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Interactions of the CpxA sensor kinase and cognate CpxR response regulator from Yersinia pseudotuberculosis
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). (Matthew Francis)
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). (Matthew Francis)
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). (Matthew Francis)
2012 (engelsk)Inngår i: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 5, nr 1, s. 536-Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
BioMed Central, 2012. Vol. 5, nr 1, s. 536-
Emneord [en]
BACTH assay, lambda cI homodimerization assay, homodimer, heterodimer, linker, coiled-coil, winged helix-turn-helix, phosphorylation
HSV kategori
Forskningsprogram
mikrobiologi; molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-60582DOI: 10.1186/1756-0500-5-536OAI: oai:DiVA.org:umu-60582DiVA, id: diva2:561247
Forskningsfinansiär
Swedish Research CouncilTilgjengelig fra: 2012-10-23 Laget: 2012-10-17 Sist oppdatert: 2018-06-08bibliografisk kontrollert
Inngår i avhandling
1. Controlling virulence in Yersinia pseudotuberculosis through accumulation of phosphorylated CpxR
Åpne denne publikasjonen i ny fane eller vindu >>Controlling virulence in Yersinia pseudotuberculosis through accumulation of phosphorylated CpxR
2014 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Alternativ tittel[sv]
Reglering av virulens hos Yersinia pseudotuberculosis genom ackumulering av fosforylerat CpxR-protein
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. 

sted, utgiver, år, opplag, sider
Umeå: Umeå university, 2014. s. 63
Emneord
Y. pseudotuberculosis, CpxA, CpxR, invasin, RovA, RovM, T3SS, virulence, transcriptional regulation
HSV kategori
Forskningsprogram
molekylärbiologi; mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-97320 (URN)978-91-7601-163-8 (ISBN)
Disputas
2015-01-22, Norrlands universitetssjukhus, Auditorium E04, Unod R1, Umeå universitet, Umeå, 09:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2014-12-19 Laget: 2014-12-15 Sist oppdatert: 2018-06-07bibliografisk kontrollert

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