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Liu, Junfa
Publications (7 of 7) 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
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
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
Carlsson, K. E., Liu, J., Edqvist, P. J. & Francis, M. S. (2007). Extracytoplasmic-stress-responsive pathways modulate type III secretion in Yersinia pseudotuberculosis.. Infect Immun, 75(8), 3913-24
Open this publication in new window or tab >>Extracytoplasmic-stress-responsive pathways modulate type III secretion in Yersinia pseudotuberculosis.
2007 (English)In: Infect Immun, ISSN 0019-9567, Vol. 75, no 8, p. 3913-24Article in journal (Refereed) Published
Abstract [en]

Three signal transduction pathways, the two-component systems CpxRA and BaeSR and the alternative sigma factor sigma(E), respond to extracytoplasmic stress that facilitates bacterial adaptation to changing environments. At least the CpxRA and sigma(E) pathways control the production of protein-folding and degradation factors that counter the effects of protein misfolding in the periplasm. This function also influences the biogenesis of multicomponent extracellular appendages that span the bacterial envelope, such as various forms of pili. Herein, we investigated whether any of these regulatory pathways in the enteropathogen Yersinia pseudotuberculosis affect the functionality of the Ysc-Yop type III secretion system. This is a multicomponent molecular syringe spanning the bacterial envelope used to inject effector proteins directly into eukaryotic cells. Disruption of individual components revealed that the Cpx and sigma(E) pathways are important for Y. pseudotuberculosis type III secretion of Yops (Yersinia outer proteins). In particular, a loss of CpxA, a sensor kinase, reduced levels of structural Ysc (Yersinia secretion) components in bacterial membranes, suggesting that these mutant bacteria are less able to assemble a functional secretion apparatus. Moreover, these bacteria were no longer capable of localizing Yops into the eukaryotic cell interior. In addition, a cpxA lcrQ double mutant engineered to overproduce and secrete Yops was still impaired in intoxicating cells. Thus, the Cpx pathway might mediate multiple influences on bacterium-target cell contact that modulate Yersinia type III secretion-dependent host cell cytotoxicity.

Keywords
Bacterial Proteins/genetics/metabolism/physiology, Cell Survival, Gene Deletion, Hela Cells, Humans, Membrane Proteins/*metabolism, Organelles/physiology, Protein Folding, Protein Kinases/genetics/physiology, Protein Transport/physiology, Sigma Factor/genetics/physiology, Signal Transduction/genetics/*physiology, Transcription Factors/genetics/physiology, Yersinia pseudotuberculosis/*pathogenicity/*physiology
Identifiers
urn:nbn:se:umu:diva-16667 (URN)17517869 (PubMedID)
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2018-06-09Bibliographically approved
Carlsson, K. E., Liu, J., Edqvist, P. J. & Francis, M. S. (2007). Influence of the Cpx extracytoplasmic-stress-responsive pathway on Yersinia sp.-eukaryotic cell contact.. Infect Immun, 75(9), 4386-99
Open this publication in new window or tab >>Influence of the Cpx extracytoplasmic-stress-responsive pathway on Yersinia sp.-eukaryotic cell contact.
2007 (English)In: Infect Immun, ISSN 0019-9567, Vol. 75, no 9, p. 4386-99Article in journal (Refereed) Published
Abstract [en]

The extracytoplasmic-stress-responsive CpxRA two-component signal transduction pathway allows bacteria to adapt to growth in extreme environments. It controls the production of periplasmic protein folding and degradation factors, which aids in the biogenesis of multicomponent virulence determinants that span the bacterial envelope. This is true of the Yersinia pseudotuberculosis Ysc-Yop type III secretion system. However, despite using a second-site suppressor mutation to restore Yop effector secretion by yersiniae defective in the CpxA sensor kinase, these bacteria poorly translocated Yops into target eukaryotic cells. Investigation of this phenotype herein revealed that the expression of genes which encode several surface-located adhesins is also influenced by the Cpx pathway. In particular, the expression and surface localization of invasin, an adhesin that engages beta1-integrins on the eukaryotic cell surface, are severely restricted by the removal of CpxA. This reduces bacterial association with eukaryotic cells, which could be suppressed by the ectopic production of CpxA, invasin, or RovA, a positive activator of inv expression. In turn, these infected eukaryotic cells then became susceptible to intoxication by translocated Yop effectors. In contrast, bacteria harboring an in-frame deletion of cpxR, which encodes the cognate response regulator, displayed an enhanced ability to interact with cell monolayers, as well as elevated inv and rovA transcription. This phenotype could be drastically suppressed by providing a wild-type copy of cpxR in trans. We propose a mechanism of inv regulation influenced by the direct negative effects of phosphorylated CpxR on inv and rovA transcription. In this fashion, sensing of extracytoplasmic stress by CpxAR contributes to productive Yersinia sp.-eukaryotic cell interactions.

Identifiers
urn:nbn:se:umu:diva-16664 (URN)17620356 (PubMedID)
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2018-06-09Bibliographically approved
Edqvist, P. J., Aili, M., Liu, J. & Francis, M. S. (2007). Minimal YopB and YopD translocator secretion by Yersinia is sufficient for Yop-effector delivery into target cells.. Microbes and infection, 9(2), 224-233
Open this publication in new window or tab >>Minimal YopB and YopD translocator secretion by Yersinia is sufficient for Yop-effector delivery into target cells.
2007 (English)In: Microbes and infection, ISSN 1286-4579, E-ISSN 1769-714X, Vol. 9, no 2, p. 224-233Article in journal (Refereed) Published
Abstract [en]

Pathogenic Yersinia sp. utilise a common type III secretion system to translocate several anti-host Yop effectors into the cytosol of target eukaryotic cells. The secreted YopB and YopD translocator proteins are essential for this process, forming pores in biological membranes through which the effectors are thought to gain access to the cell interior. The non-secreted cognate chaperone, LcrH, also plays an important role by ensuring pre-secretory stabilisation and efficient secretion of YopB and YopD. This suggests that LcrH-regulated secretion of the translocators could be used by Yersinia to control effector translocation levels. We collected several LcrH mutants impaired in chaperone activity. These poorly bound, stabilised and/or secreted YopB and YopD in vitro. However, these mutants generally maintained stable substrates during a HeLa cell infection and these infected cells were intoxicated by translocated effectors. Surprisingly, this occurred in the absence of detectable YopB- and YopD-dependent pores in eukaryotic membranes. A functional type III translocon must therefore only require minuscule amounts of secreted translocator proteins. Based on these observations, LcrH dependent control of translocation via regulated YopB and YopD secretion would need to be exquisitely tight.

Keywords
Bacterial Outer Membrane Proteins/*biosynthesis, Bacterial Proteins/biosynthesis/genetics/*physiology, Hela Cells, Humans, Molecular Chaperones/genetics/*physiology, Mutagenesis, Point Mutation, Pore Forming Cytotoxic Proteins/biosynthesis, Protein Binding, Protein Transport/genetics, Virulence Factors/*metabolism, Yersinia pseudotuberculosis/genetics/metabolism/*pathogenicity
Identifiers
urn:nbn:se:umu:diva-16668 (URN)10.1016/j.micinf.2006.11.010 (DOI)17223369 (PubMedID)
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2018-06-09Bibliographically 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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