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Antiphagocytosis by Yersinia pseudotuberculosis: role of the YopH target proteins
Umeå University, Faculty of Medicine, Molecular Biology.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The enteropathogenic bacterium Yersinia pseudotuberculosis binds to β1 integrins on a host cell via its surface protein invasin. This event stimulates signal transduction to the actin cytoskeleton of the eukaryotic cell, which allows the cell to engulf the bacterium that is attached to its surface. However, the pathogen Y. pseudotuberculosis can evade such phagocytosis by injecting virulence effectors that interfere with the antipathogenic machinery of the host cells. One of these virulence effectors is the tyrosine phosphatase YopH. Through its enzymatic activity, YopH blocks phagocytosis by affecting the signalling that is associated with cytoskeletal rearrangements.

Cas is a substrate of YopH in both professional and non-professional phagocytes. We showed that YopH binds to the central substrate domain of Cas and that this interaction is required for YopH to target focal adhesion structures in host cells. We also demonstrated that YopH binds another substrate, FAK, through Cas. Moreover, we suggested that targeting of Cas is necessary for the cytotoxic effects mediated by YopH.

The protein Fyb is specific to immune cells, and it has been identified as a substrate of YopH in macrophages. We discovered that both the N-terminal substrate-binding domain and the C-terminal catalytic region of YopH bind Fyb in a phosphotyrosine-dependent manner. Moreover, we observed that both the substrate-binding domain and the phosphatase activity of YopH are essential for the effects of this protein on macrophages, which include dephosphorylation of Fyb, blocking of phagocytosis, and cytotoxicity.

The role of Fyb in macrophages is largely unknown, although there is evidence that this protein is involved in integrin-linked actin organization. We identified a novel interaction partner of Fyb, mAbp1, which is a protein that binds to F-actin. Studies in vitro indicated that mAbp1 binds to the N terminus of Fyb via a C-terminal SH3 domain. We also found that both Fyb and mAbp1 co-localize with F-actin at the leading edges of macrophages. Further studies suggested that mAbp1 influences the spreading of macrophages and the antiphagocytosis mediated by pathogenic Yersinia. These results support a role for Fyb in signalling that affects F-actin dynamics, and they also provide additional insight into the mechanisms involved. Fyb has been shown to form a complex with SKAP-HOM, another substrate of YopH in macrophages. Our data implied that the level of SKAP-HOM protein depends on the presence of Fyb, but the function of the Fyb/SKAP-HOM complex in macrophages has not been determined. However, since Fyb is the only known haematopoietic-specific substrate of YopH, it is possible that Fyb is involved in other antimicrobial functions.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi , 2006. , 65 p.
Series
Doctoral thesis / Umeå University, Department of Molecular Biology, 1072
Keyword [en]
Molecular biology, Cas, Fyb, mAbp1, SKAP-HOM, Yersinia pseudotuberculosis, YopH
Keyword [sv]
Molekylärbiologi
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-957ISBN: 91-7264-219-X (print)OAI: oai:DiVA.org:umu-957DiVA: diva2:145183
Public defence
2007-02-01, Major Groove, NUS, 6L, Department of Molecular Biology, Umea University, Umea, 09:00 (English)
Opponent
Supervisors
Available from: 2006-12-21 Created: 2006-12-21 Last updated: 2009-10-30Bibliographically approved
List of papers
1. Disruption of target cell adhesion structures by the Yersinia effector YopH requires interaction with the substrate domain of p130Cas.
Open this publication in new window or tab >>Disruption of target cell adhesion structures by the Yersinia effector YopH requires interaction with the substrate domain of p130Cas.
Show others...
2005 (English)In: European Journal of Cell Biology, ISSN 0171-9335, Vol. 84, no 4, 477-489 p.Article in journal (Refereed) Published
Abstract [en]

The docking protein p130Cas has, together with FAK, been found as a target of the Yersinia virulence effector YopH. YopH is a protein tyrosine phosphatase that is delivered into host cells via the bacterial type III secretion machinery, and the outcome of its activity is inhibition of host cell phagocytosis. In the present study using p130Cas-/- cells, and p130Cas-/- cells expressing variants of GFPp130Cas, we show that this docking protein, via its substrate domain, is responsible for subcellular targeting of YopH in eukaryotic cells. Since YopH inhibits phagocytosis, p130Cas was expected to be critical for signalling mediating bacterial internalization. However, p130Cas-/- cells did not exhibit reduced capacity to internalize Yersinia. On the other hand, when a dominant negative variant of p130Cas was expressed in these cells, the phagocytic capacity was severely impaired. Moreover, the p130Cas-/- cells displayed a marked reduced sensitivity towards YopH-mediated detachment compared to wild-type cells. Transfecting these cells with full-length p130Cas rendered cells hypersensitive to both mechanical and Yersinia-mediated detachment. This hypersensitivity was not seen upon transfection with the dominant negative substrate domain-deleted variant of p130Cas. This implicates p130Cas as a prominent regulator of cell adhesion, where its substrate-binding domain has a significant function.The docking protein p130Cas has, together with FAK, been found as a target of the Yersinia virulence effector YopH. YopH is a protein tyrosine phosphatase that is delivered into host cells via the bacterial type III secretion machinery, and the outcome of its activity is inhibition of host cell phagocytosis. In the present study using p130Cas-/- cells, and p130Cas-/- cells expressing variants of GFPp130Cas, we show that this docking protein, via its substrate domain, is responsible for subcellular targeting of YopH in eukaryotic cells. Since YopH inhibits phagocytosis, p130Cas was expected to be critical for signalling mediating bacterial internalization. However, p130Cas-/- cells did not exhibit reduced capacity to internalize Yersinia. On the other hand, when a dominant negative variant of p130Cas was expressed in these cells, the phagocytic capacity was severely impaired. Moreover, the p130Cas-/- cells displayed a marked reduced sensitivity towards YopH-mediated detachment compared to wild-type cells. Transfecting these cells with full-length p130Cas rendered cells hypersensitive to both mechanical and Yersinia-mediated detachment. This hypersensitivity was not seen upon transfection with the dominant negative substrate domain-deleted variant of p130Cas. This implicates p130Cas as a prominent regulator of cell adhesion, where its substrate-binding domain has a significant function.

Keyword
Animals, Bacterial Outer Membrane Proteins/*physiology, Cell Adhesion, Crk-Associated Substrate Protein, Fibroblasts/cytology/metabolism/microbiology, Focal Adhesion Kinase 1, Focal Adhesion Protein-Tyrosine Kinases, Focal Adhesions, Mice, Phagocytosis, Protein Binding, Protein Structure; Tertiary, Protein-Tyrosine Kinases/metabolism, Protein-Tyrosine-Phosphatase/*physiology, Proteins/genetics/*physiology, Retinoblastoma-Like Protein p130, Virulence, Yersinia/*pathogenicity
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-16647 (URN)10.1016/j.ejcb.2004.11.009 (DOI)15900707 (PubMedID)
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2010-04-22Bibliographically approved
2. Interaction between the Yersinia tyrosine phosphatase YopH and its macrophage substrate, Fyn-binding protein, Fyb
Open this publication in new window or tab >>Interaction between the Yersinia tyrosine phosphatase YopH and its macrophage substrate, Fyn-binding protein, Fyb
2005 (English)In: Journal of Molecular Biology and Biotechnology, ISSN 1464-1801, E-ISSN 1660-2412, Vol. 9, no 3-4, 214-223 p.Article in journal (Refereed) Published
Abstract [en]

Pathogenic Yersinia species can evade phagocytosis by injecting virulence effectors that interfere with the phagocytic machinery of host cells. One of these virulence effectors is the protein tyrosine phosphatase YopH. Through its enzymatic activity, YopH interferes with the initial phagocytic process by affecting signalling for cytoskeletal rearrangements. Fyb (Fyn-binding protein), which is an immune cell-specific adaptor protein, has been identified as a substrate of YopH in macrophages. In this study, the interaction between YopH and Fyb is studied. We show that YopH binds to Fyb via different regions in both phosphotyrosine-dependent and phosphotyrosine-independent ways. The phosphotyrosine substrate binding N-terminal part (1-130) of YopH as well as the C-terminal catalytic region binds to Fyb in a phosphotyrosine-dependent manner. We also show that a central part of YopH (130-260) interacts with the Fyb C-terminus (548-783) in a phosphotyrosine-independent manner. Further, we demonstrate that the N-terminal binding region of YopH is important for YopH-mediated functions on macrophages such as dephosphorylation of Fyb, blockage of phagocytosis, and cytotoxic effects. Copyright (c) 2005 S. Karger AG, Basel.

Place, publisher, year, edition, pages
Wymondham, Norfolk: Horizon Scientific Press, 2005
Keyword
Adaptor Proteins; Signal Transducing/genetics/*metabolism, Animals, Bacterial Outer Membrane Proteins/genetics/*metabolism, Cell Line, Immunoprecipitation, Macrophages/*microbiology, Mice, Microscopy; Fluorescence, Phagocytosis, Phosphorylation, Phosphotyrosine/metabolism, Protein Binding, Protein Interaction Mapping, Protein-Tyrosine-Phosphatase/genetics/*metabolism, Yersinia/*enzymology/pathogenicity
Identifiers
urn:nbn:se:umu:diva-16644 (URN)10.1159/000089649 (DOI)16415594 (PubMedID)
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2011-03-25Bibliographically approved
3. Fyn binding protein, Fyb, interacts with mammalian actin binding protein, mAbp1.
Open this publication in new window or tab >>Fyn binding protein, Fyb, interacts with mammalian actin binding protein, mAbp1.
2005 (English)In: FEBS Letters, ISSN 0014-5793, Vol. 579, no 11, 2339-2347 p.Article in journal (Refereed) Published
Abstract [en]

The immune cell specific protein Fyn-T binding protein (Fyb) has been identified as a target of the Yersinia antiphagocytic effector Yersinia outer protein H (YopH), but its role in macrophages is unknown. By using Fyb domains as bait to screen a mouse lymphoma cDNA library, we identified a novel interaction partner, mammalian actin binding protein 1 (mAbp1). We show that mAbp1 binds the Fyb N-terminal via its C-terminally located src homology 3 domain. The interaction between Fyb and mAbp1 is detected in macrophage lysates and the proteins co-localize with F-actin in the leading edge. Hence, mAbp1 is likely to constitute a downstream effector of Fyb involved in F-actin dynamics.

Keyword
Actins/metabolism, Adaptor Proteins; Signal Transducing/chemistry/genetics/*metabolism, Amino Acid Motifs, Animals, Bacterial Outer Membrane Proteins/metabolism, Cell Line, Mice, Microfilament Proteins/genetics/*metabolism, Phosphorylation, Phosphotyrosine/metabolism, Protein Binding, Protein Transport, Protein-Tyrosine-Phosphatase/metabolism, Proto-Oncogene Proteins/metabolism, Proto-Oncogene Proteins c-fyn, Two-Hybrid System Techniques, src Homology Domains/genetics, src-Family Kinases/metabolism
Identifiers
urn:nbn:se:umu:diva-16652 (URN)10.1016/j.febslet.2005.03.031 (DOI)15848169 (PubMedID)
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2009-10-30Bibliographically approved
4. Mammalian actin-binding protein 1 influences spreading of macrophages
Open this publication in new window or tab >>Mammalian actin-binding protein 1 influences spreading of macrophages
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5609 (URN)
Available from: 2006-12-21 Created: 2006-12-21 Last updated: 2010-01-13Bibliographically approved

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