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Coiled-coils in the YopD translocator family: A predicted structure unique to the YopD N-terminus contributes to full virulence of Yersinia pseudotuberculosis
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). (Matthew Francis)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). (Matthew Francis)
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, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
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2012 (English)In: Infection, Genetics and Evolution, ISSN 1567-1348, E-ISSN 1567-7257, Vol. 12, no 8, 1729-1742 p.Article in journal (Refereed) Published
Abstract [en]

Pathogenic Yersinia all harbor a virulence plasmid-encoded Ysc–Yop T3SS. In this system, translocator function is performed by the hydrophobic proteins YopB and YopD. With the goal to better understand how YopD orchestrates yop-regulatory control, translocon pore formation and Yop effector translocation, we performed an in silico prediction of coiled-coil motifs in YopD and YopD-like sequences from other bacteria. Of interest was a predicted N-terminal coiled-coil that occurred solely in Yersinia YopD sequences. To investigate if this unique feature was biologically relevant, two in cis point mutations were generated with a view to disrupting this putative structure. Both mutants maintained full T3SS function in vitro in terms of environmental control of Yops synthesis and secretion, effector toxin translocation and evasion of phagocytosis and killing by cultured immune cells. However, these same mutants were attenuated for virulence in a murine oral-infection model. The cause of this tardy disease progression is unclear. However, these data indicate that any structural flaw in this element unique to the N-terminus will subtly compromise an aspect of YopD biology. Sub-optimal T3SSs are then formed that are unable to fortify Yersinia against attack by the host innate and adaptive immune response.

Place, publisher, year, edition, pages
Elsevier, 2012. Vol. 12, no 8, 1729-1742 p.
Keyword [en]
Pathogen-Host Interaction, Virulence Factor, Viruelnce, Bacteria
National Category
Medical and Health Sciences
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-59466DOI: 10.1016/j.meegid.2012.07.016OAI: oai:DiVA.org:umu-59466DiVA: diva2:552394
Funder
Swedish Research Council, 2009-5628
Available from: 2012-09-19 Created: 2012-09-14 Last updated: 2017-12-07Bibliographically approved
In thesis
1. YopD translocator function in Yersinia pseudotuberculosis type III secretion
Open this publication in new window or tab >>YopD translocator function in Yersinia pseudotuberculosis type III secretion
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type III secretion systems (T3SS) are a common feature of Gram-negative bacteria, allowing them to inject anti-host effectors into the interior of infected eukaryotic cells. By this mechanism, these virulence factors help the bacteria to modulate eukaryotic cell function in its favor and subvert host innate immunity. This promotes a less hostile environment in which infecting bacteria can colonize and cause disease.

In pathogenic Yersinia, a crucial protein in this process is YopD. YopD is a T3S substrate that, together with YopB, forms a translocon pore in the host cell membrane through which the Yop effectors may gain access to the target-cell cytosol. The assembly of the translocator pore in plasma membranes is considered a fundamental feature of all T3SSs. How the pore is formed, what determines the correct size and ultimately the stoichiometry between YopD YopB, is still unknown. Portions of YopD are also observed inside HeLa cells. Moreover, YopD functions together with its T3S chaperone, LcrH, to control Yops synthesis in the bacterial cytoplasm. The multifunctional YopD may influence all these processes by compartmentalizing activities into discrete modular domains along the protein length. Therefore, understanding how particular domains and/or residues within these regions coordinate multiple functions of the protein will provide a platform to improve our knowledge of the molecular mechanisms behind translocation through T3SSs.

Comprehensive site-directed mutagenesis of the YopD C-terminal amphipathic α-helix domain, pinpointed hydrophobic residues as important for YopD function. Some YopD variants were defective in self-assembly and in the ability to interact with the needle tip protein, LcrV, which were required to facilitate bacterial T3S activity. A similar mutagenesis approach was used to understand the role of the two predicted coiled-coils located at the N-terminal and C-terminal region of YopD. The predicted N-terminal element that occurs solely in the Yersinia YopD translocator family is essential for optimal T3SS and full disease progression. The predicted YopD C-terminal coiled-coil shapes a functional translocon inserted into host cell membranes. This translocon was seen to be a dynamic structure facilitating at least two roles during effectors delivery into cells; one to guarantee translocon pore insertion into target cell membranes and the other to promote targeted activity of internalized effector toxins.

In Yersinia expression of yop genes and secretion of the corresponding polypeptides is tightly regulated at a transcriptional and post-transcriptional level. If T3S chaperones of the translocator class are known to influence transcriptional output of T3SS genes in other bacteria, we show that in Yersinia the class II T3S chaperone LcrH has no such effect on the LcrF transcriptional activator activity. We also demonstrate that there are possibly additional yop-regulatory roles for the LcrH chaperone besides forming a stable complex with YopD to impose post-transcriptional silencing on Yops synthesis. This mechanism that relies upon an active T3SS, might act independently of both YopD and the regulatory element LcrQ.

In conclusion, this work has sought to delineate the encrypted functions of the YopD translocator that contribute to Yersinia T3SS-dependent pathogenesis. Contributions of the YopD cognate chaperone LcrH in yop regulatory control are also presented.  

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2012. 222 p.
Keyword
Y. pseudotuberculosis, T3SS, translocon, YopD, coiled-coil, effector delivery, regulation, virulence
National Category
Medical and Health Sciences
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-61544 (URN)978-91-7459-483-6 (ISBN)
Public defence
2012-12-14, Major Groove, Biomedicinhuset, Byggnad 6L, Umeå University, Umeå, 09:00 (English)
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Supervisors
Available from: 2012-11-23 Created: 2012-11-19 Last updated: 2012-11-23Bibliographically approved

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Costa, Tiago R. D.Amer, Ayad A. A.Fällman, MariaFahlgren, AnnaFrancis, Matthew
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