From the
Laboratory of Experimental Cancerology, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium, the ||Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, K. L. Ledeganckstraat 35, Ghent University, B-9000 Ghent, Belgium, and the
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Received for publication, January 7, 2003 , and in revised form, April 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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The apical side of enterocytes bears tightly packed microvilli hampering the intimate adhesion of trophozoites. Disorganization of microvilli is an early morphological change caused by trophozoites (10), but its molecular basis is not understood. The purpose of this study was to investigate the molecular mechanism by which trophozoites of E. histolytica efface the brush border microvilli of enteric cells. Each microvillus contains 20 actin filaments, which are bundled and linked to the plasma membrane by actin-binding proteins such as villin, fimbrin, small espin, ezrin, and brush border myosin I (1114). Villin is a major actin-bundling protein and a member of the family of Ca2+-regulated actin-binding proteins. It consists of two similar domains of 44 kDa and a head piece domain of 8.5 kDa. In vitro, the core domain caps, severs, and nucleates actin filaments in a Ca2+-dependent way, whereas the head piece domain binds actin filaments in a Ca2+-independent way. For bundling, the intact protein is required (15). Overexpression of villin in fibroblastic CV-1 cells results in the formation of microvilli, whereas villin antisense expression impairs brush border formation in enteric Caco-2 cells (16, 17). Although microvilli of villin knock-out mice have a normal ultrastructure, villin is required in response to cell injury (14).
Our experimental model consists of 3-week-old well differentiated Caco-2 cell layers on top of which we seed trophozoites of various strains, namely pathogenic E. histolytica HM-1:IMSS, transfected E. histolytica pSA8 (18), or non-pathogenic Entamoeba dispar SAW760, and we examined villin by immunocytochemistry and Western blotting.
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EXPERIMENTAL PROCEDURES |
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CoculturesTrophozoites or bacteria were pelleted at 200 x g for 5 min, washed in DMEM + 10% fetal calf serum supplemented or not with inhibitors, and added to the Caco-2 cell layers. Cocultures were incubated at 37 °C for various incubation times. In cocultures for 0 min, trophozoites were seeded on the cell layer and removed immediately thereafter. Washing fluids were collected, and trophozoites were counted. Some cell layers were cocultured with sonicated (3 x 5 s) or heat-inactivated (30 min at 56 °C) trophozoites or with filtered (0.2 µm) conditioned medium from 60-min cocultures. Prior to some cocultures, trophozoites were treated for 2 h in TYI-S-33 supplemented with the following inhibitors: z-FA.fmk; z-FF.fmk; calpain inhibitors I, II, and III; PD 150606; cathepsin B inhibitor II; CA-074-me; and z-VAD.fmk (all were from Calbiochem). -Lactose and E64 were from Sigma, and pepstatin was from Roche Applied Science. Caco-2 cell layers were treated for 2 h in their own medium. To remove trophozoites from the cell layers following coculture, 200 mM
-lactose was added for 15 min. Alternatively, a 1:1 mixture of human non-decomplemented serum and DMEM was used to selectively kill trophozoites through the alternative complement pathway (23).
Urea LysatesCocultures were stopped by washing with ice-cold PBS and lysed with 9 M urea lysis buffer supplemented with proteinase and phosphatase inhibitors (7). Supernatants were taken for SDS-polyacrylamide gel electrophoresis. To evaluate protein degradation by proteinases released from the trophozoites during lysate preparations (colysis) (24), lysates of Caco-2 cell layers were mixed with lysates of trophozoites.
Western Blot AnalysisLysates were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Hybond) for immunostaining with a pAb against ezrin (obtained from Dr. Monique Arpin, Curie Institute, Paris, France) or -catenin (Sigma) or a monoclonal antibody against villin (ID2C3, obtained from Dr. Sylvie Robine, Curie Institute, Paris, France) (25) or gelsolin (Sigma), all of which followed by peroxidase-conjugated secondary antibodies (Amersham Biosciences). The signal was revealed by chemiluminescence (ECL, Amersham Biosciences), and intensity of bands was quantified using the program Quantity One (Bio-Rad).
Assays for EhCP ActivityGelatin zymography was done as described by Hellberg et al. (26). Trophozoites were lysed by four cycles of freeze-thawing in PBS and centrifuged at 15,000 x g. Supernatants were reduced with 10 mM DTT for 10 min at 37 °C, and equal protein concentrations (4 µg) were loaded on a 10% SDS-polyacrylamide gel copolymerized with 0.1% gelatin. After separation, gels were incubated for 1 h in 2.5% Triton X-100 at 37 °C followed by 3 h in substrate buffer (100 mM sodium acetate, pH 4.5, 1% Triton X-100, 20 mM DTT) and stained with Coomassie Brilliant Blue.
Spectrophotometrical analysis of EhCP activity was done with the synthetic substrate z-RR.pNA (Bachem) (27). Conditioned media or total freeze-thaw lysates of trophozoites, Caco-2 cell layers, or 15-min cocultures were supplemented with 2 mM DTT, and 30 µl was added to 170 µl of 0.1 mM KH2PO4, and 2 mM EDTA, pH7.0, containing 0.1 mM z-RR.pNA. After 1 h at 28 °C, the optical density was measured at 405 nm in a microplate reader (Molecular Devices Corporation). The release of p-nitroaniline is proportional to the amount of EhCP activity.
ImmunocytochemistryCaco-2 cells were seeded on 1.8-cm2 coverslips and cultured for 3 weeks. Cocultures with 15 x 104 trophozoites or with 2 x 108 bacteria and control Caco-2 cell layers were processed for immunostaining as described by Leroy et al. (7). We used rabbit pAb against ezrin and mouse monoclonal antibody against villin (ID2C3). Cells were examined under a fluorescence microscope (Dialux 20, Leitz) or a confocal microscope (Leica TCS NT, Leica Microsystems). Ezrin microvillar patterns were scored as cauliflower-like or disturbed in 50 fields from three independent experiments by three observers. The Student's t test (p < 0.05) was used for statistics.
Caspase ActivityCaspase activity was determined in accordance with Van de Craen et al. (28). Cultures were washed three times with ice-cold PBS and subsequently lysed for 5 min at room temperature in buffer containing 10 mM Hepes, pH 7.5, 220 mM mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM KH2PO4, 2 mM MgCl2, 1 mM glutathione, and 1% Nonidet P-40 supplemented with leupeptin, aprotinin, and phenylmethylsulfonyl fluoride all at 10 µg/ml. Supernatants were collected and kept on ice. Caspase activity was measured by incubation of lysates for 50 min at 30 °C with 50 µM fluorogenic substrate Ac-DEVD-amc (Peptide Institute) in the same buffer supplemented with 0.1 mM phenylmethylsulfonyl fluoride and 1 mM DTT. The release of amc was monitored for 50 min in a fluorometer (Cytofluor, PerSeptive Biosystems) at excitation and emission wavelengths of 360 and 460 nm, respectively. Purified recombinant caspase-3 or lysates from apoptotic Jurkat cells were used as positive controls.
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RESULTS |
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Amebic Proteolysis of Villin Is Not Attributed to Detergent ColysisAfter a 15-min coculture, 80% of seeded trophozoites were still attached to the Caco-2 cell layer at the moment of detergent lysis. Therefore, detergent colysis, an important caveat for conclusions about proteolysis drawn from Western blots (24), might explain the lack of further villin proteolysis during longer coculture. To prevent proteolysis during detergent colysis, 9 M urea was added to the lysis buffer. As a control, lysates of trophozoites and of Caco-2 cell layers were made separately, mixed, and analyzed on Western blot. Villin proteolysis during colysis was much lower (28%) than during coculture (77%) and hardly differed from control cultures without trophozoites (19%) (Fig. 2B). From these results, we conclude that villin proteolysis occurs during coculture and not during detergent lysis.
Proteolysis Requires Adhesion of Living Trophozoites to the Enteric CellsNeither amebic sonicates nor heat-inactivated trophozoites nor conditioned medium of cocultures had an effect on villin. Cocultures in the presence of anti-adhesive concentrations (200 mM) of -lactose (29) prevented proteolysis (Fig. 3A). Moreover, the removal of trophozoites following 15-min coculture by 15-min incubation in the presence of 200 mM
-lactose or human serum resulted in the disappearance of villin proteolytic bands. In 60-min cocultures followed by the removal of trophozoites, low amounts of villin proteolytic bands were still present (Fig. 3B). From these results, we conclude that adhesion of living trophozoites to the enterocytes is necessary to initiate and maintain villin proteolysis.
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Caspases Do Not Participate at Villin ProteolysisCaspases constitute a family of cysteine proteinases involved in programmed cell death (30). Caspase-3-like activation occurs within 30-min coculture of trophozoites with Jurkat cells in vitro (9). Therefore, the possible contribution of caspases to the specific and limited proteolysis of villin in Caco-2 cells was assessed. Using the fluorogenic substrate Ac-DEVD-amc, we were unable to detect any caspase activity in cell lysates derived from cocultures. In three independent experiments, the released fluorescence was even lower in lysates from 15-, 60-, and 180-min cocultures (79, 73, and 77%, respectively) as compared with Caco-2 cell layers alone (100%) and the same was found in cocultures with 1-week-old Caco-2 cell layers. As a readout for caspase-3 and caspase-8 activation, the proteolysis of two substrates was determined, PARP and Bid, respectively. Neither PARP nor Bid was cleaved upon coculture as evidenced by Western blot (data not shown). Neither was villin proteolysis prevented by 100 µM z-VAD.fmk, an irreversible and highly specific caspase inhibitor. Here, we demonstrate that caspases are not activated within 2 h cocultures of Caco-2 cell layers with HM-1:IMSS trophozoites and that caspases are not responsible for villin proteolysis.
Villin Proteolysis Is Mediated by Amebic Cysteine ProteinasesSeveral proteinase inhibitors were tested for their capacity to prevent villin proteolysis in coculture. Caco-2 cell layers and trophozoites were pretreated separately with proteinase inhibitors, and inhibitors were added to cocultures. Villin proteolysis was prevented by the cysteine proteinase inhibitors 200 µM E64, 100 µM z-FA.fmk, and by 20 µM z-FF.fmk (Fig. 4A) but not by 100 µM calpain inhibitor I, II, or III, 1 µM pepstatin, 100 µM PD150606, 100 µM calpeptin, 50 µM cathepsin B inhibitor II, 1 µM cathepsin D inhibitor, nor by 100 µM CA-074-me. None of the modulators was toxic at the concentrations given as assayed by propidium iodide staining. Because 100 µM z-FA.fmk inhibited villin proteolysis most efficiently, it was used in further experiments. To find out whether the proteinase responsible for villin proteolysis was of amebic or of enteric origin, Caco-2 cell layers, trophozoites, or both were pretreated with z-FA.fmk and cocultures were done in the absence of z-FA.fmk. The pretreatment of trophozoites alone with z-FA.fmk was sufficient to inhibit villin proteolysis, whereas no inhibition was observed when only the Caco-2 cell layer was pretreated (Fig. 4B, upper panel), indicating that EhCPs were involved. Villin proteolysis could not be inhibited when 100 µM z-FA.fmk was added to the lysis buffers (Fig. 4C), in agreement with the previous conclusion that villin was not degraded during detergent colysis (see Fig. 2B). To confirm that z-FA.fmk inhibits EhCPs, trophozoites were treated with 100 µM z-FA.fmk and processed for zymography (Fig. 4B, lower panel). Untreated trophozoites show a banding pattern similar to the one published by Hellberg et al. (26) where the upper band was identified as EhCP1 (48 kDa) and the lower band identified as EhCP2 (35 kDa). Pretreatment with 100 µM z-FA.fmk abolished all of the EhCP activity (Fig. 4B, lower panel). To substantiate further the participation of EhCPs at villin proteolysis, we used pSA8, a mutant strain of HM-1: IMSS, in which the overall level of EhCP activity is strongly reduced by antisense expression of EhCP5 (18) and E. dispar SAW760, a non-pathogenic strain that lacks genes encoding orthologues of EhCP5 and EhCP1 (31). Bands representing pSA8 EhCP activity on zymograms were of lower intensity as compared with HM-1:IMSS (Fig. 5A). The zymogram of a lysate from E. dispar shows one band (EdCP3), which is located between EhCP1 and EhCP2 (26). Western blots of lysates from 15-min cocultures showed a reduced proteolysis of villin by either pSA8 or SAW760 as compared with HM-1: IMSS trophozoites (Fig. 5B). The EhCP activity in cocultures (optical density of 0.396 ± 0.064) with HM-1:IMSS trophozoites was not higher than in suspensions of trophozoites alone (optical density of 0.484 ± 0.004) as was evidenced by spectrophotometric analysis using the synthetic cathepsin B substrate z-RR.pNA. Neither was there an increase in EhCP activity in conditioned medium from cocultures (optical density of 0.144 ± 0.02) as compared with conditioned medium from trophozoites alone (optical density of 0.161 ± 0.01).
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Changes in Villin Immunocytochemical PatternSo far, we demonstrated that villin proteolysis was prevented in cocultures with trophozoites, showing a reduced EhCP activity. We wondered whether in such cocultures the prevention of villin proteolysis resulted in a better protection of microvilli. Therefore, 3-week-old Caco-2 cell layers were cocultured during 1 h with E. dispar SAW760 or E. histolytica HM-1:IMSS trophozoites pretreated or not with z-FA.fmk and processed for ezrin immunocytochemistry. The microvillar pattern as revealed by ezrin immunostaining was the same as by villin immunostaining but had less cytoplasmic background and, therefore, was easier to score (Fig. 6, AB). Ezrin and villin immunocytochemistry revealed a homogeneous cauliflower-like pattern on the apical side of 3-week-old differentiated Caco-2 cell layers. Within 1 h of coculture with HM-1:IMSS trophozoites, this pattern changed into a disturbed pattern characterized by few elongated microvilli centrally and remaining microvilli at the cell periphery resembling a honeycomb (Fig. 6, A and B, HM-1).
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Immunocytochemical patterns correspond with the microvillar patterns seen on SE micrographs (10, 32). Increasing amounts of trophozoites in cocultures accelerated these microvillar alterations. Living trophozoites were required because neither heat-inactivated nor sonicated trophozoites caused those alterations. Cauliflower-like and disturbed patterns were scored in 50 fields by three different observers. Fields were either homogeneous for one pattern or heterogeneous. In heterogenous fields, both patterns were scored. In cocultures with SAW760- or z-FA.fmk-pretreated HM-1:IMSS trophozoites, the ezrin microvillar pattern remained predominantly cauliflower-like in contrast to cocultures with HM-1:IMSS trophozoites where patterns were mainly disturbed (Fig. 6, B and C).
Disturbance of Microvilli by Other Microorganisms without Villin ProteolysisWe wanted to examine whether villin proteolysis was the consequence of the disturbance of microvilli, regardless its cause. Therefore, we examined villin in cocultures with S. typhimurium and EPEC that are known to disturb microvilli of enteric cells (33, 34). The disturbance of microvilli was confirmed in these experiments through ezrin immunocytochemical staining of 1-h cocultures of Caco-2 cells with S. typhimurium, EPEC, or HM-1:IMSS trophozoites (Fig. 7A). Western blots showed no proteolysis of villin from cocultures with bacteria, in contrast to cocultures with amebic trophozoites (Fig. 7B). A comparison of these cocultures suggests that proteolysis of villin is not the consequence of the disturbance of microvilli.
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DISCUSSION |
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Proteolysis during preparation of detergent lysates called colysis (24) was not responsible for villin proteolysis as revealed by comparative quantitative analysis of Western blots from cocultures and from mixed lysates in matched experiments. Moreover, villin proteolysis could not be prevented by the addition to the lysis buffer of 100 µM z-FA.fmk, an inhibitor that was effective when added to cocultures (see Fig. 4). The finding that ezrin, gelsolin, and -catenin were not proteolysed in coculture implicates a degree of specificity for villin that is unlikely in the case of colysis. We infer from these observations that villin proteolysis occurs within the first minute of coculture and before detergent lysis.
Experiments with conditioned medium from cocultures, sonicated and heat-inactivated trophozoites, indicate that adhesion of living trophozoites is a prerequisite for villin proteolysis as described also for other molecular interactions and final host cell killing (69, 35). Amebic adhesion to host cells required the Gal/N-acetylgalactosamine-specific lectin as was confirmed by inhibition of adhesion and prevention of villin proteolysis by 200 mM -lactose. Moreover, the steady-state pattern of villin bands on Western blots necessitates the presence of trophozoites evidenced by the rapid clearance of proteolytic bands by the enterocytes upon the removal of trophozoites.
Villin proteolysis was not restricted to cocultures with differentiated Caco-2 cell layers but also occurred with other colonic cell lines (see Fig. 2A) and in ex vivo cocultures with human colonic explants.2
Caspases have been implicated in apoptotic cell death of Jurkat cells by trophozoites in vitro (9). However, these proteinases were not likely to be implicated in villin proteolysis. Caspases were not activated within 2 h of coculture of Caco-2 cells with trophozoites as evidenced by the lack of cleavage of Ac-DVED-amc, a specific caspase substrate (36). Neither could villin proteolysis be prevented by z-VAD.fmk, an irreversible and highly specific caspase inhibitor that reduced the size of amebic liver abscess in severe combined immunodeficient mice infected intrahepatically with E. histolytica trophozoites (37). The cleavage of PARP and Bid (38, 39), early events in caspase-dependent apoptotic cell death, did not occur in cocultures of Caco-2 cell layers with trophozoites. The lack of caspase activation in our cocultures might be explained by the different origin, differentiation, and intercellular organization of the enteric Caco-2 cell layers as compared with the T leukemia cells, Jurkat, that were used by others (9).
EhCPs were considered as candidate villin proteinases because of their multifunctional role in host invasion (40, 41). We evaluated the role of EhCPs using proteinase inhibitors as well as transfected and non-pathogenic amebic strains. z-FA.fmk (100 µM), an irreversible inhibitor of cathepsin B-like proteinases (42), prevented Caco-2 cell layer destruction in cocultures,2 in line with previous observations on BHK cell layers (43). Treatment of cocultures with z-FA.fmk resulted in a reduced villin proteolysis. Concordantly, cocultures with amebic strains bearing a lower EhCP activity than HM-1:IMSS showed less villin proteolysis (see Fig. 5), whereas in coculture with Rahman, an amebic strain bearing comparable EhCP activity as HM-1:IMSS (44), villin was proteolysed as much as in cocultures with HM-1:IMSS.2 The amebic origin of the EhCP activity was inferred from pretreatment of trophozoites followed by coculture in the absence of the inhibitor. Villin immunoprecipitated on beads was proteolysed by amebic lysates in the test tube with the formation of the same proteolytic bands as in cocultures,2 pointing to a direct interaction of EhCPs with villin. It remains to be elucidated how EhCPs signal to villin inside the host cell and where the cleavage sites are located. Alternatively, EhCPs might interact indirectly via a signaling cascade starting from a ligand-receptor interaction at the host cell membrane.
We found a positive correlation between the disturbance of microvilli as revealed by ezrin immunocytochemistry and villin proteolysis on Western blots in cocultures with HM-1:IMSS either untreated or treated with 100 µM z-FA.fmk or 200 mM -lactose or in cocultures with a transfected amebic strain or a non-pathogenic strain. These results demonstrate that villin proteolysis is an efficient way to alter the structure of microvilli and confirm the need for villin in the maintenance of microvilli in vitro (16, 17). Effacement of microvilli associated with villin proteolysis has been reported before in the proximal tubule as a result of renal tubular injury (45). However, villin proteolysis is not the only mechanism of effacement of microvilli. Enteric bacterial pathogens such as S. typhimurium and EPEC also efface microvilli when cocultured with enterocytes (46). The lack of villin proteolysis in such cocultures indicates that molecular mechanisms associated with the effacement of microvilli vary in different microorganisms (46, 47). Such a comparison also shows that villin proteolysis cannot be considered as the consequence of effacement of microvilli by amebic trophozoites, in line with the kinetics of both phenomena. Villin proteolysis occurred within 1 min of coculture before any microscopical damage to Caco-2 cell layers was observed, and microvilli were only disturbed at least 15 min thereafter.
Microvilli are anti-invasive as they maintain the structure and absorptive function of the intestinal barrier (48, 49). Therefore, all of the infectious enteric microorganisms have interest in effacing microvilli from the surface of the enterocytes.
In conclusion, our experiments propose a new molecular event during the early steps of amebic invasion. Investigation of the phenomena downstream of villin proteolysis may broaden our understanding of cellular microbiology (1) in general and help in further understanding the pathogenesis of amebiasis, including the crucial role of amebic cysteine proteinases.
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FOOTNOTES |
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Supported by GOA 2002 from Ghent University.
¶ Recipient of a scholarship from the Portuguese Foundation for Science and Technology BD-15980.
** Supported by the Biotech Fonds.
Present address: Dept. of Microbiology, Faculty of Medicine, Technion, Haifa 31096, Israel.
¶¶ Postdoctoral fellow with the FWO-Vlaanderen, Belgium. To whom correspondence should be addressed. Tel.: 32-9-240-30-63; Fax: 32-9-240-49-91; E-mail: ancy.leroy{at}rug.ac.be.
1 The abbreviations used are: DMEM, Dulbecco's minimum essential medium; Ac-DEVD-amc, acetyl-Asp-Glu-Val-Asp-7-amino-4-methyl coumarin; DTT, dithiothreitol; EhCP, E. histolytica cysteine proteinase; EPEC, enteropathogenic Escherichia coli; E64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; pAb, polyclonal antibody; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; z-RR.pNA, benzyloxycarbonyl-Arg-Arg.p-nitroanilide; z-FA.fmk, benzyloxycarbonyl-Phe-Ala.fluoromethylketon; z-FF.fmk, benzyloxycarbonyl-Phe-Phe.fluoromethylketon; z-VAD.fmk, benzyloxycarbonyl-Val-AlaAsp.fluoromethylketon.
2 T. Lauwaet, M. Mareel, and A. Leroy, unpublished observation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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