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Impact of Defective Translocon Assemblies on Hierarchal Yop Effector Translocation by Yersinia pseudotuberculosis
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-165173OAI: oai:DiVA.org:umu-165173DiVA, id: diva2:1369778
Available from: 2019-11-13 Created: 2019-11-13 Last updated: 2019-11-14
In thesis
1. Biogenesis, function and regulation of the type III secretion translocon of Yersinia pseudotuberculosis
Open this publication in new window or tab >>Biogenesis, function and regulation of the type III secretion translocon of Yersinia pseudotuberculosis
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Many Gram negative bacteria use type III secretion systems to cross-talk with eukaryotic cells. Type III secretion system assembly and function is tightly regulated. It initiates with assembly of a basal body-like structure, and is followed by a cytoplasmic-located substrate sorting and export platform that first engages with early substrates required for needle assembly. At the needle tip, a translocon is formed upon eukaryotic cell contact to allow the translocation of effector proteins to the host cell. The focus of this thesis is on understanding aspects of biogenesis, regulation and function of the translocon and its interaction with the host cell. Research questions are addressed in enteropathogenic Yersinia pseudotuberculosis model.

Prioritising the secretion of translocon components before effector proteins is a task given partly to the InvE/MxiC/HrpJ family of proteins. In Yersinia, homology to this protein family is partitioned over two proteins; YopN and TyeA. Certain Yersinia strains naturally produce a single YopN/TyeA polypeptide hybrid. To understand the implications of hybrid formation towards type III secretion control, a series of mutants were engineered to produce only a single hybrid peptide. Using in vitro assays revealed no difference in substrate secretion profiles between parent and mutants. Moreover, no obvious prioritisation of secretion between translocator and effector substrates was observed. Although these in vitro studies indicate that the YopN-TyeA single polypeptide is fully functionally competent, these mutants were attenuated in the mouse infection model. Hence, natural production of YopN and TyeA as a single polypeptide alone is unlikely to confer a fitness advantage to the infecting bacteria and is unlikely to orchestrate hierarchal substrate secretion.

The YopB and YopD translocon components form a pore in the host cell plasma membrane to deliver the effectors into the host cell. To better understand how YopD contributes to the biogenesis, function and regulation of the translocon pore, a series of mutants were constructed to disrupt two predicted α-helix motifs, one lying at the N-terminus and the other at the C-terminus. Based upon phenotypes associated with environmental control of Yop synthesis and secretion, effector translocation, evasion of phagocytosis, killing of immune cells and virulence in a mouse infection model, the mutants were grouped into three phenotypic classes. A particularly interesting mutant class maintained full T3SS function in vitro, but were attenuated for virulence in a murine oral-infection model. To better understand the molecular basis for these phenotypic differences, the effectiveness of RAW 264.7 cells to respond to infection by these mutants was scrutinised. Sixteen individual cytokines were profiled with mouse cytokine screen multiplex analysis. Signature cytokine profiles were observed that could again separate the different YopD mutants into distinct categories. The activation and supression of certain cytokines that function as central innate immune response modulators correlated well with the ability of mutant bacteria to modulate programmed cell death and antiphagocytosis pathways. Hence, the biogenesis of sub-optimal translocon pores alters host cell responsiveness and limits the ability of Yersinia to fortify against attack by both early and late arms of the host innate immune response.

The amount of bacteria now resistant to multiple antibiotics is alarming. By providing insights into a common virulence process, this work may ultimately facilitate the design of novel broad-acting inhibitors of type III secretion, and thereby be useful to treat an array of bacterial infections.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2019. p. 100
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2062
Keywords
Yersinia pseudotuberculosis, type III secretion system, translocation, translocon, regulation, bacteria-eukaryotic cell contact, cytokine profiling
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-165155 (URN)978-91-7855-157-6 (ISBN)
Public defence
2019-12-06, 933, Unod B 9, Norrlands universitetssjukhus, Umeå, 09:00 (English)
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Available from: 2019-11-15 Created: 2019-11-12 Last updated: 2019-11-14Bibliographically approved

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Farag, SalahFrancis, Monika K.Nadeem, AftabWai, Sun NyuntFrancis, Matthew S

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Farag, SalahFrancis, Monika K.Nadeem, AftabWai, Sun NyuntFrancis, Matthew S
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Department of Molecular Biology (Faculty of Science and Technology)Department of Medical Biochemistry and BiophysicsDepartment of Molecular Biology (Faculty of Medicine)Umeå Centre for Microbial Research (UCMR)Molecular Infection Medicine Sweden (MIMS)
Microbiology in the medical area

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