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Role of the Yersinia protein YopK in microbe-host interactions
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). (Maria Fällman)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There are three human pathogenic species of the genus Yersiniae: Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis. To cause disease, these strains inhibit several key innate defense mechanisms, including phagocytosis, the critical process for bacterial clearance. The ability of Yersinia to evade the immune defense is dependent on delivery of virulence effectors, Yersinia outer proteins (Yops), into the interacting cell by a mechanism involving the type III secretion machinery. We have shown that the virulence protein YopK plays an important role in the control of Yop effector translocation via a feedback mechanism involving another virulence protein, YopE. We also found that YopK participated in regulation of Yop effector translocation by modulating level and ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, using a yeast two-hybrid screen with YopK as a bait, the eukaryotic protein RACK1 was identified as a target for this virulence protein. We found that RACK1 was engaged upon Y. pseudotuberculosis-mediated β1-integrin activation, where it was recruited to phagocytic cups. Downregulation of RACK1 by RNAi resulted in a reduced ability of Y. pseudotuberculosis to block phagocytosis, indicating that RACK1 is required for efficient Yersinia-mediated antiphagocytosis. Based on our data, we suggest a model where Yersinia, via YopK, targets RACK1 to ensure a directed delivery of the Yop effectors to the “right place” where they bind to and inactivate their targets, resulting in efficient inhibition of phagocytosis.  

A yopK mutant strain over-delivers Yop effectors, but is still avirulent in mice, indicating that YopK is important for the fine-tuning of effector protein delivery during infection. To analyse this, we investigated the importance of YopK during in vivo infection. We found that a yopK mutant colonized Peyer’s patches and the mesenteric lymph node more rapidly compared to wild-type Y. pseudotuberculosis, but was unable to spread systemically to liver and spleen and cause full disease in mice. Further, we showed that a yopK mutant was able to colonize liver and spleen and cause full disease in mice lacking the main phagocytes, polymorphonuclear leukocytes (PMNs). We also showed that YopK was important for Yersinia-mediated silencing of the PMN response.

To summarize, we suggest that YopK is important for Yersinia to evade the PMN defense and thereby spread systemically and cause disease. YopK is proposed to do this by allowing a controlled, directed Yop effector delivery that is just sufficient to inhibit host immune defense mechanisms. The controlled and precise delivery of virulence effectors avoids inappropriate triggering of PMNs and thereby an enhanced immune response favoring the host.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2012. , 58 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1492
Keyword [en]
Yersinia, YopK, T3SS, antiphagocytosis, neutrophil, translocation, virulence
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-53585ISBN: 978-91-7459-408-9 (print)OAI: oai:DiVA.org:umu-53585DiVA: diva2:513738
Public defence
2012-04-27, Major Groove, Building 6L, Umeå University, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, K2008-58X-11222-14-3
Available from: 2012-04-04 Created: 2012-04-03 Last updated: 2012-04-04Bibliographically approved
List of papers
1. 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
2. The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function
Open this publication in new window or tab >>The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 2, e16784- p.Article in journal (Refereed) Published
Abstract [en]

Many Gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated beta1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.

Place, publisher, year, edition, pages
San Francisco: Public Library of Science, 2011
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-43212 (URN)10.1371/journal.pone.0016784 (DOI)21347310 (PubMedID)
Available from: 2011-04-22 Created: 2011-04-22 Last updated: 2017-12-11Bibliographically approved
3. Role of YopK in Yersinia resistance against polymorphonuclear leukocyte defense
Open this publication in new window or tab >>Role of YopK in Yersinia resistance against polymorphonuclear leukocyte defense
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(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-53703 (URN)
Available from: 2012-04-04 Created: 2012-04-04 Last updated: 2012-04-04Bibliographically approved

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