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Autoproteolysis and Intramolecular Dissociation of Yersinia YscU Precedes Secretion of Its C-Terminal Polypeptide YscU CC
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). (MIMS)
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Chemical Biological Center)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). (MIMS)
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Chemical Biological Center)
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, e49349Article in journal (Refereed) Published
Abstract [en]

Type III secretion system mediated secretion and translocation of Yop-effector proteins across the eukaryotic target cell membrane by pathogenic Yersinia is highly organized and is dependent on a switching event from secretion of early structural substrates to late effector substrates (Yops). Substrate switching can be mimicked in vitro by modulating the calcium levels in the growth medium. YscU that is essential for regulation of this switch undergoes autoproteolysis at a conserved N↑PTH motif, resulting in a 10 kDa C-terminal polypeptide fragment denoted YscUCC. Here we show that depletion of calcium induces intramolecular dissociation of YscUCC from YscU followed by secretion of the YscUCC polypeptide. Thus, YscUCC behaved in vivo as a Yop protein with respect to secretion properties. Further, destabilized yscU mutants displayed increased rates of dissociation of YscUCC in vitro resulting in enhanced Yop secretion in vivo at 30°C relative to the wild-type strain.These findings provide strong support to the relevance of YscUCC dissociation for Yop secretion. We propose that YscUCC orchestrates a block in the secretion channel that is eliminated by calcium depletion. Further, the striking homology between different members of the YscU/FlhB family suggests that this protein family possess regulatory functions also in other bacteria using comparable mechanisms.

Place, publisher, year, edition, pages
2012. Vol. 7, no 11, e49349
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:umu:diva-61703DOI: 10.1371/journal.pone.0049349ISI: 000311821000040PubMedID: 23185318OAI: oai:DiVA.org:umu-61703DiVA: diva2:571567
Available from: 2012-11-23 Created: 2012-11-23 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Regulation of the multi-functional protein YscU in assembly of the Yersinia type III secretion injectisome
Open this publication in new window or tab >>Regulation of the multi-functional protein YscU in assembly of the Yersinia type III secretion injectisome
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Reglering av det multifunktionella proteinet YscU i sammansättningen av Yersinias typ III-sekretionssystem
Abstract [en]

Yersinia pseudotuberculosis is a Gram-negative zoonotic pathogenic bacterium causing gastroenteritis in human and animals. It shares a conserved virulence plasmid encoding for a needle-like secretion machinery, or type III secretion system, which can be found in other pathogenic Gram-negative bacteria. The type III secretion system (T3SS) is a macromolecular assembly that enables pathogenic effector proteins (or Yersinia outer proteins, Yops) to be transported into eukaryotic host cells. This export machinery is assembled in a highly ordered stepwise mechanism. The activation of T3SS is also dependent on calcium concentration, temperature, and pH of the growth media as mimic factors for host cell’s contact. The T3SS-associated inner-membrane protein, YscU, of Yersinia is proposed to function as a substrate specificity switch protein and forms basal structure of T3SS. YscU has four α helical transmembrane domain and a soluble cytoplasmic domain YscUC which undergoes auto-proteolysis at a conserved N↑PTH motif. The auto-proteolysis process, which is required for the assembly of the injectisome and secretion of Yops, results in a 10-kDa C-terminal polypeptide fragment, denoted YscUCC and 6-kDa N-terminal fragment YscUCN. In this thesis, we showed that YscUC dissociation was important for Yops secretion and resulted in unfolded YscUCN and oligomeric YscUCC. By combination in vivo and in vitro methods, growth media conditions as calcium, temperature, and pH were indicated to control secretion by regulation of YscUC dissociation. The calcium-binding isotherm to YscUC was fit best with a one-site binding model resulting in Kd 800 µM, which is identical to calcium level that blocks secretion in vivo. YscU is also the key protein for the T3SS pH dependence, demonstrated by thermal unfolding profile and secondary structure of protein were altered between pH 7.4 and 6.0. In addition, bacterial inner membrane was proposed to assist the YscUCN folding, monitored by using lipid bilayer as a mimic environment in nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. This binding is important for Yops secretion and YscUC is anchored to bacterial membrane upon dissociation. The other substrate specificity switch protein YscP has function as a “molecular ruler” controlling length of the secretion needle. Previous genetic experiments have suggested that YscP and YscU interact physically, when mutation at defined residues on yscU (suppressor mutants) rescued Yops secretion in null-yscP mutant. In this research, direct binding of YscU and YscP was proved as weak but important interaction with Kd 430 mM by application of NMR and the binding interface of YscP was centred on the last helix of YscUC. Furthermore, we found that the YscP interaction could inhibit YscU auto-proteolysis. Studying the dissociation kinetic of suppressor YscUC variants at temperature 30 and 37oC provides strong support to a model where YscU is a temperature sensor for T3SS and YscUC dissociation is required for Yops secretion. Interestingly, the NPTH motif is conserved through most of YscU family members, meaning that role of dissociation may be conserved also in other bacterial injectisomes. To this end, the dissociation of YscU can be used as a therapeutic target in drug discovery. We attempted to identify the small-molecules that can hinder YscU dissociation. The small compound methyl(5-methyl-2-phenyl-1,3-thiazolidin-4-yl)acetate was found to be able to inhibit dissociation and to crystalize full YscUC, which has never been successfully done before. Finally, we found that the inner-rod protein YscI is binding to YscUC with a 1:1 stoichiometry as shown with pull-down assays and isothermal titration calorimetry. Taken together we have made several discoveries that expand the functional palette of YscU and all these functions were shown to have biological relevance with Yops secretion levels. In light of the strong sequence conservation between T3SS utilizing pathogenic bacteria the findings are likely to be general characters.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2017. 64 p.
Keyword
Type III secretion system (T3SS), injectisome, Yersinia pseudotuberculosis, inhibitors, Yops, YscU, YscP, YscI, disorder-to-order transition, nuclear magnetic resonance (NMR), circular dichroism (CD), isothermal titration calorimetry (ITC)
National Category
Chemical Sciences
Research subject
biological chemistry
Identifiers
urn:nbn:se:umu:diva-130134 (URN)978-91-7601-613-8 (ISBN)
Public defence
2017-02-10, KB3B1, Stora Hörsalen, KBC huset, Umeå, 10:00 (English)
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Available from: 2017-01-20 Created: 2017-01-12 Last updated: 2017-01-19Bibliographically approved

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Frost, StefanHo, OanhLogin, Frédéric HWeise, Christoph FWolf-Watz, HansWolf-Watz, Magnus
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