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Thanikkal, Edvin
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Publications (6 of 6) Show all publications
Thanikkal, E. J., Kumar Gahlot, D., Liu, J., Fredriksson Sundbom, M., Gurung, J. M., Ruuth, K., . . . Francis, M. S. (2019). The Yersinia pseudotuberculosis Cpx envelope stress system contributes to transcriptional activation of rovM. Virulence, 10(1), 37-57
Open this publication in new window or tab >>The Yersinia pseudotuberculosis Cpx envelope stress system contributes to transcriptional activation of rovM
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2019 (English)In: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 10, no 1, p. 37-57Article in journal (Refereed) Published
Abstract [en]

The Gram-negative enteropathogen Yersinia pseudotuberculosis possesses a number of regulatory systems that detect cell envelope damage caused by noxious extracytoplasmic stresses. The CpxA sensor kinase and CpxR response regulator two-component regulatory system is one such pathway. Active Cpx signalling upregulates various factors designed to repair and restore cell envelope integrity. Concomitantly, this pathway also down-regulates key determinants of virulence. In Yersinia, cpxA deletion accumulates high levels of phosphorylated CpxR (CpxR~P). Accumulated CpxR~P directly repressed rovA expression and this limited expression of virulence-associated processes. A second transcriptional regulator, RovM, also negatively regulates rovA expression in response to nutrient stress. Hence, this study aimed to determine if CpxR~P can influence rovA expression through control of RovM levels. We determined that the active CpxR~P isoform bound to the promoter of rovM and directly induced its expression, which naturally associated with a concurrent reduction in rovA expression. Site-directed mutagenesis of the CpxR~P binding sequence in the rovM promoter region desensitised rovM expression to CpxR~P. These data suggest that accumulated CpxR~P inversely manipulates the levels of two global transcriptional regulators, RovA and RovM, and this would be expected to have considerable influence on Yersinia pathophysiology and metabolism.

Place, publisher, year, edition, pages
Taylor & Francis Group, 2019
Keywords
Environmental stress responsiveness, gene expression control, metabolic networks, microbial behaviour, growth and survival, fitness
National Category
Microbiology Microbiology in the medical area
Research subject
Microbiology; Molecular Biology; Infectious Diseases
Identifiers
urn:nbn:se:umu:diva-154425 (URN)10.1080/21505594.2018.1556151 (DOI)000453473300001 ()30518290 (PubMedID)
Funder
Swedish Research Council, 2009-3660Swedish Research Council, 2014-6652
Available from: 2018-12-17 Created: 2018-12-17 Last updated: 2019-03-05Bibliographically approved
Thanikkal, E. (2014). Controlling virulence in Yersinia pseudotuberculosis through accumulation of phosphorylated CpxR. (Doctoral dissertation). Umeå: Umeå university
Open this publication in new window or tab >>Controlling virulence in Yersinia pseudotuberculosis through accumulation of phosphorylated CpxR
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[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. 

Place, publisher, year, edition, pages
Umeå: Umeå university, 2014. p. 63
Keywords
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:nbn:se:umu:diva-97320 (URN)978-91-7601-163-8 (ISBN)
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: 2018-06-07Bibliographically approved
Liu, J., Thanikkal, E., Obi, I. & Francis, M. (2012). Elevated CpxR~P levels repress the Ysc-Yop type III secretion system of Yersinia pseudotuberculosis. Research in Microbiology, 163(8), 518-530
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, p. 518-530Article 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
Keywords
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: 2018-06-08Bibliographically approved
Thanikkal, E., Mangu, J. & Francis, M. (2012). Interactions of the CpxA sensor kinase and cognate CpxR response regulator from Yersinia pseudotuberculosis. BMC Research Notes, 5(1), 536
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, p. 536-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
Keywords
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: 2018-06-08Bibliographically approved
Liu, J., Obi, I. R., Thanikkal, E. J., Kieselbach, T. & Francis, M. S. (2011). Phosphorylated CpxR Restricts Production of the RovA Global Regulator in Yersinia pseudotuberculosis. PLoS ONE, 6(8), e23314
Open this publication in new window or tab >>Phosphorylated CpxR Restricts Production of the RovA Global Regulator in Yersinia pseudotuberculosis
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 8, p. e23314-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: 2018-06-08Bibliographically approved
Thanikkal, E., Obi, I., Liu, J., Gurung, J., Dersch, P. & Francis, M.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
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(English)Manuscript (preprint) (Other academic)
Keywords
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: 2018-06-07Bibliographically approved
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