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A study of the YopD-LcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD.
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Molecular Biology.
Umeå University, Faculty of Medicine, Molecular Biology.
2000 (English)In: Molecular Microbiology, ISSN 0950-382X, Vol. 38, no 1, 85-102 p.Article in journal (Refereed) Published
Abstract [en]

The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersinia outer protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic alpha-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic alpha-helix abolished the YopD-LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.

Place, publisher, year, edition, pages
2000. Vol. 38, no 1, 85-102 p.
URN: urn:nbn:se:umu:diva-4860DOI: 10.1046/j.1365-2958.2000.02112.xPubMedID: 11029692OAI: diva2:144119
Available from: 2005-11-24 Created: 2005-11-24Bibliographically 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.
Yersinia pseudotuberculosis, bacterial pathogenesis, YopE, LcrH, virulence, effector translocation, type III secretion, regulation
National Category
urn:nbn:se:umu:diva-646 (URN)91-7305-977-3 (ISBN)
Public defence
2005-12-16, 13:00 (English)
Available from: 2005-11-24 Created: 2005-11-24 Last updated: 2010-11-19Bibliographically approved

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