umu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Multiple twists in the molecular tales of YopD and LcrH in type III secretion by Yersinia pseudotuberculosis
Umeå University, Faculty of Medicine, Molecular Biology.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The type III secretion system (T3SS) is a highly conserved secretion system among Gram negative bacteria that translocates anti-host proteins directly into the infected cells to overcome the host immune system and establish a bacterial infection. Yersinia pseudotuberculosis is one of three pathogenic Yersinia spp. that use a plasmid encoded T3SS to establish an infection. This complex multi-component Ysc-Yop system is tightly regulated in time and space. The T3SS is induced upon target cell contact and by growth in the absence of calcium. There are two kinds of substrates for the secretion apparatus, the translocator proteins that make up the pore in the eukaryotic target cell membrane, and the translocated effector proteins, that presumably pass through this pore en route to the eukaryotic cell interior.

The essential YopD translocator protein is involved in several important steps during effector translocation, such as pore formation, effector translocation. Moreover, in complex with its cognate chaperone LcrH, it maintains regulatory control of yop gene expression. To understand the molecular mechanism of YopD function, we made sequential in-frame deletions throughout the entire protein and identified discrete functional domains that made it possible to separate the role of YopD in translocation from its role in pore formation and regulation, really supporting translocation to be a multi-step process. Further site-directed mutagenesis of the YopD C-terminus, a region important for these functions, revealed no function for amino acids in the coiled-coil domain, while hydrophobic residues within the alpha-helical amphipathic domain are functionally significant for regulation, pore formation and translocation of effectors.

Unique to the T3SSs are the chaperones which are required for efficient type III protein secretion. The translocator-class chaperone LcrH binds two translocator proteins, YopB and YopD, which is necessary for their pre-secretory stabilization and their efficient secretion. We have shown that LcrH interacts with each translocator at a unique binding-site established by the folding of its three tandem tetratricopeptide repeats (TPRs). Beside the regulatory LcrH-YopD complex, LcrH complexes with YscY, a component of the Ysc-Yop T3SS, that is also essential for regulatory control. Interestingly the roles for LcrH do not end here, because it also appears to function in fine tuning the amount of effector translocation into target cells upon cell contact. Moreover, LcrH’s role in pre-secretory stability appears to be an in vitro phenomenon, since upon bacteria-host cell contact we found accumulated levels of YopB and YopD inside the bacteria in absence of a LcrH chaperone. This suggests the true function of LcrH is seen during target cell contact. In addition, these stable YopB and YopD are secreted in a Ysc-Yop independent manner in absence of a functional LcrH. We propose a role for LcrH in conferring substrate secretion pathway specificity, guiding its substrate to the cognate Ysc-Yop T3SS to secure subsequent effector translocation.

Together, this work has sought to better understand the key functions of LcrH and YopD in Yersinia pathogenicity. Using an approach based heavily on recombinant DNA technology and tissue culture infections, the complex molecular cross-talk between chaperone and its substrate, and the effect this has on the Yersinia lifestyle, are now being discovered.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknat- och Medfak) , 2007. , 88 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1074
Keyword [en]
Yersinia pseudotuberculosis, T3SS, YopD, translocation process, LcrH, class II chaperone, substrate secretion pathway specificity
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-985ISBN: 91-7264-231-9 (print)OAI: oai:DiVA.org:umu-985DiVA: diva2:145291
Public defence
2007-02-16, Major Groove, 6 L NUS, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2007-01-31 Created: 2007-01-31 Last updated: 2009-05-15Bibliographically approved
List of papers
1. The YopD translocator of Yersinia pseudotuberculosis is a multifunctional protein comprised of discrete domains.
Open this publication in new window or tab >>The YopD translocator of Yersinia pseudotuberculosis is a multifunctional protein comprised of discrete domains.
Show others...
2004 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 186, no 13, 4110-4123 p.Article in journal (Refereed) Published
Abstract [en]

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III translocon to microinject several anti-host Yop effectors into the cytosol of target eukaryotic cells. YopD has been implicated in several key steps during Yop effector translocation, including maintenance of yop regulatory control and pore formation in the target cell membrane through which effectors traverse. These functions are mediated, in part, by an interaction with the cognate chaperone, LcrH. To gain insight into the complex molecular mechanisms of YopD function, we performed a systematic mutagenesis study to search for discrete functional domains. We highlighted amino acids beyond the first three N-terminal residues that are dispensable for YopD secretion and confirmed that an interaction between YopD and LcrH is essential for maintenance of yop regulatory control. In addition, discrete domains within YopD that are essential for both pore formation and translocation of Yop effectors were identified. Significantly, other domains were found to be important for effector microinjection but not for pore formation. Therefore, YopD is clearly essential for several discrete steps during efficient Yop effector translocation. Recognition of this modular YopD domain structure provides important insights into the function of YopD.

Keyword
Bacterial Outer Membrane Proteins/chemistry/*physiology, Bacterial Proteins/physiology, Base Sequence, Hela Cells, Hemolysis, Humans, Molecular Chaperones/physiology, Molecular Sequence Data, Protein Transport, Yersinia pseudotuberculosis/*physiology
Identifiers
urn:nbn:se:umu:diva-16673 (URN)10.1128/JB.186.13.4110-4123.2004 (DOI)15205412 (PubMedID)
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2017-12-14Bibliographically approved
2. Functional insights into the YopD C-terminus through comprehensive site-directed mutagenesis
Open this publication in new window or tab >>Functional insights into the YopD C-terminus through comprehensive site-directed mutagenesis
Show others...
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5687 (URN)
Available from: 2007-01-31 Created: 2007-01-31 Last updated: 2010-01-13Bibliographically approved
3. Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion.
Open this publication in new window or tab >>Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion.
Show others...
2006 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 59, no 1, 31-44 p.Article in journal (Refereed) Published
Abstract [en]

Non-flagellar type III secretion systems (T3SSs) transport proteins across the bacterial cell and into eukaryotic cells. Targeting of proteins into host cells requires a dedicated translocation apparatus. Efficient secretion of the translocator proteins that make up this apparatus depends on molecular chaperones. Chaperones of the translocators (also called class-II chaperones) are characterized by the possession of three tandem tetratricopeptide repeats (TPRs). We wished to dissect the relations between chaperone structure and function and to validate a structural model using site-directed mutagenesis. Drawing on a number of experimental approaches and focusing on LcrH, a class-II chaperone from the Yersinia Ysc-Yop T3SS, we examined the contributions of different residues, residue classes and regions of the protein to chaperone stability, chaperone-substrate binding, substrate stability and secretion and regulation of Yop protein synthesis. We confirmed the expected role of the conserved canonical residues from the TPRs to chaperone stability and function. Eleven mutations specifically abrogated YopB binding or secretion while three mutations led to a specific loss of YopD secretion. These are the first mutations described for any class-II chaperone that allow interactions with one translocator to be dissociated from interactions with the other. Strikingly, all mutations affecting the interaction with YopB mapped to residues with side chains projecting from the inner, concave surface of the modelled TPR structure, defining a YopB interaction site. Conversely, all mutations preventing YopD secretion affect residues that lie on the outer, convex surface of the triple-TPR cluster in our model, suggesting that this region of the molecule represents a distinct interaction site for YopD. Intriguingly, one of the LcrH double mutants, Y40A/F44A, was able to maintain stable substrates inside bacteria, but unable to secrete them, suggesting that these two residues might influence delivery of substrates to the secretion apparatus.

Keyword
Amino Acid Sequence, Bacterial Outer Membrane Proteins/chemistry/metabolism, Bacterial Proteins/chemistry/*genetics/*metabolism, Binding Sites, Humans, Models; Molecular, Molecular Chaperones/chemistry/*genetics/*metabolism, Molecular Sequence Data, Mutagenesis; Site-Directed, Phenotype, Protein Binding, Protein Conformation, Repetitive Sequences; Nucleic Acid, Sequence Alignment, Two-Hybrid System Techniques, Yersinia/genetics/metabolism
Identifiers
urn:nbn:se:umu:diva-16670 (URN)10.1111/j.1365-2958.2005.04923.x (DOI)16359316 (PubMedID)
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2017-12-14Bibliographically approved
4. Examination of LcrH type III secretion chaperone function during Yersinia-eukaryotic cell contact
Open this publication in new window or tab >>Examination of LcrH type III secretion chaperone function during Yersinia-eukaryotic cell contact
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5689 (URN)
Available from: 2007-01-31 Created: 2007-01-31 Last updated: 2010-01-13Bibliographically approved
5. Mapping of a YscY binding domain within the LcrH chaperone that is required for regulation of Yersinia type III secretion
Open this publication in new window or tab >>Mapping of a YscY binding domain within the LcrH chaperone that is required for regulation of Yersinia type III secretion
Show others...
2005 (English)In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 187, no 22, 7738-7752 p.Article in journal (Refereed) Published
Abstract [en]

Type III secretion systems are used by many animal and plant interacting bacteria to colonize their host. These systems are often composed of at least 40 genes, making their temporal and spatial regulation very complex. Some type III chaperones of the translocator class are important regulatory molecules, such as the LcrH chaperone of Yersinia pseudotuberculosis. In contrast, the highly homologous PcrH chaperone has no regulatory effect in native Pseudomonas aeruginosa or when produced in Yersinia. In this study, we used LcrH-PcrH chaperone hybrids to identify a discrete region in the N terminus of LcrH that is necessary for YscY binding and regulatory control of the Yersinia type III secretion machinery. PcrH was unable to bind YscY and the homologue Pcr4 of P. aeruginosa. YscY and Pcr4 were both essential for type III secretion and reciprocally bound to both substrates YscX of Yersinia and Pcr3 of P. aeruginosa. Still, Pcr4 was unable to complement a DeltayscY null mutant defective for type III secretion and yop-regulatory control in Yersinia, despite the ability of YscY to function in P. aeruginosa. Taken together, we conclude that the cross-talk between the LcrH and YscY components represents a strategic regulatory pathway specific to Yersinia type III secretion.

Place, publisher, year, edition, pages
American Society for Microbiology, 2005
Keyword
Amino acid sequence, bacterial proteins, binding sites, DNA; bacterial, HeLa cells, humans, molecular chaperones, molecular sequence data, protein interaction mapping, protein structure; tertiary, protein transport, sequence analysis; DNA, Yersinia pseudotuberculosis
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-16672 (URN)10.1128/JB.187.22.7738-7752.2005 (DOI)000233400200021 ()16267298 (PubMedID)
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2017-12-14Bibliographically approved

Open Access in DiVA

fulltext(1524 kB)670 downloads
File information
File name FULLTEXT01.pdfFile size 1524 kBChecksum SHA-1
12970303b7a1f6a43ebc268c4b2ed4f34680d9ec7822728ad4c726c3c57db9fd164c7b6b
Type fulltextMimetype application/pdf

By organisation
Molecular Biology
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 670 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 589 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf