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The membrane localization domain is required for intracellular localization and autoregulation of YopE in Yersinia pseudotuberculosis.
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Wolf-Watz)
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Fällman)
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Fällman)
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2009 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 77, no 11, 4740-4749 p.Article in journal (Refereed) Published
Abstract [en]

Recent work has shown that a domain of YopE of Yersinia pseudotuberculosis ranging from amino acids 54 to 75 (R. Krall, Y. Zhang, and J. T. Barbieri, J. Biol. Chem. 279:2747-2753, 2004) is required for proper localization of YopE after ectopic expression in eukaryotic cells. This domain, called the membrane localization domain (MLD), has not been extensively studied in Yersinia. Therefore, an in cis MLD deletion mutant of YopE was created in Y. pseudotuberculosis. The mutant was found to secrete and translocate YopE at wild-type levels. However, the mutant was defective in the autoregulation of YopE expression after the infection of HeLa cells. Although the mutant translocated YopE at wild-type levels, it showed a delayed HeLa cell cytotoxicity. This delay was not caused by a change in GTPase activating protein (GAP) activity, since the mutant showed wild-type YopE GAP activity toward Rac1 and RhoA. The MLD mutant displayed a changed intracellular localization pattern of YopE in HeLa cells after infection, and the YopEDeltaMLD protein was found to be dispersed within the whole cell, including the nucleus. In contrast, wild-type YopE was found to localize to the perinuclear region of the cell and was not found in the nucleus. In addition, the yopEDeltaMLD mutant was avirulent. Our results suggest that YopE must target proteins other than RhoA and Rac1 and that the MLD is required for the proper targeting and hence virulence of YopE during infection. Our results raise the question whether YopE is a regulatory protein or a "true" virulence effector protein.

Place, publisher, year, edition, pages
American Society for Microbiology , 2009. Vol. 77, no 11, 4740-4749 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:umu:diva-32212DOI: 10.1128/IAI.00333-09PubMedID: 19687205OAI: diva2:301976
Available from: 2010-03-03 Created: 2010-03-03 Last updated: 2010-11-22Bibliographically approved
In thesis
1. The multifunctional GAP protein YopE of Yersinia is involved in effector translocation control and virulence
Open this publication in new window or tab >>The multifunctional GAP protein YopE of Yersinia is involved in effector translocation control and virulence
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Det multifunktionella GAP proteinet YopE från Yersinia är involverat i kontroll av effektortranslokering och virulens
Abstract [en]

The Gram-negative bacterium Yersinia pseudotuberculosis employs a type 3 secretion system (T3SS) to establish infections. The T3SS translocates a diverse set of effector proteins directly into the host cells. The coordinate action of the translocated effectors blocks the innate immune system of the host and ensures extracellular proliferation of the bacterium. YopE is an essential effector that disrupts the actin cytoskeleton of infected host cells. This cytotoxicity is caused by the inactivation of RhoGTPases by the GTPase Activating Protein (GAP) activity of YopE. YopE was demonstrated to inactivate the RhoGTPases Rac1 and RhoA in vivo. However, Rac1 and RhoA inactivation was not a prerequisite for cytotoxicity or virulence. Thus, YopE must have additional targets during infection. Surprisingly, avirulent yopE mutants had lost the control of Yop expression in the presence of target cells and they all overtranslocated effectors. It appeared as if translocated YopE was able to control Yop expression and effector translocation via a feedback inhibition mechanism. This feedback inhibition was dependent on functional GAP activity. Translocation control could also be mediated by exogenous GAP activity, suggesting that effector translocation control might be a general property of all bacterial GAP proteins. Besides YopE, the regulatory protein YopK was also found to be involved in the effector translocation control process. Clearly, as demonstrated in virulence, the roles for YopE and YopK are intimately related.                       Further, YopE possesses a membrane localization domain (MLD) required for proper localization. A yopE∆MLD mutant had lost the feedback inhibition of YopE expression and was avirulent. Hence, the effector translocation control of YopE requires both proper localization as well as functional GAP activity.                                           In addition, fish keratocytes were established as a novel model system for Y. pseudotuberculosis infections. YopE was found to be the sole effector responsible for cytotoxicity towards the keratocytes. Further, induction of cytotoxicity required fully native YopE protein which indicated that the keratocytes would be useful as a sensitive model system for further studies of YopE mediated phenotypes.

In summary, this thesis work has sought to unravel the multiple functions of translocated YopE. A novel role was elucidated where Yersinia utilizes translocated YopE to control the process of effector translocation into host cells. This regulatory control was connected to virulence in the mouse model of disease. Thus, perhaps YopE should be considered also as a regulatory protein besides being a classical effector.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2010. 56 p.
Yersinia, T3SS, YopE, GAP activity, translocation control, virulence
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
urn:nbn:se:umu:diva-37960 (URN)978-91-7459-100-2 (ISBN)
Public defence
2010-12-13, N320, Naturvetarhuset, Umeå universitet, Umeå, 10:00 (English)
Available from: 2010-11-22 Created: 2010-11-19 Last updated: 2010-11-22Bibliographically approved

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Isaksson, Elin LFahlgren, AnnaCarlsson, Sara ERosqvist, RolandWolf-Watz, Hans
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Department of Molecular Biology (Faculty of Science and Technology)Department of Molecular Biology (Faculty of Medicine)Molecular Infection Medicine Sweden (MIMS)
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