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Functional analysis of the YopE GTPase-activating protein (GAP) activity of Yersinia pseudotuberculosis
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Wolf-Watz)
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 Medical Biosciences, Pathology.
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Wolf-Watz)
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2006 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 8, no 6, 1020-1033 p.Article in journal (Refereed) Published
Abstract [en]

YopE of Yersinia pseudotuberculosis inactivates three members of the small RhoGTPase family (RhoA, Rac1 and Cdc42) in vitro and mutation of a critical arginine abolishes both in vitro GTPase-activating protein (GAP) activity and cytotoxicity towards HeLa cells, and renders the pathogen avirulent in a mouse model. To understand the functional role of YopE, in vivo studies of the GAP activity in infected eukaryotic cells were conducted. Wild-type YopE inactivated Rac1 as early as 5 min after infection whereas RhoA was down regulated about 30 min after infection. No effect of YopE was found on the activation state of Cdc42 in Yersinia-infected cells. Single-amino-acid substitution mutants of YopE revealed two different phenotypes: (i) mutants with significantly lowered in vivo GAP activity towards RhoA and Rac1 displaying full virulence in mice, and (ii) avirulent mutants with wild-type in vivo GAP activity towards RhoA and Rac1. Our results show that Cdc42 is not an in vivo target for YopE and that YopE interacts preferentially with Rac1, and to a lesser extent with RhoA, during in vivo conditions. Surprisingly, we present results suggesting that these interactions are not a prerequisite to establish infection in mice. Finally, we show that avirulent yopE mutants translocate YopE in about sixfold higher amount compared with wild type. This raises the question whether YopE's primary function is to sense the level of translocation rather than being directly involved in downregulation of the host defence.

Place, publisher, year, edition, pages
Wiley , 2006. Vol. 8, no 6, 1020-1033 p.
Keyword [en]
Animals, Bacterial Outer Membrane Proteins/*analysis/genetics/*physiology, Bacterial Translocation/physiology, DNA; Bacterial/analysis/genetics, Down-Regulation/physiology, Female, GTPase-Activating Proteins/*analysis/*physiology, Gene Expression Regulation; Bacterial, Hela Cells, Humans, L-Lactate Dehydrogenase/metabolism, Mice, Mice; Inbred C57BL, Mutation, Substrate Specificity, Virulence, Yersinia pseudotuberculosis/*chemistry/pathogenicity/*physiology, cdc42 GTP-Binding Protein/analysis/genetics/physiology, rac1 GTP-Binding Protein/analysis/genetics/physiology, rhoA GTP-Binding Protein/analysis/genetics/physiology
Identifiers
URN: urn:nbn:se:umu:diva-16693DOI: 10.1111/j.1462-5822.2005.00684.xPubMedID: 16681842OAI: oai:DiVA.org:umu-16693DiVA: diva2:156366
Available from: 2007-10-09 Created: 2007-10-09 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Role of YopE and LcrH in effector translocation, HeLa cell cytotoxicity and virulence
Open this publication in new window or tab >>Role of YopE and LcrH in effector translocation, HeLa cell cytotoxicity and virulence
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In order to establish an extra-cellular infection the gram-negative bacteria Yersinia pseudotuberculosis uses a type III secretion system (T3SS) to translocate a set of anti-host effectors into eukaryotic cells. The toxins disrupt signalling pathways important for phagocytosis, cytokine production and cell survival. Secretion and translocation via this T3SS is strictly regulated on several levels. In this context, the function of YopE and LcrH during Yersinia infections has been analysed.

YopE is an essential translocated effector that disrupts the actin cytoskeleton of infected eukaryotic cells, by inactivating small GTPases through its GTPase activating protein (GAP) activity. However, cytotoxicity can be uncoupled from in vitro GAP activity towards the RhoA, Rac1 and Cdc42 GTPases. Furthermore, in vivo studies of the YopE GAP activity revealed that only RhoA and Rac1 are targeted, but this is not a pre-requisite for Yersinia virulence. Hence, YopE must target one or more additional GTPases to cause disease in mice.

YopE was the only Yersinia effector that blocks LDH release from infected cells. Moreover, translocated YopE could regulate the level of subsequent effector translocation by a mechanism that involved the YopE GAP function and another T3S component, YopK. Loss of translocation control elevated total T3S gene expression in the presence of eukaryotic cells. This indicated the existence of a regulatory loop for feedback control of T3S gene expression in the bacteria that originates from the interior of the eukaryotic cell after effector translocation is completed. This might represent the true virulence function of YopE.

Exoenzyme S (ExoS) of Pseudomonas aeruginosa has a YopE-like GAP domain with similar activity towards RhoA, Rac1 and Cdc42. However, ExoS is unable to complement hyper-translocation resulting from loss of YopE. This indicates a unique function for YopE in translocation control in Yersinia that might be dependent on correct intracellular localisation. It follows that the Membrane Localisation Domain in YopE was important for translocation control, but dispensable for cytotoxicity and blockage of LDH release.

YopD and its cognate chaperone LcrH are negative regulatory elements of the T3S regulon and together with YopB, are involved in the effector translocation process. Randomly generated point mutants in LcrH specifically effected stability and secretion of both the YopB and YopD substrates in vitro and prevented their apparent insertion as translocon pores in the membranes of infected cells. Yet, these mutants still produced stable substrates in the presence of eukaryotic cells and most could mediate at least partial effector translocation. Thus, only minimal amounts of the YopB and YopD translocator proteins are needed for translocation and the LcrH chaperone may regulate this process from inside the bacteria.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2005. 53 p.
Keyword
Yersinia pseudotuberculosis, bacterial pathogenesis, YopE, LcrH, virulence, effector translocation, type III secretion, regulation
National Category
Dentistry
Identifiers
urn:nbn:se:umu:diva-646 (URN)91-7305-977-3 (ISBN)
Public defence
2005-12-16, 13:00 (English)
Supervisors
Available from: 2005-11-24 Created: 2005-11-24 Last updated: 2010-11-19Bibliographically approved
2. 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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Aili, MargaretaIsaksson, Elin LHallberg, BengtWolf-Watz, HansRosqvist, Roland
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