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Yersinia pseudotuberculosis type III secretion is reliant upon anauthentic N‐terminal YscX secretor domain
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Matthew S. Francis)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Matthew S. Francis)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Matthew S. Francis)
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Certain Gram‐negative bacteria use type III secretion systems to deliver effectorproteins into eukaryotic cells, serving either parasitic or mutualistic roles inside the hostcell. About 25 structural proteins are needed to assemble and deliver effector proteins.Collections of these proteins are quite well characterized, although the function ofsome continues to remain obscure. This is true for the Yersinia Ysc‐Yop systemcomponents YscX, a secreted substrate and YscY, its cognate non‐secreted chaperone.Despite recent evidence suggesting that they might coordinate Yop substrate secretion,YscX and YscY remain poorly characterized. To further investigate the function of theseproteins in the enteropathogen Y. pseudotuberculosis, we explored correlationsbetween the YscX N‐terminal segment, YscX secretion, as well as the secretion of otherYops. Analysis of a series of chimeric substrates in which the extreme YscX N‐terminushad been exchanged with equivalent functional secretion signals of other Ysc‐Yopsubstrates revealed that this segment contains non‐redundant information needed forYscX function, which includes permitting surface polymerization of the YscF needle andYops secretion. Further, in cis deletion of the YscX N‐terminus and ectopic expression ofepitope tagged YscX variants again correlated stable YscX production but not secretionto the type III secretion of Yops. Despite this, the first 5 codons were determined toconstitute a minimal signal capable of promoting secretion of the signalless ‐lactamasereporter. Hence, YscX does contain a fully equipped N‐terminal secretor domain topromote secretion of self. Nevertheless, the primary role of this N‐terminal segmentmust be to assemble an operational secretion system, and this occurs independently ofYscX secretion.

National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-70110OAI: oai:DiVA.org:umu-70110DiVA: diva2:619569
Projects
Controlling substrate export by the Ysc-Yop type III secretion system in Yersinia
Note

Submitted

Available from: 2013-05-05 Created: 2013-05-05 Last updated: 2013-05-06Bibliographically approved
In thesis
1. Controlling substrate export by the Ysc-Yop type III secretion system in Yersinia
Open this publication in new window or tab >>Controlling substrate export by the Ysc-Yop type III secretion system in Yersinia
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Several pathogenic Gram-negative bacteria invest in sophisticated type III secretion systems (T3SS) to incapacitate their eukaryotic hosts. T3SSs can secrete protein cargo outside the bacterial cell and also target many of them into the eukaryotic cell interior. Internalized proteins promote bacterial colonization, survival and transmission, and can often cause severe disease. An example is the Ysc-Yop T3SS apparatus assembled by pathogenic Yersinia spp. A correctly assembled Ysc-Yop T3SS spans the Yersinia envelope and also protrudes from the bacterial surface. Upon host cell contact, this system is competent to secrete hydrophobic translocators that form a translocon pore in the host cell membrane to complete the delivery channel bridging both bacterial and host cells. Newly synthesized effector Yops may pass through this channel to gain entry into the host cell cytosol.As type III secretion (T3S) substrates function sequentially during infection, it is hypothesized that substrate export is temporally controlled to ensure that those required first are prioritized for secretion. On this basis three functional groups are classified as early (i.e. structural components), middle (i.e. translocators) and late (i.e. effectors). Factors considered to orchestrate the T3S of substrates are many, including the intrinsic substrate secretion signal sequences, customized chaperones, and recognition/sorting platforms at the base of the assembled T3SS. Investigating the interplay between these elements is critical for a better understanding of the molecular mechanisms governing export control during Yersinia T3S.To examine the composition of the N-terminal T3S signals of the YscX early substrate and the YopD middle substrate, these segments were altered by mutagenesis and the modified substrates analyzed for their T3S. Translational fusions between these signals and a signalless β-Lactamase were used to determine their optimal length required for efficient T3S. This revealed that YscX and YopD export is most efficiently supported by their first 15 N-terminal residues. At least for YopD, this is a peptide signal and not base upon information in the mRNA sequence. Moreover, features within and upstream of this segment contribute to their translational control. In parallel, bacteria were engineered to produce substrate chimeras where the N-terminal segments were exchanged between substrates of different classes in an effort to examine the temporal dynamics of T3S. In several cases, Yersinia producing chimeric substrates were defective in T3S activity, which could be a consequence of disturbing a pre-existing hierarchal secretion mechanism.YopN and TyeA regulatory molecules can be naturally produced as a 42 kDa YopN-TyeA hybrid, via a +1 frame shift event somewhere at the 5’-end of yopN. To study this event, Yersinia were engineered to artificially produce this hybrid, and these maintained in vitro T3S control of both middle and late substrates. However, modestly diminished directed targeting of effectors into eukaryotic cells correlated to virulence attenuation in vivo. Upon further investigation, a YopN C-terminal segment encompassing residues 278 to 287 was probably responsible, as this region is critical for YopN to control T3S, via enabling a specific interaction with TyeA.Investigated herein were molecular mechanisms to orchestrate substrate export by the T3SS of Yersinia. While N-terminal secretion signals may contribute to specific substrate order, the YopN and TyeA regulatory molecules do not appear to distinguish between the different substrate classes.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2013. 77 p.
Series
Doctoral thesis / Umeå University, Department of Molecular Biology
Keyword
Y. pseudotuberculosis, T3SS, YscX, YopD, assembly, translation control, temporal secretion.
National Category
Medical and Health Sciences
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-70113 (URN)978-91-7459-566-6 (ISBN)
Public defence
2013-05-29, Norrlands universitetssjukhus, Biomedicinhuset, Byggnad 6L, Major Groove, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2013-05-08 Created: 2013-05-05 Last updated: 2013-12-12Bibliographically approved

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Amer, AyadGurung, JyotiFrancis, Matthew

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