containing Rad50, which are detectable by immunofluorescence (26). Therefore, if CDT exposure results in the formation of DSBs, it is likely that the formation of Rad50 foci should result as well.

Asp-222 in CdtB is required for CDT toxicity to cultured cells. C. jejuni 81-176 CDT was expressed in Escherichia coli XL1-Blue (Stratagene, La Jolla, Calif.) with the expression plasmid pTrc18 (43). The inactive CDT mutant (CDT-CdtBD222A) was constructed by subjecting pTrc18 to site-directed mutagenesis with the mutagenic oligonucleotides 5'-CTCTTGCTTATGCAATTACAGGAAATTC-3' and 5'-TCCCTCCGCTTGCTTGAGTTGCTGC-3' according to the ExSite protocol (Stratagene) as described by Hassane et al. (17). Asp-222 is predicted to be critical for CdtB's DNase activity (9). The conversion of Asp-222 to Ala-222 was shown to inhibit both CdtB-mediated in vivo DNA degradation and toxicity in a yeast model (17). Crude extracts derived from E. coli XL1-Blue expressing all three cdt wild-type genes from pTrc18 (CDT) or from pTrc18-CdtBD222A (CDT-CdtBD222A) and an extract containing the expression vector alone without an insert sequence, pTrc99A, were prepared and quantified for protein content as previously described (43). We tested these extracts for their ability to cause cell cycle arrest when added exogenously to HeLa cells as described before (43). As expected, only addition of CDT produced an accumulation of G2/M cells (Fig. 1A); addition of CDT-CdtBD222A did not (Fig. 1B). The results for the vector control were essentially identical to those shown for the mutant holotoxin (not shown).



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  		FIG. 1. Conversion of Asp-222 to Ala-222 in CdtB inhibits CDT toxicity. Shown is the DNA content of HeLa cells at 24 h after intoxication with CDT (A) or CDT-CdtBD222A (B). An accumulation of G2/M cells occurs only with CDT treatment. x axis, DNA content; y axis, cell number. Left arrowhead (each panel), cells with G1 DNA content; right arrowhead, cells with G2/M DNA content.

Formation of Rad50 foci occurs as a result of CDT exposure. We tested the effects of these extracts on primary human fetal fibroblasts (IMR-90) obtained from the American Type Culture Collection (Manassas, Va.). IMR-90 cells were previously shown to form Rad50 foci in response to DSB-inducing agents (26).

Cells between passages 11 and 16 were grown in 16-well Lab-tek chamber slides (Nalge Nunc International, Naperville, Ill.) to 40% confluence in minimal essential medium (CellGro, Herndon, Va.) supplemented with 10% fetal bovine serum (FBS), Earle's salts, sodium bicarbonate, sodium pyruvate, nonessential amino acids, and antibiotics (penicillin and streptomycin) in accordance with their manufacturer's recommendations (Invitrogen, Carlsbad, Calif.).

Cells were incubated in the presence of 0.25-mg/ml extract containing either CDT or CDT-CdtBD222A or in the presence of the control extract for 90 min. Afterwards, cells were washed twice with phosphate-buffered saline (PBS) and supplied with fresh medium. At 22 h postintoxication, cells were fixed and permeabilized for 10 min at -20°C in 3:2 (vol/vol) methanol-acetone and then processed for immunofluorescence using PBS-5% heat-inactivated goat serum as a blocking solution. Monoclonal anti-Rad50 was incubated with cells at room temperature at a 1:200 dilution in blocking solution (GeneTex, San Antonio, Tex.) for 1 h in a humidified chamber. Staining with a secondary antibody (1:100 dilution of goat anti-mouse Alexa-594; Molecular Probes, Eugene, Oreg.) was performed for 30 min in blocking solution at room temperature. Cells were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, Calif.) plus DAPI (4',6'-diamidino-2-phenylindole).

Rad50 was detected almost exclusively in the nucleus, as determined by colocalization with DAPI (not shown), in all experiments. As shown in Fig. 2B and C, cells treated with the CDT-CdtBD222A or control extract demonstrated diffuse Rad50 distribution throughout the nucleus. However, in CDT-treated cells, Rad50 became distributed focally (A). The fact that CDT-CdtBD222A did not cause focus formation illustrates the dependence of focus formation on the DNase activity of CdtB and not a previously uncharacterized function of CdtA or CdtC.



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  		FIG. 2. IMR-90 cells treated with CDT form foci independent of cell division. (A to C) Results for cells grown in 10% FBS; (D to F) results for cells grown in serum-free medium. (A and D) Cells treated with CDT. Note the formation of Rad50 foci; (B and E) cells treated with CDT-CdtBD222A; (C and F) cells treated with the vector control. The cells treated with the controls (B, C, E, and F) did not induce focus formation. Representative nuclei are shown.

Cell cycle progression is not required for formation of Rad50 foci. Cortes-Bratti et al. (7) observed both G1 and G2 arrest when primary lung fibroblasts were treated with a related CDT from Haemophilus ducreyi. This observation suggested that, possibly, cell cycle progression was not needed for cellular intoxication. We deprived cells of serum to test whether Rad50 foci would form under those conditions when cells were exposed to CDT.

Serum starvation was performed for 48 h. This treatment caused cells to remain predominantly in G0/G1 phase (not shown). Extract that contained either CDT or CDT-CdtBD222A or the control extract was applied to the cells in serum-free medium (SFM) at a final concentration 0.25 mg/ml for a period of 90 min, after which it was washed off. Cells were stained for Rad50 as described above at 22 h postintoxication. Treatment of serum-starved cells with CDT extract caused Rad50 foci to form, in contrast to treatment with CDT-CdtBD222A or control extract, where foci did not form (Fig. 2D to F). This finding signified that noncycling cells were also susceptible to intoxication with CDT.

The number of Rad50 foci present in the cells treated as described above was quantified. Nuclei that contained at least five discernible foci were scored positive. At a minimum, 400 cells were assessed for nuclear foci per experimental condition. It was found that the number of focus-positive cells did not vary significantly in relation to whether cells were dividing. As Fig. 3 shows, approximately 40% of cells treated with CDT developed foci regardless of whether cells were proliferating (i.e., grown in the presence or absence of serum). In contrast, cells that were treated with CDT-CdtBD222A or the vector control extract developed nuclear foci at a frequency of under 3%. This finding suggests that CDT can cause DNA damage regardless of whether cell cycle progression is occurring.



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  		FIG. 3. Cells treated with CDT demonstrate a high percentage of nuclei containing Rad50 foci. IMR-90 cells grown in medium supplemented with 10% fetal calf serum (FCS) are compared to cells grown in SFM. y axis, percentage of cells that were scored focus positive (at least five discernible foci); error bars, standard deviations of four experiments.

Cell cycle analysis. We assessed the DNA content of IMR-90 cells treated with CDT or CDT-CdtBD222A extract. Cells were harvested and then stained with propidium iodide as described previously (43). At least 10,000 events were recorded per experiment, each of which was performed in triplicate at least three times. IMR-90 cells were grown in serum-containing medium and treated with CDT-CdtBD222A or CDT extract (0.25 mg per ml) for 90 min. CDT-CdtBD222A-treated cells failed to accumulate in any particular phase of the cell cycle (Fig. 4A), while CDT treatment resulted in a biphasic cell cycle arrest in which G1- and G2-arrested cells were observed (Fig. 4B). The observed biphasic arrest is consistent with the presence of functional p53, as would be expected for normal cells such as IMR-90 cells; in most CDT studies, which used cell lines lacking p53, cells arrested exclusively in G2 (5, 8, 29, 34-36, 38, 43).



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  		FIG. 4. CDT-induced cell cycle arrest in synchronized and nonsynchronized cells. IMR-90 cells treated with CDT-CdtBD222A or CDT are compared. x axes, DNA content; y axes, cell number. Left arrowhead (each panel), cells with G1 DNA content; right arrowhead cells with G2/M DNA content. (A and B) Asynchronous IMR-90 cells treated with CDT-CdtBD222A (A) or CDT (B); (C and D) serum-starved cells treated with CDT-CdtBD222A (C) or CDT (D); (E and F) serum-stimulated cells treated with CDT-CdtBD222A (E) or CDT (F) at 24 h after readdition of FBS; (G and H) serum-stimulated cells treated with CDT-CdtBD222A (G) or CDT (H) at 48 h after readdition of FBS. Concentration of extracts is 0.25 mg per ml.

We next confirmed whether serum-starved cells treated with CDT remained in G1 phase at 24 h postintoxication. Cells treated with CDT- or CDT-CdtBD222A-containing extracts for 24 h in serum-free medium remained in G1 as expected (Fig. 4C and D). This indicated that treatment with extracts did not induce cells to progress through the cell cycle.

Since all cells remained in G0/G1 phase under these serum-free conditions in the presence of extracts, we then ascertained whether CDT-treated cells could resume cell cycle progression once restimulated to grow by simultaneously washing off the extract-containing SFM three times with PBS, followed by addition of FBS to a final concentration of 10%. We determined whether cell cycle progression resumed by examining the DNA content of the serum-stimulated cells at 24 h after the addition of serum. Cells treated with CDT-CdtBD222A-containing extract reentered the cell cycle, as indicated by the observable increase in S-phase cells (Fig. 4E). In contrast, CDT-treated cells remained in G1 (Fig. 4F). At 48 h after stimulation with serum, CDT-CdtBD222A-treated cells continued to progress through all phases of the cell cycle (Fig. 4G) while CDT-treated cells remained in G1 (Fig. 4H). This observation suggests that cell cycle progression is not required for intoxication and that normal cells do not necessarily preferentially arrest in G2, contrary to hypotheses put forward recently (20, 21). However, this finding is consistent with the presence of DSB-induced Rad50 foci in CDT-treated, serum-starved cells as we observed above (Fig. 2D) and with related observations by Li et al. (23), where the effects of the H. ducreyi CDT on immature dendritic cells were examined.

Formation of {gamma}-H2AX in CDT-treated cells. The histone protein H2AX is rapidly phosphorylated at DSBs (31, 32). The resulting phosphoprotein ({gamma}-H2AX) mediates recruitment of repair complexes to sites where DSBs occur, including complexes that contain Rad50. Previous stoichiometric analyses have suggested that several thousand {gamma}-H2AX molecules are formed about each DSB (24, 28). Given this stoichiometry, detection of {gamma}-H2AX provides a more sensitive means to detect the apparently subtle DNA damage caused by CDT.

We tested whether treatment of cells with exogenous CDT could result in {gamma}-H2AX formation by immunoblot analysis using a {gamma}-H2AX-specific monoclonal antibody (Upstate Biotechnology, Waltham, Mass.). Cells were treated as described above with extract of CDT or CDT-CdtBD222A or the vector control extract. At 22 h postintoxication, lysates of these cells (alongside untreated cells) were prepared in accordance with the antibody manufacturer's protocol. Immunoblotting was performed according to the ECF protocol (Amersham Biosciences, Piscataway, N.J.), and the resulting bands were visualized with a Storm phosphorimager (Amersham). Results were normalized to ß-actin. As shown in Fig. 5, {gamma}-H2AX formation occurred only with CDT treatment whereas cells treated with CDT-CdtBD222A and control extracts were similar to untreated cells. This finding provides further indication that CDT induces DNA damage in a manner dependent on the DNase activity of CdtB.



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  		FIG. 5. Formation of {gamma}-H2AX in response to CDT treatment. Proteins were immunoblotted with a {gamma}-H2AX-specific antibody or a ß-actin-specific antibody. Lane 1, untreated cells; lane 2, cells treated with vector control extract; lane 3, cells treated with the CDT extract; lane 4, cells treated with the CDT-CdtBD222A extract. Equivalent numbers of CDT-treated IMR-90 cells or control extract-treated cells were added to each lane.

The detection of cellular DNA repair responses after exposure to C. jejuni CDT supports the hypothesis that CDT causes DNA damage and is consistent with the apparent in vivo DNase activity of CdtB. Notably, CDT-CdtBD222A was completely unable to promote the induction of repair responses in our studies. Thus, the effects seen here are entirely dependent on the enzymatic activity of CdtB rather than being an undetermined function of CdtA or CdtC. Relocalization of Rad50 into focal structures in response to toxin exposure, presumably near sites of DNA damage, was observed. Recent findings by Li et al. (23) demonstrate the presence of Mre11 foci by using the CDT from H. ducreyi. Those findings are in agreement with the data here obtained using C. jejuni CDT, as Mre11 is contained in the same complex containing Rad50. We further demonstrate the persistence of DNA lesions until 22 h postintoxication, as indicated by the presence of {gamma}-H2AX, which Li et al. (23) demonstrated at earlier time points, suggesting that lesions resulting from C. jejuni CDT are persistent. We noted, however, in the present study that cell death did not occur until at least 72 h after addition of CDT. Indeed, up to that time, cells continued to grow, becoming gradually more "distended." Moreover, at 22 h postintoxication, IMR-90 cells failed to demonstrate phosphatidylserine exposition, as determined by staining with annexin V-phycoerythrin and 7-aminoactinomycin (Pharmingen, San Diego, Calif.) according to the manufacturer's protocol (Fig. 6). Hence, it is unlikely that the observations reported here result from apoptosis. The observation that CDT forms DSBs is biologically significant in that a range of tumors are thought to arise from this type of lesion, providing a "hit" that predisposes individuals to cancer. Whether cancer predisposition is truly a consequence of CDT exposure or perhaps C. jejuni infection awaits further study.



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  		FIG. 6. CDT-associated apoptosis not apparent by 22 h postintoxication. IMR-90 cells were stained with annexin V-phycoerythrin (PE) and 7-aminoactinomycin (7-AAD). Dot plots are shown. (A) Untreated control cells; (B) cells treated with the CDT extract; (C) cells treated with the CDT-CdtBD222A extract; (D) cells treated with the positive control, camptothecin, for 22 h. Quadrants are drawn for comparison to untreated control cells (A).


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ACKNOWLEDGMENTS
 
This work was supported in part by NIH grant AI48590 to C.L.P.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky Medical Center, MS415, 800 Rose St., Lexington, KY 40536-0298. Phone: (859) 323-5313. Fax: (859) 257-8994. E-mail: cpicket{at}uky.edu. Back

Editor: J. T. Barbieri


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Infection and Immunity, January 2003, p. 541-545, Vol. 71, No. 1
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.1.541-545.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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