Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin

Gut Pathogens - Tập 4 Số 1 - 2012
Manja Boehm1, Benjamin Hoy2, Manfred Rohde3, Nicole Tegtmeyer1, Kristoffer T. Bæk4, Omar A. Oyarzábal5, Lone Brøndsted4, Silja Weßler2, Steffen Backert6
1From the School for Medicine and Medical Science, University College Dublin, Belfield Campus, Dublin, Ireland
2The Division of Microbiology, University Salzburg, A-5020, Salzburg, Austria
3The Department of Medical Microbiology, Helmholtz Center for Infection Research, Inhoffen Str. 7, D-38124, Braunschweig, Germany
4The Department of Veterinary Disease Biology, University Copenhagen, Stigbøjlen 4, DK-1870, Frederiksberg C, Denmark
5Institute for Environmental Health, Inc., 15300 Bothell Way NE Lake Forest Park, Seattle, WA, 98155, USA
6University College Dublin, UCD School of Biomolecular and Biomedical Sciences, Science Center West L231, Belfield Campus, Dublin, Ireland

Tóm tắt

Abstract Background

Campylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.

 Results

In the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria.

Conclusion

These results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

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Tài liệu tham khảo

World Health Organization: Global burden of disease (GBD) 2002 estimates. WHO, 2004, Geneva, Switzerland,http://www.who.int/topics/global_burden_of_disease/en/

Young KT, Davis LM, DiRita VJ: Campylobacter jejuni: molecular biology and pathogenesis. Nat Rev Microbiol. 2007, 5: 665-679. 10.1038/nrmicro1718.

Nachamkin I: Szymanski CM, Blaser MJ: Campylobacter. ASM Press, Washington, DC, 2008.

Oyarzabal OA, Backert S: Microbial Food Safety: An Introduction. Springer Verlag, Heidelberg (Germany), in press

Blaser MJ, Engberg J: Clinical aspects of Campylobacter jejuni and Campylobacter coli infections. Campylobacter. Edited by: Nachamkin I, Szymanski CM, Blaser MJ. 2008, ASM Press, Washington, DC, 99-121. 3

Ketley JM: Pathogenesis of enteric infection by Campylobacter. Microbiology. 1997, 143: 5-21. 10.1099/00221287-143-1-5.

Wooldridge KG, Ketley JM: Campylobacter-host cell interactions. Trends Microbiol. 1997, 5: 96-102. 10.1016/S0966-842X(97)01004-4.

Dasti JI, Tareen AM, Lugert R, Zautner AE, Gross U: Campylobacter jejuni: a brief overview on pathogenicity-associated factors and disease-mediating mechanisms. Int J Med Microbiol. 2010, 300: 205-211. 10.1016/j.ijmm.2009.07.002.

van Spreeuwel JP, Duursma GC, Meijer CJ, Bax R, Rosekrans PC, Lindeman J: Campylobacter colitis: histological immunohistochemical and ultrastructural findings. Gut. 1985, 26: 945-951. 10.1136/gut.26.9.945.

Oelschlaeger TA, Guerry P, Kopecko DJ: Unusual microtubule-dependent endocytosis mechanisms triggered by Campylobacter jejuni and Citrobacter freundii. Proc Natl Acad Sci USA. 1993, 90: 6884-6888. 10.1073/pnas.90.14.6884.

Wooldridge KG, Williams PH, Ketley JM: Host signal transduction and endocytosis of Campylobacter jejuni. Microb Pathog. 1997, 21: 299-305.

Pei Z, Burucoa C, Grignon B, Baqar S, Huang XZ, Kopecko DJ, Bourgeois AL, Fauchere JL, Blaser MJ: Mutation in the peb1A locus of Campylobacter jejuni reduces interactions with epithelial cells and intestinal colonization of mice. Infect Immun. 1998, 66: 938-943.

Konkel ME, Monteville MR, Rivera-Amill V, Joens LA: The pathogenesis ofCampylobacter jejuni-mediated enteritis. Curr Issues Intest Microbiol. 2001, 2: 55-71.

Poly F, Guerry P: Pathogenesis of Campylobacter. Curr Opin Gastroenterol. 2008, 24: 27-31. 10.1097/MOG.0b013e3282f1dcb1.

Euker TP, Konkel ME: The cooperative action of bacterial fibronectin-binding proteins and secreted proteins promote maximal Campylobacter jejuni invasion of host cells by stimulating membrane ruffling. Cell Microbiol. 2012, 14: 226-238. 10.1111/j.1462-5822.2011.01714.x.

Moser I, Schroeder W, Salnikow J: Campylobacter jejuni major outer membrane protein and a 59-kDa protein are involved in binding to fibronectin and INT 407 cell membranes. FEMS Microbiol Lett. 1997, 157: 233-238. 10.1111/j.1574-6968.1997.tb12778.x.

Konkel ME, Gray SA, Kim BJ, Garvis SG, Yoon JJ: Identification of the enteropathogens Campylobacter jejuni and Campylobacter coli based on the cadF virulence gene and its product. Clin Microbiol. 1999, 37: 510-517.

Monteville MR, Yoon JE, Konkel ME: Maximal adherence and invasion of INT 407 cells by Campylobacter jejuni requires the CadF outer-membrane protein and microfilament reorganization. Microbiology. 2003, 149: 153-165. 10.1099/mic.0.25820-0.

Krause-Gruszczynska M, Rohde M, Hartig R, Genth H, Schmidt G, Keo T, Koenig W, Miller WG, Konkel ME, Backert S: Role of small Rho GTPases Rac1 and Cdc42 in host cell invasion of Campylobacter jejuni. Cell Microbiol. 2007, 9: 2431-2444. 10.1111/j.1462-5822.2007.00971.x.

Krause-Gruszczynska M, Boehm M, Rohde M, Tegtmeyer N, Takahashi S, Buday L, Oyarzabal OA, Backert S: The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2. Cell Commun Signal. 2011, 9: 32-10.3389/fcimb.2011.00017.

Boehm M, Krause-Gruszczynska M, Rohde M, Tegtmeyer N, Takahashi S, Oyarzabal OA, Backert S: Major host factors involved in epithelial cell invasion of Campylobacter jejuni: Role of fibronectin, integrin beta1, FAK, Tiam-1 and DOCK180 in activating Rho GTPase Rac1. Front Cell Infect Microbiol. in press

Kazmierczak BI, Mostov K, Engel JN: Interaction of bacterial pathogens with polarized epithelium. Annu Rev Microbiol. 2001, 55: 407-435. 10.1146/annurev.micro.55.1.407.

Tegtmeyer N, Wittelsberger R, Hartig R, Wessler S, Martinez-Quiles N, Backert S: Serine phosphorylation of cortactin controls focal adhesion kinase activity and cell scattering induced by Helicobacter pylori. Cell Host Microbe. 2011, 9: 520-531. 10.1016/j.chom.2011.05.007.

Balkovetz DF, Katz J: Bacterial invasion by a paracellular route: divide and conquer. Microbes Infect. 2003, 5: 613-619. 10.1016/S1286-4579(03)00089-3.

Konkel ME, Mead DJ, Hayes SF, Cieplak W: Translocation of Campylobacter jejuni across human polarized epithelial cell monolayer cultures. J Infect Dis. 1992, 166: 308-315. 10.1093/infdis/166.2.308.

Grant CCR, Konkel ME, Cieplak W, Tompkins LS: Role of flagella in adherence, internalization, and translocation of Campylobacter jejuni in nonpolarized and polarized epithelial cell cultures. Infect Immun. 1993, 61: 1764-1771.

Brás AM, Ketley JM: Transcellular translocation of Campylobacter jejuni across human polarised epithelial monolayers. FEMS Microbiol Lett. 1999, 179: 209-215.

Monteville MR, Konkel ME: Fibronectin-facilitated invasion of T84 eukaryotic cells by Campylobacter jejuni occurs preferentially at the basolateral cell surface. Infect Immun. 2002, 70: 6665-6671. 10.1128/IAI.70.12.6665-6671.2002.

Hu L, Tall BD, Curtis SK, Kopecko DJ: Enhanced microscopic definition of Campylobacter jejuni 81–176 adherence to, invasion of, translocation across, and exocytosis from polarized human intestinal Caco-2 cells. Infect Immun. 2008, 76: 5294-5304. 10.1128/IAI.01408-07.

Kalischuk LD, Inglis GD, Buret AG: Campylobacter jejuni induces transcellular translocation of commensal bacteria via lipid rafts. Gut Pathog. 2009, 1: 2-10.1186/1757-4749-1-2.

Ó Cróinín T, Backert S: Host epithelial cell invasion by Campylobacter jejuni: trigger or zipper mechanism?. Front Cell Infect Microbiol. 10.3389/fcimb.2012.00025. in press

Hoy B, Löwer M, Weydig C, Carra G, Tegtmeyer N, Geppert T, Schröder P, Sewald N, Backert S, Schneider G, Wessler S: Helicobacter pylori HtrA is a new secreted virulence factor that cleaves E-cadherin to disrupt intercellular adhesion. EMBO Rep. 2010, 11: 798-804. 10.1038/embor.2010.114.

Hoy B, Geppert T, Boehm M, Reisen F, Plattner P, Gadermaier G, Sewald N, Ferreira F, Briza P, Schneider G, Backert S, Wessler S: Distinct roles of secreted HtrA proteases from Gram-negative pathogens in cleaving the junctional protein and tumor suppressor E-cadherin. J Biol Chem. in press

Löwer M, Weydig C, Metzler D, Reuter A, Starzinski-Powitz A, Wessler S, Schneider G: Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA. PLoS One. 2008, 3: e3510-10.1371/journal.pone.0003510.

Brøndsted L, Andersen MT, Parker M, Jørgensen K, Ingmer H: The HtrA protease of Campylobacter jejuni is required for heat and oxygen tolerance and for optimal interaction with human epithelial cells. Appl Environ Microbiol. 2005, 71: 3205-3212. 10.1128/AEM.71.6.3205-3212.2005.

Bæk KT, Vegge CS, Brøndsted L: HtrA chaperone activity contributes to host cell binding in Campylobacter jejuni. Gut Pathog. 2011, 3: 13-10.1186/1757-4749-3-13.

Baek KT, Vegge CS, Skórko-Glonek J, Brøndsted L: Different contributions of HtrA protease and chaperone activities to Campylobacter jejuni stress tolerance and physiology. Appl Environ Microbiol. 2011, 77: 57-66. 10.1128/AEM.01603-10.

Krojer T, Sawa J, Schäfer E, Saibil HR, Ehrmann M, Clausen T: Structural basis for the regulated protease and chaperone function of DegP. Nature. 2008, 453: 885-890. 10.1038/nature07004.

Kim DY, Kim KK: Structure and function of HtrA family proteins, the key players in protein quality control. J Biochem Mol Biol. 2005, 38: 266-274. 10.5483/BMBRep.2005.38.3.266.

Clausen T, Southan C, Ehrmann M: The HtrA family of proteases: implications for protein composition and cell fate. Mol Cell. 2002, 10: 443-455. 10.1016/S1097-2765(02)00658-5.

Bumann D, Aksu S, Wendland M, Janek K, Zimny-Arndt U, Sabarth N, Meyer TF, Jungblut PR: Proteome analysis of secreted proteins of the gastric pathogen Helicobacter pylori. Infect Immun. 2002, 70: 3396-3403. 10.1128/IAI.70.7.3396-3403.2002.

Wroblewski LE, Shen L, Ogden S, Romero-Gallo J, Lapierre LA, Israel DA, Turner JR, Peek RM: Helicobacter pylori dysregulation of gastric epithelial tight junctions by urease-mediated myosin II activation. Gastroenterology. 2009, 136: 236-246. 10.1053/j.gastro.2008.10.011.

Cossart P, Sansonetti PJ: Bacterial invasion: the paradigms of enteroinvasive pathogens. Science. 2004, 304: 242-248. 10.1126/science.1090124.

Rottner K, Stradal TE, Wehland J: Bacteria-host-cell interactions at the plasma membrane: stories on actin cytoskeleton subversion. Dev Cell. 2005, 9: 3-17. 10.1016/j.devcel.2005.06.002.

Backert S, König W: Interplay of bacterial toxins with host defense: molecular mechanisms of immunomodulatory signaling. Int J Med Microbiol. 2005, 295: 519-530. 10.1016/j.ijmm.2005.06.011.

Gerlach RG, Hensel M: Salmonella pathogenicity islands in host specificity, host pathogen-interactions and antibiotics resistance of Salmonella enterica. Berl Munch Tierarztl Wochenschr. 2007, 120: 317-327.

Stecher B, Hardt WD: The role of microbiota in infectious disease. Trends Microbiol. 2008, 16: 107-114. 10.1016/j.tim.2007.12.008.

Tsolis RM, Young GM, Solnick JV, Bäumler AJ: From bench to bedside: stealth of enteroinvasive pathogens. Nat Rev Microbiol. 2008, 6: 883-892. 10.1038/nrmicro2012.

Everest PH, Goossens H, Butzler JP, Lloyd D, Knutton S, Ketley JM, Williams PH: Differentiated Caco-2 cells as a model for enteric invasion by Campylobacter jejuni and C. coli. J Med Microbiol. 1992, 37: 319-325. 10.1099/00222615-37-5-319.

Harvey P, Battle T, Leach S: Different invasion phenotypes of Campylobacter isolates in Caco-2 cell monolayers. J Med Microbiol. 1999, 48: 461-469. 10.1099/00222615-48-5-461.

Brás AM, Ketley JM: Transcellular translocation of Campylobacter jejuni across human polarised epithelial monolayers. FEMS Microbiol Lett. 1999, 179: 209-215.

Chen ML, Ge Z, Fox JG, Schauer DB: Disruption of tight junctions and induction of proinflammatory cytokine responses in colonic epithelial cells by Campylobacter jejuni. Infect Immun. 2006, 74: 6581-6589. 10.1128/IAI.00958-06.

Wine E, Chan VL, Sherman PM: Campylobacter jejuni mediated disruption of polarized epithelial monolayers is cell-type specific, time dependent, and correlates with bacterial invasion. Pediatr Res. 2008, 64: 599-604. 10.1203/PDR.0b013e31818702b9.

Pogacar MS, Klancnik A, Mozina SS, Cencic A: Attachment, invasion, and translocation of Campylobacter jejuni in pig small-intestinal epithelial cells. Foodborne Pathog Dis. 2010, 7: 589-595. 10.1089/fpd.2009.0301.

Biswas D, Niwa H, Itoh K: Infection with Campylobacter jejuni induces tyrosine-phosphorylated proteins into INT-407 cells. Microbiol Immunol. 2004, 48: 221-228.

Hu L, McDaniel JP, Kopecko DJ: Signal transduction events involved in human epithelial cell invasion by Campylobacter jejuni 81–176. Microb Pathog. 2006, 40: 91-100. 10.1016/j.micpath.2005.11.004.

Konkel ME, Garvis SD, Tipton S, Anderson DE, Cieplak W: Identification and molecular cloning of a gene encoding a fibronectin binding protein (CadF) from Campylobacter jejuni. Mol Microbiol. 1997, 24: 953-963. 10.1046/j.1365-2958.1997.4031771.x.

Goon S, Ewing CP, Lorenzo M, Pattarini D, Majam G, Guerry P: σ28-regulated nonflagella gene contributes to virulence of Campylobacter jejuni 81–176. Infect Immun. 2006, 74: 769-772. 10.1128/IAI.74.1.769-772.2006.

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