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Fish scale keratocytes constitute a sensitive model system for YopE mediated phenotypes in Yersinia pseudotuberculosis infections
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Hans Wolf-Watz)
2010 (English)Manuscript (preprint) (Other academic)
Abstract [en]

The bacterial pathogen Yersinia pseudotuberculosis employs a type 3 secretion system (T3SS) to deliver virulence associated effectors directly into the host cell. Four of the translocated effectors affect the actin cytoskeleton of the cell, demonstrating the importance of inducing actin rearrangements for the establishment of a successful Yersinia infection. To further examine the role of the effectors in actin targeting, we have established a novel model system using fish scale keratocytes. Fish scale keratocytes are rapidly migrating cells present on the surface of teleost skin. The keratocytes are part of the quick wound repair mechanism of fish and are also able to internalize bacteria. The cell consists of a large extended lamellipodium, a two-dimensional actin network regulated by RhoGTPases.                                         By using live cell microscopy, we identified that wild-type Y. pseudotuberculosis caused a cytotoxic effect towards the keratocytes already within 10 minutes of infection. In contrast, a bacterial strain lacking the T3SS was rapidly internalized by the keratocytes. Further, YopE was found to be the sole effector responsible for this cytotoxic effect. YopE inactivates multiple small RhoGTPases via its GAP activity (GTPase activating protein) to induce cytotoxicity in a wide variety of cell types, including HeLa cells. Several domains of YopE are important for proper GAP function. When a number of earlier isolated point mutants of YopE were examined, none of the mutants could induce cytotoxicity towards keratocytes. Thus, this finding is in sharp contrast to earlier observations in HeLa cells, where all but one mutation caused cytotoxicity. In conclusion, the keratocytes appear to be more sensitive towards YopE mediated effects than HeLa cells and would therefore constitute a relevant model system for Yersinia infections for further studies.

Place, publisher, year, edition, pages
2010. , 56 p.
Keyword [en]
Yersinia, infection, fish keratocytes, YopE, cytotoxicity
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-37900ISBN: 978-91-7459-100-2 (print)OAI: oai:DiVA.org:umu-37900DiVA: diva2:371042
Available from: 2010-11-19 Created: 2010-11-18 Last updated: 2013-06-12Bibliographically 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.
Keyword
Yersinia, T3SS, YopE, GAP activity, translocation control, virulence
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
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)
Opponent
Supervisors
Available from: 2010-11-22 Created: 2010-11-19 Last updated: 2010-11-22Bibliographically approved

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