Electroporation of Functional Bacterial Effectors into Mammalian Cells

Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; Savchenko, Alexei; Skarina, Tatiana; Cui, Hong; Cort, John R.; Adkins, Joshua N.; Brown, Roslyn N.

doi:10.3791/52296

Title: Electroporation of Functional Bacterial Effectors into Mammalian Cells

Abstract

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

Authors:

Sontag, Ryan L. ^[1]; Mihai, Cosmin ^[2]; Orr, Galya ^[2]; Savchenko, Alexei ^[3]; Skarina, Tatiana ^[3]; Cui, Hong ^[3]; Cort, John R. ^[1]; Adkins, Joshua N. ^[1]; Brown, Roslyn N. ^[4]

Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Biological Sciences Division
Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Science Lab.
Univ. of Toronto, ON (Canada). Ontario Center for Structural Proteomics
Washington State Univ., Pullman, WA (United States). Center for Bioproducts and Bioenergy

Publication Date:: Mon Jan 19 00:00:00 EST 2015

Research Org.:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States). Environmental Molecular Sciences Laboratory (EMSL)

Sponsoring Org.:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:: 1184956

Report Number(s):: PNNL-SA-102481
Journal ID: ISSN 1940-087X; jove; 42696; 400412000

Grant/Contract Number:: AC05-76RL01830

Resource Type:: Accepted Manuscript

Journal Name:: Journal of Visualized Experiments

Additional Journal Information:: Journal Issue: 95; Journal ID: ISSN 1940-087X

Publisher:: MyJoVE Corp.

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; electroporation; protein; transfection; expression; localization; confocal microscopy; Salmonella; effector; Immunology; Issue 95; Environmental Molecular Sciences Laboratory

Citation Formats


                    Sontag, Ryan L., Mihai, Cosmin, Orr, Galya, Savchenko, Alexei, Skarina, Tatiana, Cui, Hong, Cort, John R., Adkins, Joshua N., and Brown, Roslyn N. Electroporation of Functional Bacterial Effectors into Mammalian Cells.  United States: N. p., 2015. 
Web.  doi:10.3791/52296.

Copy to clipboard


                    Sontag, Ryan L., Mihai, Cosmin, Orr, Galya, Savchenko, Alexei, Skarina, Tatiana, Cui, Hong, Cort, John R., Adkins, Joshua N., & Brown, Roslyn N. Electroporation of Functional Bacterial Effectors into Mammalian Cells.  United States.  https://doi.org/10.3791/52296

Copy to clipboard


                    Sontag, Ryan L., Mihai, Cosmin, Orr, Galya, Savchenko, Alexei, Skarina, Tatiana, Cui, Hong, Cort, John R., Adkins, Joshua N., and Brown, Roslyn N. Mon .  
"Electroporation of Functional Bacterial Effectors into Mammalian Cells".  United States.  https://doi.org/10.3791/52296.  https://www.osti.gov/servlets/purl/1184956.

Copy to clipboard


                    
@article{osti_1184956,

  title        = {Electroporation of Functional Bacterial Effectors into Mammalian Cells},

  author       = {Sontag, Ryan L. and Mihai, Cosmin and Orr, Galya and Savchenko, Alexei and Skarina, Tatiana and Cui, Hong and Cort, John R. and Adkins, Joshua N. and Brown, Roslyn N.},

  abstractNote = {Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.},

  doi          = {10.3791/52296},

  journal      = {Journal of Visualized Experiments},

  number       = 95,

  volume       = ,

  place        = {United States},

  year         = {Mon Jan 19 00:00:00 EST 2015},

  month        = {Mon Jan 19 00:00:00 EST 2015}

}

Copy to clipboard

Journal Article:

Free Publicly Available Full Text

Accepted Manuscript (DOE)

Publisher's Version of Record

https://doi.org/10.3791/52296

Other availability

Search WorldCat to find libraries that may hold this journal

Citation Metrics:

Cited by: 2 works

Citation information provided by
Web of Science

Save / Share:

Export Metadata

Save to My Library

Works referencing / citing this record:

Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter
journal, July 2016

Sontag, Ryan L.; Nakayasu, Ernesto S.; Brown, Roslyn N.
mSystems, Vol. 1, Issue 4
DOI: 10.1128/msystems.00032-15

Similar Records in DOE PAGES and OSTI.GOV collections:

Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

Journal Article Qiu, Jiazhang ; Sheedlo, Michael J. ; Yu, Kaiwen ; ... - Nature (London)

Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or asmore »« less
https://doi.org/10.1038/nature17657
Bacterial IMPDH gene used for the selection of mammalian cell transfectants.

Journal Article Baccam, M ; Huberman, E ; Energy Systems - BioTechiques

Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or require unique conditions. Here we describe a simple and versatile dominant selectable marker that involves bacterial IMP dehydrogenase (IMPDH), an enzyme essential for the replication of mammalian and bacterial cells. Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant to the toxic effect of themore »« less
The essential effector SCRE1 in Ustilaginoidea virens suppresses rice immunity via a small peptide region

Journal Article Zhang, Nan ; Yang, Jiyun ; Fang, Anfei ; ... - Molecular Plant Pathology

Summary The biotrophic fungal pathogen Ustilaginoidea virens causes rice false smut, a newly emerging plant disease that has become epidemic worldwide in recent years. The U. virens genome encodes many putative effector proteins that, based on the study of other pathosystems, could play an essential role in fungal virulence. However, few studies have been reported on virulence functions of individual U. virens effectors. Here, we report our identification and characterization of the secreted cysteine‐rich protein SCRE1, which is an essential virulence effector in U. virens . When SCRE1 was heterologously expressed in Magnaporthe oryzae , the protein was secreted and translocatedmore »« less
Cited by 36
https://doi.org/10.1111/mpp.12894
Prediction of bacterial E3 ubiquitin ligase effectors using reduced amino acid peptide fingerprinting

Journal Article McDermott, Jason E. ; Cort, John R. ; Nakayasu, Ernesto S. ; ... - PeerJ

Background Although pathogenic Gram-negative bacteria lack their own ubiquitination machinery, they have evolved or acquired virulence effectors that can manipulate the host ubiquitination process through structural and/or functional mimicry of host machinery. Many such effectors have been identified in a wide variety of bacterial pathogens that share little sequence similarity amongst themselves or with eukaryotic ubiquitin E3 ligases. Methods To allow identification of novel bacterial E3 ubiquitin ligase effectors from protein sequences we have developed a machine learning approach, the SVM-based Identification and Evaluation of Virulence Effector Ubiquitin ligases (SIEVE-Ub). We extend the string kernel approach used previously to sequencemore »« less
Cited by 4
https://doi.org/10.7717/peerj.7055
Subcellular Localization of Pseudomonas syringae pv. tomato Effector Proteins in Plants

Journal Article Aung, Kyaw ; Xin, Xiufang ; Mecey, Christy ; ... - Methods in Molecular Biology (Online)

Animal and plant pathogenic bacteria use type III secretion systems to translocate proteinaceous effectors to subvert innate immunity of their host organisms. Type III secretion/effector system is a crucial pathogenicity factor in many bacterial pathogens of plants and animals. Pseudomonas syringae pv. tomato (Pst) DC3000 injects a total of 36 protein effectors, which target a variety of host proteins. Studies of a subset of Pst DC3000 effectors demonstrated that bacterial effectors, once inside the host cell, are localized to different subcellular compartments, including plasma membrane, cytoplasm, mitochondria, chloroplast, and Trans-Golgi network, to carry out their virulence functions. Identifying the subcellularmore »« less
Cited by 11
https://doi.org/10.1007/978-1-4939-6649-3_12

Full Text Available

Similar Records

Title: Electroporation of Functional Bacterial Effectors into Mammalian Cells

Abstract

Citation Formats

Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter journal, July 2016

Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter
journal, July 2016