Antisense Inhibition of Entamoeba histolytica Cysteine Proteases Inhibits Colonic Mucus Degradation
2006; Elsevier BV; Volume: 130; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2005.11.012
ISSN1528-0012
AutoresDarcym Moncada, Kathy Keller, Serge Ankri, David Mirelman, Kris Chadee,
Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoBackground & Aims: The exact role Entamoeba histolytica cysteine proteases play in overcoming the colonic mucus barrier, as a prerequisite to epithelial cell disruption, is not known. Herein, we determined whether E histolytica trophozoites expressing the antisense transcript to cysteine protease 5 (EhCP5) could degrade colonic mucin and destroy epithelial cells. Methods: Cysteine protease–deficient amoebae were generated by antisense inhibition of EhCP5, and assayed for proteolytic activity against [35S]cysteine-labeled mucin from LS 174T, and HT-29F Cl.16E cells. Recombinant EhCP5 mucinase activity was also assessed. Disruption of an intact mucus barrier and epithelial cell invasion by amoebae were measured using high mucin producing LS 174T and HT-29 Cl.16E monolayers or Chinese hamster ovary (CHO) cells devoid of a mucus barrier. Results: Trophozoites with reduced cysteine protease activity were ineffective at degrading [35S]cysteine-labeled colonic mucin compared to wild-type amoebae by >60%. However, bioactive recombinant EhCP5 degraded >45% of purified native mucin, which was specifically inhibited by the cysteine proteinase (CP) inhibitor, E-64. Cysteine protease–deficient trophozoites could not overcome a protective intact mucus barrier and disrupt LS 174T or HT-29F Cl.16 cell monolayers; however, they readily adhere to and disrupt CHO monolayers devoid of a mucus barrier. Conclusions: These findings unravel a central role for E histolytica CPs as key virulence factors in disrupting an intact mucus barrier in the pathogenesis of intestinal amoebiasis. Background & Aims: The exact role Entamoeba histolytica cysteine proteases play in overcoming the colonic mucus barrier, as a prerequisite to epithelial cell disruption, is not known. Herein, we determined whether E histolytica trophozoites expressing the antisense transcript to cysteine protease 5 (EhCP5) could degrade colonic mucin and destroy epithelial cells. Methods: Cysteine protease–deficient amoebae were generated by antisense inhibition of EhCP5, and assayed for proteolytic activity against [35S]cysteine-labeled mucin from LS 174T, and HT-29F Cl.16E cells. Recombinant EhCP5 mucinase activity was also assessed. Disruption of an intact mucus barrier and epithelial cell invasion by amoebae were measured using high mucin producing LS 174T and HT-29 Cl.16E monolayers or Chinese hamster ovary (CHO) cells devoid of a mucus barrier. Results: Trophozoites with reduced cysteine protease activity were ineffective at degrading [35S]cysteine-labeled colonic mucin compared to wild-type amoebae by >60%. However, bioactive recombinant EhCP5 degraded >45% of purified native mucin, which was specifically inhibited by the cysteine proteinase (CP) inhibitor, E-64. Cysteine protease–deficient trophozoites could not overcome a protective intact mucus barrier and disrupt LS 174T or HT-29F Cl.16 cell monolayers; however, they readily adhere to and disrupt CHO monolayers devoid of a mucus barrier. Conclusions: These findings unravel a central role for E histolytica CPs as key virulence factors in disrupting an intact mucus barrier in the pathogenesis of intestinal amoebiasis. Gastrointestinal mucus acts as the first line of host defense against enteric parasites by preventing the attachment of microorganisms to enterocytes and directly influences the ability of pathogens to colonize the gastrointestinal tract. In addition, mucin also aids in the expulsion of intestinal microbes.1Theodoropoulos G. Hicks S.J. Corfield A.P. Miller B.G. Carrington S.D. The role of mucins in host-parasite interactions part II—helminth parasites.Trends Parasitol. 2001; 17: 130-135Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 2Hicks S.J. Theodoropoulos G. Carrington S.D. Corfield A.P. The role of mucins in host-parasite interactions. Part I-protozoan parasites.Parasitol Today. 2000; 16: 476-481Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 3Moncada D.M. Kammanadiminti S.J. Chadee K. Mucin and Toll-like receptors in host defense against intestinal parasites.Trends Parasitol. 2003; 19: 305-311Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar MUC2 is the major gel-forming mucin secreted in the colon and the monomer has a mass of ∼1.5 million Daltons.4Herrmann A. Davies J.R. Lindell G. Martensson S. Packer N.H. Swallow D.M. Carlstedt I. Studies on the "insoluble" glycoprotein complex from human colon. Identification of reduction-insensitive MUC2 oligomers and C-terminal cleavage.J Biol Chem. 1999; 274: 15828-15836Crossref PubMed Scopus (125) Google Scholar The oligosaccharide component accounts for up to 90% of the dry weight of the molecule and this dense glycosylation protects the mucin domains from proteolytic cleavage and is responsible for microbial binding and colonization. MUC2 forms polymers that, upon hydration, give rise to the visoelestic and protective properties of the mucus gel. The ability of MUC2 to form a gel depends on its nature to polymerize via the cysteine-rich D-domains by forming intramolecular disulfide bonds with corresponding termini of MUC2 monomers.4Herrmann A. Davies J.R. Lindell G. Martensson S. Packer N.H. Swallow D.M. Carlstedt I. Studies on the "insoluble" glycoprotein complex from human colon. Identification of reduction-insensitive MUC2 oligomers and C-terminal cleavage.J Biol Chem. 1999; 274: 15828-15836Crossref PubMed Scopus (125) Google Scholar, 5Asker N. Axelsson M.A. Olofsson S.O. Hansson G.C. Dimerization of the human MUC2 mucin in the endoplasmic reticulum is followed by an N-glycosylation-dependent transfer of the mono- and dimers to the Golgi apparatus.J Biol Chem. 1998; 273: 18857-18863Crossref PubMed Scopus (114) Google Scholar, 6Bell S.L. Khatri I.A. Xu G. Forstner J.F. Evidence that a peptide corresponding to the rat Muc2 C-terminus undergoes disulphide-mediated dimerization.Eur J Biochem. 1998; 253: 123-131Crossref PubMed Scopus (33) Google Scholar These domains are poorly glycosylated in comparison to the mucin domains and therefore are hypothesized to be vulnerable to proteolytic attack by enzymes released by invasive enteric pathogens. Breaching of the mucus barrier is a prerequisite to epithelial cell attachment by invasive microorganisms, and a limited number of studies have attempted to dissect the mechanisms used by pathogens to overcome the mucus layer during the course of infection. Degradation of the protein and/or oligosaccharide components of mucin by enteric pathogens is thought to be at least one strategy used by these organisms to weaken and traverse the mucus gel. Motility, as well as expression of the Zn2+-dependent metalloprotease Hap (mucinase) by Vibrio cholerae is necessary for the bacteria to translocate through intestinal mucus.7Silva A.J. Pham K. Benitez J.A. Haemagglutinin/protease expression and mucin gel penetration in El Tor biotype Vibrio cholerae.Microbiology. 2003; 149: 1883-1891Crossref PubMed Scopus (111) Google Scholar The enteric pathogens Candida albicans, Yersinia enterocolitica, Shigella flexneri, and Helicobacter pylori all produce virulence factors involved in mucus degradation.8Colina A.R. Aumont F. Deslauriers N. Belhumeur P. de Repentigny L. Evidence for degradation of gastrointestinal mucin by Candida albicans secretory aspartyl proteinase.Infect Immun. 1996; 64: 4514-4519PubMed Google Scholar, 9Mantle M. Husar S.D. Adhesion of Yersinia enterocolitica to purified rabbit and human intestinal mucin.Infect Immun. 1993; 61: 2340-2346PubMed Google Scholar, 10Henderson I.R. Czeczulin J. Eslava C. Noriega F. Nataro J.P. Characterization of pic, a secreted protease of Shigella flexneri and enteroaggregative Escherichia coli.Infect Immun. 1999; 67: 5587-5596Crossref PubMed Google Scholar, 11Slomiany B.L. Murty V.L. Piotrowski J. Liau Y.H. Sundaram P. Slomiany A. Glycosulfatase activity of Helicobacter pylori toward gastric mucin.Biochem Biophys Res Commun. 1992; 183: 506-513Crossref PubMed Scopus (35) Google Scholar Entamoeba histolytica is the etiologic agent of human amoebiasis, and the motile trophozoite form of the parasite colonizes the large bowel and invades the colonic epithelium resulting in mucosal damage and colitis. In the most severe cases of amoebiasis, the trophozoites enter the blood stream while invading mucosal tissue to cause liver abscesses, resulting in death if left untreated. The parasite produces cysteine proteinases (CPs) that are directly involved in tissue invasion through their ability to degrade extracellular matrix proteins, and play a key role in immune evasion by degrading host antibodies and complement.12Schulte W. Scholze H. Action of the major protease from Entamoeba histolytica on proteins of the extracellular matrix.J Protozool. 1989; 36: 538-543Crossref PubMed Scopus (76) Google Scholar, 13Tran V.Q. Herdman D.S. Torian B.E. Reed S.L. The neutral cysteine proteinase of Entamoeba histolytica degrades IgG and prevents its binding.J Infect Dis. 1998; 177: 508-511Crossref PubMed Scopus (65) Google Scholar, 14Reed S.L. Ember J.A. Herdman D.S. DiScipio R.G. Hugli T.E. Gigli I. The extracellular neutral cysteine proteinase of Entamoeba histolytica degrades anaphylatoxins C3a and C5a.J Immunol. 1995; 155: 266-274PubMed Google Scholar We have previously shown that E histolytica–secreted products effectively degrade colonic mucin and the degraded mucin could not inhibit amoebic adherence to target cells as well as the native molecule.15Moncada D. Keller K. Chadee K. Entamoeba histolytica cysteine proteinases disrupt the polymeric structure of colonic mucin and alter its protective function.Infect Immun. 2003; 71: 838-844Crossref PubMed Scopus (101) Google Scholar These results suggest that the polymeric structure of mucin must be maintained to protect the epithelium. The molecules involved in mucus degradation have not yet been identified, but cysteine proteases are likely to be the key virulence factors involved in disrupting the mucus barrier. Because breaching the mucus barrier is a prerequisite for invasion by the parasite, proteolytic cleavage of mucin by CPs would be sufficient to disrupt the polymeric nature of mucin and permit the parasite to make contact with the colonic epithelium. In this study, we specifically determined whether E histolytica CPs could disrupt colonic mucin by transfecting the parasite with the pSA8 plasmid expressing antisense to E histolytica cysteine proteinase 5 (EhCP5) to generate CP-deficient amoebae. This enabled us to directly examine the involvement of the CPs in the initial events of invasive amoebiasis using colonic epithelial cells that produce a protective mucus barrier. Our results show that the CPs are essential virulent components that abolish the cytoprotective function of mucin, enabling the parasite to bind and destroy colonic epithelial cells. LS 174T (ATCC; Rockville, MD) and HT-29F Cl.1616Augeron C. Laboisse C.L. Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate.Cancer Res. 1984; 44: 3961-3969PubMed Google Scholar cells were cultured to 70%–80% confluence in minimal essential medium (MEM) and DMEM medium (Invitrogen, Burlington, Ontario, Canada), respectively, supplemented with 10% fetal calf serum (Hyclone Laboratories, Logan, UT), 100 μg of streptomycin sulfate per milliliter, 100 U of penicillin per milliliter, and 20 mmol HEPES.15Moncada D. Keller K. Chadee K. Entamoeba histolytica cysteine proteinases disrupt the polymeric structure of colonic mucin and alter its protective function.Infect Immun. 2003; 71: 838-844Crossref PubMed Scopus (101) Google Scholar, 17Belley A. Keller K. Grove J. Chadee K. Interaction of LS174T human colon cancer cell mucins with Entamoeba histolyticaan in vitro model for colonic disease.Gastroenterology. 1996; 111: 1484-1492Abstract Full Text PDF PubMed Scopus (48) Google Scholar Chinese hamster ovary (CHO) cell cultures were maintained in F12 medium (Invitrogen) supplemented as previously. E histolytica HM-1:IMSS trophozoites were serially passaged through gerbil livers to maintain high virulence and were cultured axenically in TYI-S-33 medium at 36.6°C as described.17Belley A. Keller K. Grove J. Chadee K. Interaction of LS174T human colon cancer cell mucins with Entamoeba histolyticaan in vitro model for colonic disease.Gastroenterology. 1996; 111: 1484-1492Abstract Full Text PDF PubMed Scopus (48) Google Scholar Trophozoites were harvested after 72 hours at logarithmic growth phase by incubation on ice for 10 minutes followed by centrifugation (700 × g) for 5 minutes at 4°C. Secreted products were collected from trophozoites (2 × 107/mL) incubated in Hanks' balanced salt solution (Invitrogen) at 37°C for 2 hours as described elsewhere.18Yu Y. Chadee K. Entamoeba histolytica stimulates interleukin 8 from human colonic epithelial cells without parasite-enterocyte contact.Gastroenterology. 1997; 112: 1536-1547Abstract Full Text PDF PubMed Scopus (83) Google Scholar Trophozoites were transfected with the pSA8 or pEhAct-neo plasmids as described using a Bio-Rad gene Pulser (Bio-Rad Laboratories Ltd., Mississauga, Ontario, Canada).19Vines R.R. Purdy J.E. Ragland B.D. Samuelson J. Mann B.J. Petri Jr, W.A. Stable episomal transfection of Entamoeba histolytica.Mol Biochem Parasitol. 1995; 71: 265-267Crossref PubMed Scopus (60) Google Scholar The transfected parasites were allowed to recover for 48 hours prior to drug selection with G-418 sulfate (G418) (Invitrogen). The concentration of G418 was raised to 48 μg/mL over a 4-week period. Characterization of the transfectants by Southern blot analysis and for antisense expression by Northern blot analysis was performed as described elsewhere.20Ankri S. Stolarsky T. Bracha R. Padilla-Vaca F. Mirelman D. Antisense inhibition of expression of cysteine proteinases affects Entamoeba histolytica-induced formation of liver abscess in hamsters.Infect Immun. 1999; 67: 421-422Crossref PubMed Google Scholar CP activity was measured in E histolytica total cell lysates prepared by 3 freeze-thawing cycles. One unit of protease activity was defined as the μmol of substrate digested per min−1 mg−1 protein.21Leippe M. Sievertsen H.J. Tannich E. Horstmann R.D. Spontaneous release of cysteine proteinases but not of pore-forming peptides by viable Entamoeba histolytica.Parasitology. 1995; 111: 569-574Crossref PubMed Scopus (71) Google Scholar Protease activity was also monitored by zymogram analysis with a 12% polyacrylamide gel copolymerized with 0.1% gelatin (Sigma-Aldrich, Oakville, Ontario, Canada) as described elsewhere.22Hellberg A. Nickel R. Lotter H. Tannich E. Bruchhaus I. Overexpression of cysteine proteinase 2 in Entamoeba histolytica or Entamoeba dispar increases amoeba-induced monolayer destruction in vitro but does not augment amoebic liver abscess formation in gerbils.Cell Microbiol. 2001; 3: 13-20Crossref PubMed Scopus (82) Google Scholar EhCP5 was expressed in E coli strain BL21(DE3) [pAPlacIQ] using the expression vector pJC45. The recombinant protein was expressed as an insoluble histidine-tagged proenzyme and was solubilized, purified, and refolded as described elsewhere.23Hellberg A. Nowak N. Leippe M. Tannich E. Bruchhaus I. Recombinant expression and purification of an enzymatically active cysteine proteinase of the protozoan parasite Entamoeba histolytica.Protein Expr Purif. 2002; 24: 131-137Crossref PubMed Scopus (44) Google Scholar Processing of the recombinant enzyme to the mature active form was monitored by gelatin zymogram gel analysis and cleavage of the synthetic substrate benzyloxycarbonyl-l-arginyl-l-arginine-p-nitroanilide (z-Arg-Arg-pNA).21Leippe M. Sievertsen H.J. Tannich E. Horstmann R.D. Spontaneous release of cysteine proteinases but not of pore-forming peptides by viable Entamoeba histolytica.Parasitology. 1995; 111: 569-574Crossref PubMed Scopus (71) Google Scholar Adherence of E histolytica to CHO cells was performed using a standard protocol as described with modifications.24Chadee K. Petri Jr, W.A. Innes D.J. Ravdin J.I. Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica.J Clin Invest. 1987; 80: 1245-1254Crossref PubMed Scopus (219) Google Scholar Trophozoites were washed in M199s media (Invitrogen) supplemented with 5.7 mmol of cysteine, 25 mmol of HEPES, and 0.5% BSA (Sigma-Aldrich). The CHO cells were resuspended in media and incubated at a concentration of 20:1 with amoebae in a total volume of 1 mL of M199s. The cells were pelleted by centrifugation at 600 × g for 5 minutes at 4°C and incubated at the same temperature for 2 hours. Rosette formation is defined as the percentage of amoebae adherent to 3 or more CHO cells and was determined by counting >100 amoebae per condition from a total of 6 tubes per condition performed 3 times. Labeling of mucin with [35S]cysteine allows for tracking of the poorly glycosylated regions of MUC2. For the preparation of radiolabeled mucin, LS 174T and HT-29 Cl.16 cells were grown in medium containing 2 μCi/mL of [35S]cysteine (specific activity >1000 Ci/mmol; MP Biomedicals Inc., Irvine, CA) and the secreted mucin was purified as described.15Moncada D. Keller K. Chadee K. Entamoeba histolytica cysteine proteinases disrupt the polymeric structure of colonic mucin and alter its protective function.Infect Immun. 2003; 71: 838-844Crossref PubMed Scopus (101) Google Scholar, 17Belley A. Keller K. Grove J. Chadee K. Interaction of LS174T human colon cancer cell mucins with Entamoeba histolyticaan in vitro model for colonic disease.Gastroenterology. 1996; 111: 1484-1492Abstract Full Text PDF PubMed Scopus (48) Google Scholar Labeled mucin was incubated with either parasite cell lysate, secreted products, or recombinant EhCP5 in DPBS at 37°C. For analysis by sodium dodecyl sulfate (SDS)-PAGE, the samples were concentrated and resuspended in loading buffer (50 mmol Tris-HCl [pH 6.8], 10 mmol dithiothreitol; E-64, trans(epoxysuccinyl)-l-leucylamino-4-guanidinobutane, 2% SDS, 0.1% bromophenol blue, and 10% glycerol) and separated by SDS-PAGE with a 4% stacking and a 7% running gel. The dried gels were exposed to Kodak ZAR-5 film with an intensifying screen for 1–2 weeks at −70°C. The degradation was measured by densitometric analysis of high–molecular-weight stacking gel mucin as previously described using the NIH Image software (http://rsb.info.nih.gov/nih-image/).15Moncada D. Keller K. Chadee K. Entamoeba histolytica cysteine proteinases disrupt the polymeric structure of colonic mucin and alter its protective function.Infect Immun. 2003; 71: 838-844Crossref PubMed Scopus (101) Google Scholar, 25Belley A. Chadee K. Prostaglandin E(2) stimulates rat and human colonic mucin exocytosis via the EP(4) receptor.Gastroenterology. 1999; 117: 1352-1362Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Additional analyses of the digests were also conducted by Sepharose 4B gel filtration (column; 30 × 0.75 cm, Bio-Rad Laboratories) and 30 fractions of 0.5 mL were collected and subjected to liquid scintillation counting.15Moncada D. Keller K. Chadee K. Entamoeba histolytica cysteine proteinases disrupt the polymeric structure of colonic mucin and alter its protective function.Infect Immun. 2003; 71: 838-844Crossref PubMed Scopus (101) Google Scholar LS 174T, HT-29F Cl.16, and CHO cell monolayer destruction assays were performed with wild-type amoebae as well as pEhActNeo and pSA8 transfectants. Cell lines were seeded onto 24-well plates (Corning, NY) and grown to 80% confluency. E histolytica trophozoites were resuspended in M199s media supplemented as above, to a concentration of 1 × 105 amoebae/mL and when stated, parasites were incubated with 100 μmol E-64. The monolayers were washed 3 times with DPBS (37°C) and 1 mL of the trophozoite suspension was added to each well. The cells were incubated at 37°C for 3 hours to assess CHO and HT-29F Cl.16 cell monolayer destruction and 2, 3, 4, and 6 hours for LS 174T monolayer destruction assays. Trophozoites were removed by incubation at 4°C and by washing the monolayers three times with ice-cold DPBS. The remaining intact monolayers were fixed with 2.5% glutaraldehyde (Sigma-Aldrich) in DPBS and stained with 0.1% methylene blue in 100-mmol borate buffer pH 8.0. The stain was extracted from the cells in 1 mL of 1 N HCl and incubation at 37°C for 30 minutes. The absorbance for each well was measured at 660 nm and the amount of destruction was determined by calculating [A660 control wells − A660 experimental wells]/[A660 control wells] × 100.17Belley A. Keller K. Grove J. Chadee K. Interaction of LS174T human colon cancer cell mucins with Entamoeba histolyticaan in vitro model for colonic disease.Gastroenterology. 1996; 111: 1484-1492Abstract Full Text PDF PubMed Scopus (48) Google Scholar Survival of amoebae was determined using the Trypan blue exclusion assay. LS 174T and HT-29F Cl.16 cell lines were incubated with 1 μCi/ml [6-3H]glucosamine hydrochloride (40 Ci/mmol; MP Biomedicals Inc., Irvine, CA) for 48 hours and washed 3 times with MEM or DMEM/F-12 medium, respectively, prior to addition of test substances. Epithelial cells grown on 24-well plates were incubated with 20 μmol calcium ionophore A 23187 (Sigma-Aldrich), secreted products from E histolytica, M199s medium, or whole trophozoites (1 × 105) separated by a Millicell-HA culture plate insert with a pore size of 0.45 μmol (Millipore, Bedford, MA) for LS 174T secretion studies. Secreted [3H]-activity was determined for 100-μmol aliquots of medium collected after 4 hours of incubation by liquid scintillation counting as previously described.15Moncada D. Keller K. Chadee K. Entamoeba histolytica cysteine proteinases disrupt the polymeric structure of colonic mucin and alter its protective function.Infect Immun. 2003; 71: 838-844Crossref PubMed Scopus (101) Google Scholar The secreted mucin was analyzed by Sepharose 4B gel filtration as described previously. Data (mean ± SD) were analyzed using the Student t test with Prism 4 (GraphPad Software Inc., San Diego, CA) A P value of <.05% was considered statistically significant. To determine whether E histolytica trophozoites were successfully transfected with the pSA8 plasmid or the parental pEhAct-neo plasmid, Southern blot analysis was performed on trophozoite total genomic DNA. Genomic DNA isolated from the pEhAct-neo transfectants hybridized with the EhCP5 probe to reveal a band corresponding with the genomic copy of the gene, whereas pSA8 genomic DNA hybridized with the probe to reveal 2 bands, one corresponding to the genomic copy and the other, the episomal EhCP5 gene of plasmid origin (data not shown). Northern blot analysis confirmed that EhCP5 antisense transcripts were expressed in the pSA8 transfectants and were not present in the control transfectants (data not shown). Moreover, antisense expression increased in trophozoites grown in high concentrations of G418, and total CP activity against z-Arg-Arg-pNA was reduced by ∼90% in both the pSA8 transfectants grown in 48 or 60 μg/mL of G418. EhCP5 antisense mRNA inhibited EhCP5, as well as the expression of other cysteine proteases, which may be due to a high degree of sequence homology and conservation of all residues critical for protease function.20Ankri S. Stolarsky T. Bracha R. Padilla-Vaca F. Mirelman D. Antisense inhibition of expression of cysteine proteinases affects Entamoeba histolytica-induced formation of liver abscess in hamsters.Infect Immun. 1999; 67: 421-422Crossref PubMed Google Scholar, 26Bruchhaus I. Jacobs T. Leippe M. Tannich E. Entamoeba histolytica and Entamoeba dispardifferences in numbers and expression of cysteine proteinase genes.Mol Microbiol. 1996; 22: 255-263Crossref PubMed Scopus (159) Google Scholar We have previously shown that mucin degradation by E histolytica–secreted components was markedly inhibited by the cysteine protease inhibitor E-64.15Moncada D. Keller K. Chadee K. Entamoeba histolytica cysteine proteinases disrupt the polymeric structure of colonic mucin and alter its protective function.Infect Immun. 2003; 71: 838-844Crossref PubMed Scopus (101) Google Scholar To specifically determine the role of CPs in mucus gel disruption and epithelial cell invasion, we examined whether pSA8 transfected amoebae could degrade colonic mucin. Owing to the high molecular weight and abundant glycosylation of mucin, the native molecule remains in the 4% stacking gel when separated by SDS-PAGE, allowing degradation to be calculated by densitometric analysis of radiolabeled mucin. Figure 1A shows that the pEhAct-neo transfectants readily degrade the [35S]cysteine labeled N- and/or C-terminal flanking regions of LS 174T cell mucin similar to the wild-type amoebae (100% degradation). In contrast, the pSA8 strain showed a marked decrease in mucinase activity with trophozoites grown in 12, 24, and 48 μg/mL of G418-degrading high–molecular-weight mucin by only approximately 20% compared with the control transfectants (Figure 1A). Interestingly, pSA8 transfectants grown in 12 and 24 μg/mL of G418 with 6 μg/mL.27Ankri S. Stolarsky T. Mirelman D. Antisense inhibition of expression of cysteine proteinases does not affect Entamoeba histolytica cytopathic or haemolytic activity but inhibits phagocytosis.Mol Microbiol. 1998; 28: 777-785Crossref PubMed Scopus (107) Google Scholar Degradation products were also analyzed by Sepharose 4B gel filtration (Figure 1B). Native mucin collected from LS 174T cells elutes in the void volume (Vo) of a Sepharose 4B column calibrated with blue dextran (fractions 6–11). Incubation of labeled mucin with pEhAct-neo or wild-type lysate resulted in a net reduction of [35S]-labeled Vo mucin and a subsequent increase in lower molecular weight mucin fragments in the included fractions. pSA8 transfectants exhibited a significant loss of mucinase activity as seen by high levels of intact mucin eluting in the Vo and a lack of degradation fragments, indicative of CP-mediated disruption of the N- and/or C-terminal flanking regions of mucin. Degradation studies were also conducted using [35S]cysteine-labeled mucin from HT-29F Cl.16 cells to verify that the mucinase activity is not specific to LS 174T cell mucin. As shown in Figure 1C, E histolytica secretory components effectively degraded [35S]cysteine-labeled mucin collected from HT-29F Cl.16 cells when analyzed by SDS-PAGE and autoradiography. Furthermore, mucin degradation was significantly inhibited (∼50%) by E-64, indicating that CPs were responsible for the majority of the degradation. Analysis of the digests by sepharose 4B (S4B) gel filtration also revealed a similar elution pattern as LS 174T mucins under the same conditions (Figure 1D). Wild-type trophozoites overcome a mucus barrier and destroy LS 174T cell monolayers by 80% ± 3% within 3 hours of incubation (data not shown). However, when wild-type trophozoites were preincubated with the specific CP inhibitor, E-64, it prevented disruption of the mucus barrier and subsequent monolayer destruction by 75% ± 5%. These results suggest that CPs are used by the parasite to disrupt the mucus gel. To address whether CP activity was important in overcoming the protective mucin barrier, we tested the ability of the pEhAct-neo and pSA8 strains of the parasite to invade colonic epithelial cells. As shown in Figure 2A, the pEhAct-neo strain (12 μg/mL G418) readily overcomes the mucus barrier and kills LS 174T cells in a time-dependent manner. In contrast, the pSA8 transfectants grown in the presence of increasing concentrations of G418 (12, 24, and 48 μg/mL) (pSA8-12, -24, -48) showed decreased protease activity and a corresponding decrease in monolayer destruction. In the CP-deficient amoebae, only the pSA8-12 trophozoites were capable of causing a significant increase in monolayer destruction of 34% after 6 hours incubation; this strain retains only ∼45% of the CP activity of WT amoebae. The pSA8-24 and -48 transfectants with less than 20% and 10% of total cysteine protease activity, respectively, could not destroy an LS 174T monolayer with an intact mucus barrier after 6 hours of incubation. Monolayer destruction studies were also conducted using HT-29F Cl.16 cells (Figure 2B). As seen with the LS 174T cells, the pSA8 transfectants exhibited a marked decrease in their ability to migrate through an intact mucus barrier and disrupt HT-29F Cl.16 cell monolayers. This decrease in destruction was shown to correlate with a reduction in cysteine protease activity with pSA8-12, -24, and -48 destroying only 52%, 25%, and 11% of monolayers, respectively, compared to 83% for control transfectants after 3 hours of incubation. Because CPs are critically important in disrupting the mucus gel, we tested whether wild-type and CP-deficient amoebae could equally destroy a CHO cell monolayer devoid of a protective mucus barrier. CHO cells are more susceptible to invasion than LS 174T cells because they do not secrete gel-forming mucins and were used to determine the actual contribution of the CPs in mucus disruption versus epithelial cell invasion. As shown in Figure 2C, the pEhAct-neo strain destroyed 74% of a CHO monolayer, whereas the pSA8-12 destroyed 73% within 3 hours, and a 39% increase in monolayer destruction compared with LS 174T monolayer invasion for the same time point. Although the pSA8-24 and -48 were unable to significantly destroy an LS 174T monolayer, both transfectants retained their ability to destroy a CHO monolayer (Figure 2C), with the pSA8-24 destroying 57% and th
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