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Effects of Helicobacter hepaticus on the proteome of HEp-2 cells

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Abstract

Helicobacter hepaticus infects the bowel and biliary tree of several animals, producing inflammation. Colonisation of mouse livers can induce hepatocellular carcinomas. The effects of H. hepaticus on the proliferation and global protein expression of human HEp-2 cells were studied by examining the changes in the protein profiles of cells exposed to the bacterium. HEp-2 cells were grown for four days under a microaerobic atmosphere or under the same conditions in co-cultures with H. hepaticus at various inoculum densities. Enlargement, distension and elongation of HEp-2 cells were observed in co-cultures with H. hepaticus. The number of live cells declined by only an order of magnitude at bacterial inocula of ∼109 cfu/ml, but were reduced to less than 103 cells/ml at ∼1010 cfu/ml bacteria inocula. Protein expression by HEp-2 cells was investigated employing two-dimensional gel electrophoresis. In cells grown with or without bacteria, 17 differentially expressed proteins were identified by tandem mass spectrometry. These proteins participated in several biological functions including amino acid metabolism, cell growth and proliferation, stress response, protein translation and modification, etc. The onset of a catastrophic killing of HEp-2 cells at a bacterial density of ∼109 cfu/ml suggested a multimodal action for H. hepaticus infection, and the modulation of the expression of proteins involved in different biological functions showed that the presence of H. hepaticus has broad effects on the physiology of HEp-2 cells.

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References

  • Aragon V, Chao K, Drayfus LA (1997) Effect of cytolethal distending toxin on F-actin assembly and cell division in Chinese hamster ovary cells. Infect Immun 65:3774–3780

    PubMed  CAS  Google Scholar 

  • Avenaud P, Marais A, Monteiro L, Le Bail B, Bioulac Sage P, Balabaud C, Megraud F (2000) Detection of Helicobacter species in the liver of patients with and without primary liver carcinoma. Cancer 89:1431–1439

    Article  PubMed  CAS  Google Scholar 

  • Blaser MJ, Parsonnet J (1994) Parasitism by the “slow” bacterium Helicobacter pylori leads to altered gastric homeostasis and neoplasia. J Clin Invest 94:4–8

    Article  PubMed  CAS  Google Scholar 

  • Bolton AJ, Osborne MP, Stephen J (2000) Comparative study of the invasiveness of Salmonella serotypes Typhimurium, Choleraesuis and Dublin for Caco-2 cells, HEp-2 cells and rabbit ileal epithelia. J Med Microbiol 49:503–511

    PubMed  CAS  Google Scholar 

  • Boutin SR, Rogers AB, Shen Z, Fry RC, Love JA, Nambiar PR, Suerbaum S, Fox JG (2004) Hepatic temporal gene expression profiling in Helicobacter hepaticus-infected A/JCr Mice. Toxicol Pathol 32:678–693

    Article  PubMed  Google Scholar 

  • Brattsand G, Marklund U, Nylander K, Roos G, Gullberg M (1994) Cell-cycle regulated phosphorylation of oncoprotein 18 on Ser16, Ser25 and Ser38. Eur J Biochem 220:359–368

    Article  PubMed  CAS  Google Scholar 

  • Brentani RR (1989) The molecular basis of the metastatic phenotype. Crit Rev Oncog 1:247–260

    PubMed  CAS  Google Scholar 

  • Bulajic M, Maisonneuve P, Schnieder-Brachert W, Muller P, Reishcl U, Stimec B, Lehn N, Lowenfels AB, Lohr M (2002) Helicobacter pylori and the risk of benign and malignant biliary tract disease. Cancer 95:1946–1953

    Article  PubMed  Google Scholar 

  • Cervinka M, Cerman J, Rudolf E (2003) The role of apoptosis in pituitary adenomas in the field of conventionally used therapeutic approaches. Ann N Y Acad Sci 1010:520–524

    Article  PubMed  CAS  Google Scholar 

  • Correa P (1992) Human gastric carcinogenesis: a multistep and multifactorial process—first American cancer society award on cancer epidemiology and prevention. Cancer Res 52:6735–6750

    PubMed  CAS  Google Scholar 

  • Curmi PA, Gavet O, Charbaut E, Ozon S, Lachkar-Colmerauer S, Manceau V, Siavoshian S, Maucuer A, Sobel A (1999) Stathmin and its phosphoprotein family: general properties, biochemical and functional interaction with tubulin. Cell Struct Funct 24:345–357

    Article  PubMed  CAS  Google Scholar 

  • Dore MP, Realdi G, Mura D, Graham DY, Sepulveda AR (2002) Helicobacter infection in patients with HCV-related chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Dig Dis Sci 47:1638–1643

    Article  PubMed  Google Scholar 

  • Dovas A, Couchman JR (2005) Rho GDI: multiple functions in the regulation of Rho family GTPase activities. Biochem J 390:1–9

    Article  PubMed  CAS  Google Scholar 

  • Du L, Fukushima S, Sallmyr A, Manthorpe R, Bredberg A (2002) Exposure of HEp-2 cells to stress conditions influences antinuclear antibody reactivity. Clin Diagn Lab Immunol 9:287–294

    Article  PubMed  CAS  Google Scholar 

  • Fan XG, Peng XN, Huang Y, Yakoob J, Wang ZM, Chen YP (2002) Helicobacter species ribosomal DNA recovered from the liver tissue of Chinese patients with primary hepatocellular carcinoma. Clin Infec Dis 35:1555–1557

    Article  CAS  Google Scholar 

  • Fox JG, Dewhirst FE, Shen Z, Feng Y, Taylor NS, Paster BJ, Ericson RL, Lau CN, Correa P, Araya JC, Roa I (1998) Hepatic Helicobacter species identified in bile and gallbladder tissue from Chileans with chronic cholecystitis. Gastroenterology 114:755–763

    Article  PubMed  CAS  Google Scholar 

  • Fox JG, Dewhirst FE, Tully JG, Paster BJ, Yan L, Taylor NS, Collins MJ Jr, Gorelick PL, Ward JM (1994) Helicobacter hepaticus sp Nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J Clin Microbiol 32:1238–1245

    PubMed  CAS  Google Scholar 

  • Fox JG, Li X, Yan L, Cahill RJ, Hurley R, Lewis R, Murphy JC (1996) Chronic proliferative hepatitis in A/JCr mice associated with persistent Helicobacter hepaticus infection: a model of Helicobacter-induced carcinogenesis. Infec Immun 64:1548–1558

    CAS  Google Scholar 

  • Fox EM, Raftery M, Goodchild A, Mendz GL (2007) Campylobacter jejuni response to ox-bile stress. FEMS Immun Med Microbiol 49:165–172

    Article  CAS  Google Scholar 

  • Fukuda K, Tamotsu K, Yoshitsugu T, Noritsugu T, Tomoo K, Sumihiro M, Junichiro F, Takashi K (2002) Comparative analysis of Helicobacter DNA and biliary pathology in patients with and without hepatobiliary cancer. Carcinogenesis 23:1927–1932

    Article  PubMed  CAS  Google Scholar 

  • Garcia A, Erdman SE, Xu S, Feng Y, Rogers AB, Schrenzel MD, Murphy JC, Fox JG (2002) Hepatobiliary inflammation, neoplasia, and argyrophilic bacteria in a ferret colony. Vet Pathol 39:173–179

    Article  PubMed  CAS  Google Scholar 

  • Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 17:994–999

    Article  PubMed  CAS  Google Scholar 

  • Hailey JR, Haseman JK, Bucher JR, Radovsky AE, Malarkey DE, Miller RT, Nyska A, Maronpot RR (1998) Impact of Helicobacter hepaticus infection in B6C3F1 mice from twelve National Toxicology Program two-year carcinogenesis studies. Toxicol Pathol 26:602–611

    PubMed  CAS  Google Scholar 

  • Halligan DB, Ruotti V, Jin W, Laffoon S, Twigger SN, Dratz EA (2004) ProMost (Protein Modification Screening Tool): a web-based tool for mapping protein modifications on two-dimensional gels. Nucleic Acid Res 32:638–644

    Article  CAS  Google Scholar 

  • Ihrig M, Schrenzel MD, Fox JG (1999) Differential susceptibility to hepatic inflammation and proliferation in AXB recombinant inbred mice chronically infected with Helicobacter hepaticus. Am J Pathol 155:571–582

    PubMed  CAS  Google Scholar 

  • Johnson WM, Lior H (1988) A new heat-labile cytolethal distending toxin (CDT) produced by Campylobacter spp. Microb Pathogen 4:115–126

    Article  CAS  Google Scholar 

  • Larsson N, Melander H, Marklund U, Osterman O, Gullberg M (1995) G21M transition requires multisite phosphorylation of oncoprotein 18 by two distinct protein kinase systems. J Biol Chem 270:14175–14183

    Article  PubMed  CAS  Google Scholar 

  • Li C, Hong Y, Tan YX, Zhou H, Ai JH, Li SJ, Zhang L, Xia QC, Wu JR, Wang HY, Zeng R (2004) Accurate qualitative and quantitative proteomic analysis of clinical hepatocellular carcinoma using laser capture microdissection coupled with isotope-coded affinity tag and two-dimensional liquid chromatography mass spectrometry. Mol Cell Proteomics 3:399–409

    Article  PubMed  CAS  Google Scholar 

  • Lin Z, Crockett DK, Jenson SD, Lim MS, Elenitoba-Johnson KS (2004) Quantitative proteomic and transcriptional analysis of the response to the p38 mitogen-activated protein kinase inhibitor SB203580 in transformed follicular lymphoma cells. Mol Cell Proteomics 3:820–833

    Article  PubMed  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  • Luo XN, Mookerjee B, Ferrari A, Misty S, Atweh GF (1994) Regulation of phosphoprotein p18 in leukemia cells. Cell cycle regulated phosphorylation by p34cdc2 kinase. J Biol Chem 269:10312–10318

    PubMed  CAS  Google Scholar 

  • Mandell L, Moran A, Cocchiarella A, Houghton J, Taylor N, Fox JG, Wang TC, Kurt-Jones EA (2004) Intact Gram-negative Helicobacter pylori, Helicobacter felis, and Helicobacter hepaticus bacteria activate innate immunity via toll-like receptor 2 but not toll-like receptor 4. Infect Immun 72:6446–6454

    Article  PubMed  CAS  Google Scholar 

  • Marklund U, Osterman O, Melander H, Bergh A, Gullberg M (1994) The phenotype of a “Cdc2 kinase target site-deficient” mutant of oncoprotein 18 reveals a role of this protein in cell cycle control. J Biol Chem 269:30626–30635

    PubMed  CAS  Google Scholar 

  • Matsukura N, Yokomuro S, Yamada S, Tajiri T, Sundo T, Hadama T, Kamiya S, Naito Z, Fox JG (2002) Association between Helicobacter bilis in bile and biliary tract malignancies: H. bilis in bile from Japanese and Thai patients with benign and malignant diseases in the biliary tract. Jpn J Cancer Res 93:842–847

    PubMed  CAS  Google Scholar 

  • Nahmias AJ, Eickhoff TC (1961) Staphylococcal infection in hospitals—recent developments in epidemiological and laboratory investigations. New Eng J Med 265:74–81

    Article  PubMed  CAS  Google Scholar 

  • Niimi S, Oshizawa T, Yamaguchi T, Harashima M, Seki T, Ariga T, Kawanishi T, Hayakawa T (2003) Specific expression of annexin III in rat-small-hepatocytes. Biochem Biophys Res Commun 300:770–774

    Article  PubMed  CAS  Google Scholar 

  • Nilsson H, Mulchandani R, Tranberg KG, Stenram U, Wadstrom T (2001) Helicobacter species identified in liver from patients with cholangiocarcinoma and hepatocellular carcinoma. Gastroenterology 120:323–324

    Article  PubMed  CAS  Google Scholar 

  • Nilsson HO, Taneera J, Castedal M, Glatz E, Olsson R, Wadstrom T (2000) Identification of Helicobacter pylori and other Helicobacter species by PCR, hybridization, and partial DNA sequencing in human liver samples from patients with primary sclerosing cholangitis or primary biliary cirrhosis. J Clin Microbiol 38:1072–1076

    PubMed  CAS  Google Scholar 

  • Parsonnet J, Hansen S, Rodriguez L, Gelb AB, Wamke RA, Jellum E, Orentreich N, Vogelman JH, Friedman GD (1994) Helicobacter pylori infection and gastric lymphoma. N Engl J Med 330:1267–1271

    Article  PubMed  CAS  Google Scholar 

  • Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:1–10

    Article  Google Scholar 

  • Pellicano R, Mladenova I, Dimitrova SM, Bruno CM, Sciacca C, Rizzetto M (2004) The epidermiology of hepatitis C virus infection. An update for clinicians. Gastroenterol Dietol 50:1–7

    CAS  Google Scholar 

  • Polager S, Ginsberg D (2003) E2F mediates sustained G2 arrest and down-regulation of stathmin and AIM-1 expression in response to genotoxic stress. J Biol Chem 278:1443–1449

    Article  PubMed  CAS  Google Scholar 

  • Rabilloud T, Vuillard L, Gilly C, Lawrence JJ (1994) Silver-staining of proteins in polyacrylamide gels: a general overview. Cell Mol Biol 40:57–75

    PubMed  CAS  Google Scholar 

  • Silva CP, Pereira-Lima JC, Oliveira AG, Guerra JB, Marques DL, Sarmanho L, Cabral MM (2003) Association of the presence of Helicobacter in gallbladder tissue with cholelithiasis and cholecystitis. J Clin Microbiol 41:5615–5618

    Article  PubMed  Google Scholar 

  • Strahler JR, Lamb BJ, Ungar DR, Fox DA, Hanash SM (1992) Cell cycle progression is associated with distinct patterns of phosphorylation of Op18. Biochem Biophys Res Commun 185:197–203

    Article  PubMed  CAS  Google Scholar 

  • Verhoef C, de Man RA, Zondervan PE, Eijkemans MJ, Tilanus HW, Ijzermans JN (2003) Good outcomes after resection of large hepatocellular carcinoma in the non-cirrhotic liver. Dig Surg 21:380–386

    Article  PubMed  Google Scholar 

  • Vicentini AP, Gesztesi LR, Franco MF, de Souza W, de Moraes JZ, Travassos LR, Lopes JD (1994) Binding of Paracoccidioides brasiliensis to laminin through surface glycoprotein gp43 leads to enhancement of fungal pathogenesis. Infect Immun 62:1465–1469

    PubMed  CAS  Google Scholar 

  • Ward JM, Anver MR, Haines DC, Melhorn JM, Gorelick P, Yan L, Fox JG (1996) Inflammatory large bowel disease in immunodeficient mice naturally infected with Helicobacter hepaticus. Lab Anim Sci 46:15–20

    PubMed  CAS  Google Scholar 

  • Ward JM, Fox JG, Anver MR, Haines DC, George CV, Collins MJ Jr, Gorelick PL, Nagashima K, Gonda MA, Gilden RV (1994) Chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species. J Natl Cancer Inst 86:1222–1227

    Article  PubMed  CAS  Google Scholar 

  • Yanagisawa N, Geironson L, Al-Soud WA, Ljungh S (2005) Expression of matrix metalloprotease-2,-7 and -9 on human colon, liver and bile duct cell lines by enteric and gastric Helicobacter species. FEMS Immuno Med Microbiol 44:197–204

    Article  CAS  Google Scholar 

  • Yoshida S, Sasakawa C (2003) Exploring host microtubule dynamics: a new aspect of bacterial invasion. Trends Microbiol 11:139–143

    Article  PubMed  CAS  Google Scholar 

  • Zang L, Palmer TD, Hancock WS, Sgroi DC, Karger BL (2004) Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and 16O/18O isotopic labeling. J Proteome Res 3:604–612

    Article  PubMed  CAS  Google Scholar 

  • Zhang L, Day A, McKenzie G, Mitchell H (2006) Nongastric Helicobacter species detected in the intestinal tract of children. J Clin Microbiol 44:2276–2279

    Article  PubMed  CAS  Google Scholar 

  • Zhou G, Li H, DeCamp D, Chen S, Shu H, Gong Y, Flaig M, Gillespie JW, Hu N, Taylor PR, Emmert-Buck MR, Liotta LA, Petricoin EF 3rd, Zhao Y (2002) 2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers. Mol Cell Proteomics 1:117–124

    Google Scholar 

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Acknowledgements

This work was supported by the Cancer Council of New South Wales and the Australian Research Council. Mr. Arinze S. Okoli acknowledges the support of an Endeavour International Postgraduate Research Scholarship. The authors are thankful to Mr. Johnny Q. V. Tu for technical assistance.

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Authors and Affiliations

  1. School of Medical Sciences, The University of New South Wales, Sydney, NSW, 2052, Australia

    Arinze S. Okoli, Edward M. Fox & George L. Mendz

  2. Bioanalytical Mass Spectrometry Facility, The University of New South Wales, Sydney, NSW, 2052, Australia

    Mark J. Raftery

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  1. Arinze S. Okoli
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  2. Edward M. Fox
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  3. Mark J. Raftery
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  4. George L. Mendz
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Correspondence to George L. Mendz.

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Okoli, A.S., Fox, E.M., Raftery, M.J. et al. Effects of Helicobacter hepaticus on the proteome of HEp-2 cells. Antonie van Leeuwenhoek 92, 289–300 (2007). https://doi.org/10.1007/s10482-007-9155-5

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  • DOI: https://doi.org/10.1007/s10482-007-9155-5

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