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Role of YopE and LcrH in effector translocation, HeLa cell cytotoxicity and virulence
Umeå University, Faculty of Medicine, Molecular Biology.
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 [en]
Yersinia pseudotuberculosis, bacterial pathogenesis, YopE, LcrH, virulence, effector translocation, type III secretion, regulation
National Category
Dentistry
Identifiers
URN: urn:nbn:se:umu:diva-646ISBN: 91-7305-977-3 (print)OAI: oai:DiVA.org:umu-646DiVA: diva2:144121
Public defence
2005-12-16, 13:00 (English)
Supervisors
Available from: 2005-11-24 Created: 2005-11-24 Last updated: 2010-11-19Bibliographically approved
List of papers
1. In vitro GAP activity towards RhoA, Rac1 and Cdc42 is not a prerequisite for YopE induced HeLa cell cytotoxicity.
Open this publication in new window or tab >>In vitro GAP activity towards RhoA, Rac1 and Cdc42 is not a prerequisite for YopE induced HeLa cell cytotoxicity.
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2003 (English)In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 34, no 6, 297-308 p.Article in journal (Refereed) Published
Abstract [en]

The YopE cytotoxin of Yersinia is an essential virulence determinant that is translocated into the eukaryotic target cell via a plasmid-encoded type III secretion system. YopE possess a GTPase activating protein activity that in vitro has been shown to down regulate RhoA, Rac1, and Cdc42. Translocated YopE induces de-polymerisation of the actin microfilament structure in the eukaryotic cell which results in a rounding up of infected cells described as a cytotoxic effect. Here, we have investigated the importance of different regions of YopE for induction of cytotoxicity and in vitro GAP activity. Sequential removal of the N- and C-terminus of YopE identified the region between amino acids 90 and 215 to be necessary for induction of cytotoxicity. Internal deletions containing the essential arginine at position 144 resulted in a total loss of cytotoxic response. In-frame deletions flanking the arginine finger defined a region important for the cytotoxic effect to amino acids 166–183. Four triple-alanine substitution mutants in this region, YopE166-8A, 169-71A, 175-7A and 178-80A were still able to induce cytotoxicity on HeLa cells although they did not show any in vitro GAP activity towards RhoA, Rac1 or Cdc42. A substitution mutant in position 206-8A showed the same phenotype, ability to induce cytotoxic response but no in vitro GAP activity. We speculate that YopE may have additional unidentified targets within the eukaryotic cell.

Identifiers
urn:nbn:se:umu:diva-4857 (URN)10.1016/S0882-4010(03)00063-9 (DOI)12782482 (PubMedID)
Available from: 2005-11-24 Created: 2005-11-24 Last updated: 2017-12-14Bibliographically approved
2. Functional analysis of the YopE GTPase-activating protein (GAP) activity of Yersinia pseudotuberculosis
Open this publication in new window or tab >>Functional analysis of the YopE GTPase-activating protein (GAP) activity of Yersinia pseudotuberculosis
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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
Keyword
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:nbn:se:umu:diva-16693 (URN)10.1111/j.1462-5822.2005.00684.x (DOI)16681842 (PubMedID)
Available from: 2007-10-09 Created: 2007-10-09 Last updated: 2017-12-14Bibliographically approved
3. Regulation of Yersinia Yop-effector delivery by translocated YopE
Open this publication in new window or tab >>Regulation of Yersinia Yop-effector delivery by translocated YopE
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2008 (English)In: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 298, no 3-4, 183-192 p.Article in journal (Refereed) Published
Abstract [en]

The bacterial pathogen Yersinia pseudotuberculosis uses a type III secretion (T3S) system to translocate Yop effectors into eukaryotic cells. Effectors are thought to gain access to the cytosol via pores formed in the host cell plasma membrane. Translocated YopE can modulate this pore formation through its GTPase-activating protein (GAP) activity. In this study, we analysed the role of translocated YopE and all the other known Yop effectors in the regulation of effector translocation. Elevated levels of Yop effector translocation into HeLa cells occurred by YopE-defective strains, but not those defective for other Yop effectors. Only Yersinia devoid of YopK exhibits a similar hyper-translocation phenotype. Since both yopK and yopE mutants also failed to down-regulate Yop synthesis in the presence of eukaryotic cells, these data imply that translocated YopE specifically regulates subsequent effector translocation by Yersinia through at least one mechanism that involves YopK. We suggest that the GAP activity of YopE might be working as an intra-cellular probe measuring the amount of protein translocated by Yersinia during infection. This may be a general feature of T3S-associated GAP proteins, since two homologues from Pseudomonas aeruginosa, exoenzyme S (ExoS) and exoenzyme T (ExoT), can complement the hyper-translocation phenotypes of the yopE GAP mutant.

Place, publisher, year, edition, pages
Elsevier, 2008
Keyword
YopE; GAP activity; ExoS; ExoT, feedback inhibition, translocation, type III secretion
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-20424 (URN)10.1016/j.ijmm.2007.04.007 (DOI)17597003 (PubMedID)
Available from: 2009-03-19 Created: 2009-03-19 Last updated: 2017-12-13
4. A study of the YopD-LcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD.
Open this publication in new window or tab >>A study of the YopD-LcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD.
2000 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, 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.

Identifiers
urn:nbn:se:umu:diva-4860 (URN)10.1046/j.1365-2958.2000.02112.x (DOI)11029692 (PubMedID)
Available from: 2005-11-24 Created: 2005-11-24 Last updated: 2017-12-14Bibliographically approved
5. Minimal secretion of the YopB and YopD translocators is sufficient for Yop-effector translocation by Yersinia.
Open this publication in new window or tab >>Minimal secretion of the YopB and YopD translocators is sufficient for Yop-effector translocation by Yersinia.
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4861 (URN)
Available from: 2005-11-24 Created: 2005-11-24 Last updated: 2010-01-13Bibliographically approved

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Citation style
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