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
Controlling virulence in Yersinia pseudotuberculosis through accumulation of phosphorylated CpxR
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå university. (Matthew Francis)ORCID iD: 0000-0002-5529-4021
2014 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Reglering av virulens hos Yersinia pseudotuberculosis genom ackumulering av fosforylerat CpxR-protein (Swedish)
Abstract [en]

Like many Gram-negative bacteria, the food-borne pathogen Yersinia pseudotuberculosis harbours different regulatory mechanisms to maintain an intact bacterial envelope especially during exposure to extracytoplasmic stress (ECS). The CpxA-CpxR two component regulatory system is one such ECS-responsive regulatory mechanism. Activation of CpxA-CpxR two-component regulatory system (TCRS) accumulates phosphorylated CpxR (CpxR~P), which not only up-regulates various factors that are designed to maintain envelope integrity, but also down-regulates key determinants of bacterial virulence.

Y. pseudotuberculosis establishes close host cell contact in part through the expression of the invasin adhesin. Invasin expression is positively regulated by the transcriptional regulator RovA, which in turn is negatively regulated in response to nutrient stress by a second transcriptional regulator RovM. In Y. pseudotuberculosis, loss of CpxA phosphatase activity accumulates CpxR~P, and this represses both rovA and inv transcription directly, or indirectly via activation of rovM transcription. It is now of interest to understand the molecular mechanism behind how CpxR~P regulates gene transcription both positively and negatively.

A type III secretion system (T3SS) is a highly conserved multi-protein secretion system used by many Gram-negative bacteria to secrete protein cargo that counteracts the effects of a host cell emitted anti-bacterial activity. A typical set of proteins that make-up a functional T3SS includes structural proteins, translocators, effectors and regulatory proteins. Accumulation of CpxR~P was shown to repress the plasmid encoded Ysc-Yop T3SS of Y. pseudotuberculosis. Although yet to be confirmed experimentally, promoter-CpxR~P binding studies indicate multiple modes of regulatory control that for example, could influence levels of the plasmid-encoded Ysc-Yop system transcriptional activator, LcrF, and the chromosomal encoded negative regulators YmoA and YtxR. 

Regulatory processes of TCRS involve transient molecular interactions between different proteins and also protein with DNA. Protein-protein interaction studies using the BACTH assay showed that it can be useful in analysing the molecular interactions involving the N-terminal domain of CpxR, while the λcI homodimerization assay can be useful in analysing molecular interactions involving the C-terminal domain of CpxR. Therefore, in combination with other biochemical and physiological tests, these hybrid-based assays can be useful in dissecting molecular contacts that can be helpful in exploring the mechanism behind CpxR~P mediated transcriptional regulation.

In conclusion, this work uncovered direct involvement of CpxR~P in down-regulating virulence in Yersinia pseudotuberculosis. It also utilised genetic mutation and explored different protein-protein interaction assays to begin to investigate the mechanism behind the positive and negative regulation of gene expression mediated through active CpxR~P. 

Place, publisher, year, edition, pages
Umeå: Umeå university , 2014. , 63 p.
Keyword [en]
Y. pseudotuberculosis, CpxA, CpxR, invasin, RovA, RovM, T3SS, virulence, transcriptional regulation
National Category
Microbiology Biochemistry and Molecular Biology
Research subject
Molecular Biology; Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-97320ISBN: 978-91-7601-163-8 (print)OAI: oai:DiVA.org:umu-97320DiVA: diva2:771931
Public defence
2015-01-22, Norrlands universitetssjukhus, Auditorium E04, Unod R1, Umeå universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2014-12-19 Created: 2014-12-15 Last updated: 2014-12-17Bibliographically approved
List of papers
1. Phosphorylated CpxR Restricts Production of the RovA Global Regulator in Yersinia pseudotuberculosis
Open this publication in new window or tab >>Phosphorylated CpxR Restricts Production of the RovA Global Regulator in Yersinia pseudotuberculosis
Show others...
2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 8, e23314- p.Article in journal (Refereed) Published
Abstract [en]

Background: RovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels are kept in check by a sophisticated layering of distinct transcriptional and post-transcriptional regulatory mechanisms. In the enteropathogen Y. pseudotuberculosis, we have previously reported that the extracytoplasmic stress sensing CpxA-CpxR two-component regulatory system modulates rovA expression.

Methodology/Principal Findings: In this study, we characterized CpxR phosphorylation (CpxR similar to P) in vitro, and determined that phosphorylation was necessary for CpxR to efficiently bind to the PCR-amplified upstream regulatory region of rovA. The precise CpxR similar to P binding site was mapped by a nuclease protection assay and directed mutagenesis confirmed that in vivo binding to the rovA promoter inhibits transcription. Reduced RovA production was most pronounced following CpxR, P accumulation in the Yersinia cytoplasm during chronic Cpx pathway activation and by the indiscriminate phosphodonor action of acetyl phosphate.

Conclusions/Significance: Cpx pathway activation restricts levels of the RovA global regulator. The regulatory influence of CpxR similar to P must therefore extend well beyond periplasmic quality control in the Yersinia envelope, to include genes involved in environmental survival and pathogenicity.

Place, publisher, year, edition, pages
Public Library of Science, 2011
National Category
Biological Sciences
Identifiers
urn:nbn:se:umu:diva-46172 (URN)10.1371/journal.pone.0023314 (DOI)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2011-08-29 Created: 2011-08-29 Last updated: 2017-12-08Bibliographically approved
2. The Yersinia pseudotuberculosis Cpx envelope stress system contributes to transcription activation of rovM
Open this publication in new window or tab >>The Yersinia pseudotuberculosis Cpx envelope stress system contributes to transcription activation of rovM
Show others...
(English)Manuscript (preprint) (Other academic)
Keyword
extracytoplasmic stress, two-component signal transduction, gene regulation, virulence, nutrient availability
National Category
Microbiology
Research subject
Microbiology; Molecular Biology
Identifiers
urn:nbn:se:umu:diva-97337 (URN)
Available from: 2014-12-15 Created: 2014-12-15 Last updated: 2014-12-17Bibliographically approved
3. Elevated CpxR~P levels repress the Ysc-Yop type III secretion system of Yersinia pseudotuberculosis
Open this publication in new window or tab >>Elevated CpxR~P levels repress the Ysc-Yop type III secretion system of Yersinia pseudotuberculosis
2012 (English)In: Research in Microbiology, ISSN 0923-2508, E-ISSN 1769-7123, Vol. 163, no 8, 518-530 p.Article in journal (Refereed) Published
Abstract [en]

One way that Gram-negative bacteria respond to extracytoplasmic stress is through the CpxA-CpxR system. An activated CpxA sensor kinase phosphorylates the CpxR response regulator to instigate positive auto-amplification of Cpx pathway activation, as well as synthesis of various bacterial survival factors. In the absence of CpxA, human enteropathogenic Yersinia pseudotuberculosis accumulates high CpxR~P levels aided by the action of low molecular weight phosphodonors such as acetyl~P. Critically, these bacteria are also defective for plasmid encoded Ysc-Yop-dependent type III synthesis and secretion, an essential determinant of virulence. Herein, we investigated whether elevated CpxR~P levels account for lost Ysc-Yop function. Decisively, reducing CpxR~P in Yersinia defective for CpxA phosphatase activity - through incorporating second-site suppressor mutations in ackA-pta or cpxR - dramatically restored Ysc-Yop T3S function. Moreover, the repressive effect of accumulated CpxR~P is a direct consequence of binding to the promoter regions of the T3S genes. Thus, Cpx pathway activation has two consequences in Yersinia; one, to maintain quality control in the bacterial envelope, and the second, to restrict ysc-yop gene expression to those occasions where it will have maximal effect.

Place, publisher, year, edition, pages
Elsevier: , 2012
Keyword
Extracytoplasmic stress, CpxA, AckA, Pta, virulence
National Category
Microbiology Microbiology in the medical area
Research subject
Microbiology; Molecular Biology
Identifiers
urn:nbn:se:umu:diva-60454 (URN)10.1016/j.resmic.2012.07.010 (DOI)
Funder
Swedish Research Council
Available from: 2012-10-19 Created: 2012-10-13 Last updated: 2017-12-07Bibliographically approved
4. Interactions of the CpxA sensor kinase and cognate CpxR response regulator from Yersinia pseudotuberculosis
Open this publication in new window or tab >>Interactions of the CpxA sensor kinase and cognate CpxR response regulator from Yersinia pseudotuberculosis
2012 (English)In: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 5, no 1, 536- p.Article in journal (Refereed) Published
Abstract [en]

Background

The CpxA sensor kinase-CpxR response regulator two-component regulatory system is a sentinel of bacterial envelope integrity. Integrating diverse signals, it can alter the expression of a wide array of components that serve to shield the envelope from damage and to promote bacterial survival. In bacterial pathogens such as Yersinia pseudotuberculosis, this also extends to pathogenesis. CpxR is thought to dimerize upon phosphorylation by the sensor kinase CpxA. This phosphorylation enables CpxR binding to specific DNA sequences where it acts on gene transcription. As Cpx pathway activation is dependent on protein-protein interactions, we performed an interaction analysis of CpxR and CpxA from Y. pseudotuberculosis.

Results

CpxR full-length and truncated versions that either contained or lacked a putative internal linker were all assessed for their ability to homodimerize and interact with CpxA. Using an adenylate cyclase-based bacterial two hybrid approach, full-length CpxR readily engaged with CpxA. The CpxR N-terminus could also homodimerize with itself and with a full-length CpxR. A second homodimerization assay based upon the lamda cI repressor also demonstrated that the CpxR C-terminus could homodimerize. While the linker was not specifically required, it enhanced CpxR homodimerization. Mutagenesis of cpxR identified the aspartate at residue 51, putative N-terminal coiled-coil and C-terminal winged-helix-turn-helix domains as mediators of CpxR homodimerization. Scrutiny of CpxA full-length and truncated versions revealed that dimerization involved the N-terminus and an internal dimerization and histidine phosphotransfer domain.

Conclusions

This interaction analysis mapped regions of CpxR and CpxA that were responsible for interactions with self or with each other. When combined with other physiological and biochemical tests both hybrid-based assays can be useful in dissecting molecular contacts that may underpin Cpx pathway activation and repression.

Place, publisher, year, edition, pages
BioMed Central, 2012
Keyword
BACTH assay, lambda cI homodimerization assay, homodimer, heterodimer, linker, coiled-coil, winged helix-turn-helix, phosphorylation
National Category
Biochemistry and Molecular Biology Microbiology Microbiology in the medical area
Research subject
Microbiology; Molecular Biology
Identifiers
urn:nbn:se:umu:diva-60582 (URN)10.1186/1756-0500-5-536 (DOI)
Funder
Swedish Research Council
Available from: 2012-10-23 Created: 2012-10-17 Last updated: 2017-12-07Bibliographically approved

Open Access in DiVA

fulltext(952 kB)265 downloads
File information
File name FULLTEXT01.pdfFile size 952 kBChecksum SHA-512
e0aaa844ddcd6ca1a42f9a5bb674919faeb57f8b383d1e217d8c8fe333f42315648cd6d9429049eddd3a8bde3c3321fb9eb0f4c16920cedb027fee992fdc7fd3
Type fulltextMimetype application/pdf
Spikblad(33 kB)11 downloads
File information
File name SPIKBLAD01.pdfFile size 33 kBChecksum SHA-512
0a7a73a699add6880534aef1351f68ac2901b99d0aeb5e915ccda13ec430429f9c5c92957db73b78472c1c88f462f00304b05266bb2821055d6ecb6cf4f34796
Type spikbladMimetype application/pdf

Search in DiVA

By author/editor
Thanikkal, Edvin
By organisation
Department of Molecular Biology (Faculty of Science and Technology)
MicrobiologyBiochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar
Total: 265 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: 434 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