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Elevated CpxR~P levels repress the Ysc-Yop type III secretion system of 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). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). (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)
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). (Matthew Francis)
2012 (Engelska)Ingår i: Research in Microbiology, ISSN 0923-2508, E-ISSN 1769-7123, Vol. 163, nr 8, s. 518-530Artikel i tidskrift (Refereegranskat) 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.

Ort, förlag, år, upplaga, sidor
Elsevier, 2012. Vol. 163, nr 8, s. 518-530
Nyckelord [en]
Extracytoplasmic stress, CpxA, AckA, Pta, virulence
Nationell ämneskategori
Mikrobiologi Mikrobiologi inom det medicinska området
Forskningsämne
mikrobiologi; molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-60454DOI: 10.1016/j.resmic.2012.07.010OAI: oai:DiVA.org:umu-60454DiVA, id: diva2:560353
Forskningsfinansiär
VetenskapsrådetTillgänglig från: 2012-10-19 Skapad: 2012-10-13 Senast uppdaterad: 2018-06-08Bibliografiskt granskad
Ingår i avhandling
1. Controlling virulence in Yersinia pseudotuberculosis through accumulation of phosphorylated CpxR
Öppna denna publikation i ny flik eller fönster >>Controlling virulence in Yersinia pseudotuberculosis through accumulation of phosphorylated CpxR
2014 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Alternativ titel[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. 

Ort, förlag, år, upplaga, sidor
Umeå: Umeå university, 2014. s. 63
Nyckelord
Y. pseudotuberculosis, CpxA, CpxR, invasin, RovA, RovM, T3SS, virulence, transcriptional regulation
Nationell ämneskategori
Mikrobiologi Biokemi och molekylärbiologi
Forskningsämne
molekylärbiologi; mikrobiologi
Identifikatorer
urn:nbn:se:umu:diva-97320 (URN)978-91-7601-163-8 (ISBN)
Disputation
2015-01-22, Norrlands universitetssjukhus, Auditorium E04, Unod R1, Umeå universitet, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2014-12-19 Skapad: 2014-12-15 Senast uppdaterad: 2018-06-07Bibliografiskt granskad

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Förlagets fulltexthttp://www.sciencedirect.com/science/article/pii/S0923250812001052

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Liu, JunfaThanikkal, EdvinObi, IkennaFrancis, Matthew

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Liu, JunfaThanikkal, EdvinObi, IkennaFrancis, Matthew
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Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet)Umeå Centre for Microbial Research (UCMR)
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Research in Microbiology
MikrobiologiMikrobiologi inom det medicinska området

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