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Perturbation of the epithelial barrier by enteric pathogens
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Gastrointestinal infections in humans have been associated with a number of diseased condition, including stomach ulcers, gastroenteritis, Crohn's disease, and rheumatic arthritis. Such infections often cause altered intestinal permeability through perturbation of the tight junctions that hold epithelial cells together. The objective of the present studies was to detennine whether the enteric pathogens Salmonella, Yersinia, and Rotavirus can disrupt the integrity of the epithelial barrier, and, if so, how this is achieved. Another aim was to elucidate regulation of the epithelial batrier in relation to the structure of the cytoskeleton.

To accomplish these goals, we assessed the mechanism of enhanced cytotoxicity of Yersinia YopE and the response to this protein by its target in the epithelial bartier, both of which require contact between the bacteria and the eukaryotic cells. YopK appeared to control Yop effector delivery by regulating the size of the translocation pore, and enhanced translocation was accompanied by decreased transepithelial resistance and disruption of barrier function. We also examined the interaction of Yersinia with polarized MDCK cells to detemrine the target of these bacteria. We found that wild-type Yersinia adhered apically to the tight junction areas, and, in adjacent cells, these contact points displayed ß1 integrins and tight junction proteins that allowed localized invasin-mediated binding and translocation of cytotoxins. Studying signal transduction pathways involved in the disruption of barrier function by Salmonella typhimurium, we found that infection with the wild-type strain increased the level of activated. Rac1 and Cdc42 small G-proteins and caused them to accumulate apically in MDCK cells, and this was prevented by appropriate inhibitors. Activation of these proteins was a prerequisite of disruption of barrier integrity by S. typhimurium. We also considered specific effects of the rota virus non-structural protein NSP4 on the function of tight junctions. NSP4 has been desctibed as the first viral enterotoxin, and we found that incubation of noncontluent MDCK-1 cells with NSP4 prevented development of the permeability barrier, as well as lateral targeting of the tight junction-associated zonula occludence-1 protein.

In conclusion, our results provide strong evidence that the studied pathogens perturb the epithelial barrier by binding to specific cell receptors to deliver cytotoxins (Yesinia); by interfering with cell signaling pathways (Salmonella); and by impairing normal formation of tight junctions (NSP4).

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2001. , 58 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 702
Keyword [en]
enteric pathogens: signal transduction, barrier function
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-28644Local ID: 13800ISBN: 91-7373-146-3 (print)OAI: oai:DiVA.org:liu-28644DiVA: diva2:249455
Public defence
2001-12-03, Berzeliussalen, Universitetssjukhuset, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-11-22Bibliographically approved
List of papers
1. YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane
Open this publication in new window or tab >>YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane
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1997 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 24, no 1, 73-91 p.Article in journal (Refereed) Published
Abstract [en]

Introduction of anti-host factors into eukaryotic cells by extracellular bacteria is a strategy evolved by several Gram-negative pathogens. In these pathogens, the transport of virulence proteins across the bacterial membranes is governed by closely related type III secretion systems. For pathogenic Yersinia, the protein transport across the eukaryotic cell membrane occurs by a polarized mechanism requiring two secreted proteins, YopB and YopD. YopB was recently shown to induce the formation of a pore in the eukaryotic cell membrane, and through this pore, translocation of Yop effectors is believed to occur (Håkansson et al., 1996b). We have previously shown that YopK of Yersinia pseudotuberculosis is required for the development of a systemic infection in mice. Here, we have analysed the role of YopK in the virulence process in more detail. A yopK-mutant strain was found to induce a more rapid YopE-mediated cytotoxic response in HeLa cells as well as in MDCK-1 cells compared to the wild-type strain. We found that this was the result of a cell-contact-dependent increase in translocation of YopE into HeLa cells. In contrast, overexpression of YopK resulted in impaired translocation. In addition, we found that YopK also influenced the YopB-dependent lytic effect on sheep erythrocytes as well as on HeLa cells. A yopK-mutant strain showed a higher lytic activity and the induced pore was larger compared to the corresponding wild-type strain, whereas a strain overexpressing YopK reduced the lytic activity and the apparent pore size was smaller. The secreted YopK protein was found not to be translocated but, similar to YopB, localized to cell-associated bacteria during infection of HeLa cells. Based on these results, we propose a model where YopK controls the translocation of Yop effectors into eukaryotic cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80196 (URN)10.1046/j.1365-2958.1997.3211681.x (DOI)
Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2017-12-07Bibliographically approved
2. Apically Exposed, Tight Junction-Associated β1-Integrins Allow Binding and YopE-Mediated Perturbation of Epithelial Barriers by Wild-Type Yersinia Bacteri
Open this publication in new window or tab >>Apically Exposed, Tight Junction-Associated β1-Integrins Allow Binding and YopE-Mediated Perturbation of Epithelial Barriers by Wild-Type Yersinia Bacteri
2000 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 68, no 9, 5335-5343 p.Article in journal (Refereed) Published
Abstract [en]

Using polarized epithelial cells, primarily MDCK-1, we assessed the mode of binding and effects on epithelial cell structure and permeability of Yersinia pseudotuberculosis yadA-deficient mutants. Initially, all bacteria except the invasin-deficient (inv) mutant adhered apically to the tight junction areas. These contact points of adjacent cells displayed β1-integrins together with tight junction-associated ZO-1 and occludin proteins. Indeed, β1-integrin expression was maximal in the tight junction area and then gradually decreased along the basolateral membranes. Wild-type bacteria also opened gradually the tight junction to paracellular permeation of different-sized markers, viz., 20-, 40-, and 70-kDa dextrans and 45-kDa ovalbumin, as well as to their own translocation between adjacent cells in intimate contact with β1-integrins. The effects on the epithelial cells and their barrier properties could primarily be attributed to expression of the Yersinia outer membrane protein YopE, as the yopE mutant bound but caused no cytotoxicity. Moreover, the apical structure of filamentous actin (F-actin) was disturbed and tight junction-associated proteins (ZO-1 and occludin) were dispersed along the basolateral membranes. It is concluded that the Yersinia bacteria attach to β1-integrins at tight junctions. Via this localized injection of YopE, they perturb the F-actin structure and distribution of proteins forming and regulating tight junctions. Thereby they promote paracellular translocation of bacteria and soluble compounds.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26055 (URN)10.1128/IAI.68.9.5335-5343.2000 (DOI)10514 (Local ID)10514 (Archive number)10514 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
3. Disruption of Barrier Integrity by Salmonella typhimurium Requires Activation of Cdc42 and Rac1 in Epithelial Cells
Open this publication in new window or tab >>Disruption of Barrier Integrity by Salmonella typhimurium Requires Activation of Cdc42 and Rac1 in Epithelial Cells
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Epithelial cells that line the human intestinal mucosa constitute the initial sites of host invasion by bacterial pathogens. A number of bacteria, such as Salmonella and Yersinia, have been shown to disrupt the integrity of the epithelial barrier, although little is known about the mechanisms underlying that effect. We found that polarized MDCK-1 epithelial cells infected with invasive Salmonella typhimurium SLI344 exhibited marked changes in F-actin organization and a rapid decrease in transepithelial electrical resistance (TER). In contrast, infection with isogenic noninvasive mutants (hilA, prgH, and sipC) increased the TER in these cells. Pretreating MDCK-1 cells with the tyrosine kinase inhibitor genistein or the PI-3 kinase inhibitor wortmannin did not affect invasion and subsequent perturbation of the epithelial barrier by S. typhimurium. Instead, the specific geranylgeranyltransferase-1 inhibitor GGTI-298, but not the farnesyltransferase inhibitor FTI-277, clearly reversed the capacity of S. typhimurium to disrupt the epithelial barrier. The best-known substrates for GGTI-298 include Rho family GTPases, Racl and Cdc42. Infection with wild-type S. typhimurium increased the level of activated Racl and Cdc42 and caused these proteins to accumulate apically in MDCK-1 cells. GGTI-298-induced inactivation of Racl and Cdc42 prevented alteration of the tight and adherens junction-associated proteins Z0-1, occludin, and E-cadherin in MDCK-1 cells infected with invasive Salmonella. These results indicate that activation of Racl and Cdc42, but not tyrosine kinase or PI-3 kinase, is essential for disruption of barrier integrity by S. typhimurium in polarized MDCK-1 cells.

Keyword
Salmonella typhimurium, epithelium, tight junctions, Rac1, Cdc42
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80200 (URN)
Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2012-08-22Bibliographically approved
4. NSP4 Enterotoxin of Rotavirus Induces Paracellular Leakage in Polarized Epithelial Cells
Open this publication in new window or tab >>NSP4 Enterotoxin of Rotavirus Induces Paracellular Leakage in Polarized Epithelial Cells
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2001 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 75, no 3, 1540-1546 p.Article in journal (Refereed) Published
Abstract [en]

The nonstructural NSP4 protein of rotavirus has been described as the first viral enterotoxin. In this study we have examined the effect of NSP4 on polarized epithelial cells (MDCK-1) grown on permeable filters. Apical but not basolateral administration of NSP4 was found to cause a reduction in the transepithelial electrical resistance, redistribution of filamentous actin, and an increase in paracellular passage of fluorescein isothiocyanate-dextran. Significant effects on transepithelial electrical resistance were noted after a 20- to 30-h incubation with 1 nmol of NSP4. Most surprisingly, the epithelium recovered its original integrity and electrical resistance upon removal of NSP4. Preincubation of nonconfluent MDCK-1 cells with NSP4 prevented not only development of a permeability barrier but also lateral targeting of the tight-junction-associated Zonula Occludens-1 (ZO-1) protein. Taken together, these data indicate new and specific effects of NSP4 on tight-junction biogenesis and show a novel effect of NSP4 on polarized epithelia.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26053 (URN)10.1128/​JVI.75.3.1540-1546.2001 (DOI)10512 (Local ID)10512 (Archive number)10512 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved

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