This work was supported by the Israel Science Foundation.

I. Designing a Yeast Expression System for Type III Effectors
Calibrating a yeast system appropriate for expression of the type III effector(s) of interest is an important task and may require some trial and error. Factors of major relevance that should be considered and optimized when designing such a system are: 1) the promoter driving expression of the effector(s), 2) the copy number of the effector gene, 3) the epitope tag used to verify protein expression, and 4) the yeast strain.
<p...

<ol>
	<li>Siggers, K. A., Lesser, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+Saccharomyces+cerevisiae:+a+versatile+model+system+for+the+identification+and+characterization+of+bacterial+virulence+proteins.">The yeast Saccharomyces cerevisiae: a versatile model system for the identification and characterization of bacterial virulence proteins.</a> Cell Host Microbe. 4, 8-15 (2008).</li><li>Parsons, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+chemical-genetic+and+genetic+interaction+data+links+bioactive+compounds+to+cellular+target+pathways.">Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways.</a> Nat. Biotechnol. 22, 62-69 (2004).</li><li>Munkvold, K. R., Martin, M. E., Bronstein, P. A., Collmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+the+Pseudomonas+syringae+pv.+tomato+DC3000+type+III+secretion+system+effector+repertoire+reveals+several+effectors+that+are+deleterious+when+expressed+in+Saccharomyces+cerevisiae.">A survey of the Pseudomonas syringae pv. tomato DC3000 type III secretion system effector repertoire reveals several effectors that are deleterious when expressed in Saccharomyces cerevisiae.</a> Mol. Plant-Microbe Interact. 21, 490-502 (2008).</li><li>Curak, J., Rohde, J., Stagljar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+as+a+tool+to+study+bacterial+effectors.">Yeast as a tool to study bacterial effectors.</a> Curr. Opin. Microbiol. 12, 18-23 (2009).</li><li>Slagowski, N. L., Kramer, R. W., Morrison, M. F., LaBaer, J., Lesser, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+genomic+yeast+screen+to+identify+pathogenic+bacterial+proteins.">A functional genomic yeast screen to identify pathogenic bacterial proteins.</a> PLoS Pathog. 4, e9-e9 (2008).</li><li>Huang, J., Lesser, C. F., Lory, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+role+of+the+CopN+protein+in+Chlamydia+pneumoniae+intracellular+growth.">The essential role of the CopN protein in Chlamydia pneumoniae intracellular growth.</a> Nature. 456, 112-115 (2008).</li></ol>

In this presentation, we illustrated how to use the budding yeast Saccharomyces cerevisiae as a heterologous system for the expression of type III bacterial effector proteins and how to identify effector-induced growth inhibition phenotypes. Importantly, these phenotypes can be utilized in genetic screens to identify suppressors of the negative impact of effectors on yeast growth. Suppressors may represent either direct targets of the effector studied or proteins that participate in cellular processes affected b...

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<td>Sarstedt</td>
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<td>Inoculation loop</td>
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identification of growth inhibition phenotypes induced by expression of bacterial type iii effectors in yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

Watch this Scientific Journal Video about Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast at JoVE.com

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

identification-growth-inhibition-phenotypes-induced-expression

Research

JoVE Journal

Biology

16.6K ビュー.  Tel Aviv University. このビデオでは、我々は酵母における細菌のタイプIIIエフェクターの発現とエフェクター誘導性成長抑制の表現型を同定するための手順を説明します。そのような表現型は、その後、エフェクター機能と目標を明らかにするために悪用される可能性があります。

ビデオ: Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Bacterial Gene Expression Analysis Using Microarrays

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and easy to use expression system.
As a single-celled microorganism P. pastoris is easy to manipulate and grows rapidly on inexpensive media at high cell densities. Being a eukaryote, P. pastoris is able to perform many of the post-translational modifications performed by higher eukaryotic cells and the obtained recombinant proteins undergo protein folding, proteolytic processing, disulfide bond formation and glycosylation [1].
As a methylotrophic yeast P. pastoris is capable of metabolizing methanol as its sole carbon source. The strong promoter for alcohol oxidase, AOX1, is tightly regulated and induced by methanol and it is used for the expression of the gene of interest. Accordingly, the expression of the foreign protein can be induced by adding methanol to the growth medium [2; 3].
Another important advantage is the secretion of the recombinant protein into the growth medium, using a signal sequence to target the foreign protein to the secretory pathway of P. pastoris. With only low levels of endogenous protein secreted to the media by the yeast itself and no added proteins to the media, a heterologous protein builds the majority of the total protein in the medium and facilitates following protein purification steps [3; 4].
The vector used here (pPICZ&alpha;A) contains the AOX1 promoter for tightly regulated, methanol-induced expression of the gene of interest; the &alpha;-factor secretion signal for secretion of the recombinant protein, a Zeocin resistance gene for selection in both E. coli and Pichia and a C-terminal peptide containing the c-myc epitope and a polyhistidine (6xHis) tag for detection and purification of a recombinant protein. We also show western blot analysis of the recombinant protein using the specific Anti-myc-HRP antibody recognizing the c-myc epitope on the parent vector.

Expression of Recombinant Proteins in the Methylotrophic Yeast Pichia pastoris

The protocol describes protein expression using the methylotrophic yeast Pichia pastoris. The preparation of electrocompetent yeast cells, transformation of the vector with the gene of interest into P. pastoris and yeast DNA purification are also performed. Western blot analysis and protein purification build the last steps in this protein expression protocol.

Protein expression in the microbial eukaryotic host Pichia pastoris offers the possibility to generate high amounts of recombinant protein in a fast and ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter &beta;1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the "classical" ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.

For development of RNA interference (RNAi)-based therapies, a novel strategy was developed, transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells. Here, tkRNAi was successfully applied for reversal of classical ABCB1-mediated multidrug resistance (MDR) of cancer cells.

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's disease, and Huntington's disease 1. Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity 2. At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy&#x2019;s disease 3.
The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins 3-8. Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. 
A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. 
Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.

This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

Quantification of Orofacial Phenotypes in Xenopus

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial development will allow for more rigorous analysis of orofacial phenotypes upon abrogation with substances that can genetically or molecularly manipulate gene expression or protein function. Using two dimensional images of the embryonic heads, traditional size dimensions-such as orofacial width, height and area- are measured. In addition, a roundness measure of the embryonic mouth opening is used to describe the shape of the mouth. Geometric morphometrics of these two dimensional images is also performed to provide a more sophisticated view of changes in the shape of the orofacial region. Landmarks are assigned to specific points in the orofacial region and coordinates are created. A principle component analysis is used to reduce landmark coordinates to principle components that then discriminate the treatment groups. These results are displayed as a scatter plot in which individuals with similar orofacial shapes cluster together. It is also useful to perform a discriminant function analysis, which statistically compares the positions of the landmarks between two treatment groups. This analysis is displayed on a transformation grid where changes in landmark position are viewed as vectors. A grid is superimposed on these vectors so that a warping pattern is displayed to show where significant landmark positions have changed. Shape changes in the discriminant function analysis are based on a statistical measure, and therefore can be evaluated by a p-value. This analysis is simple and accessible, requiring only a stereoscope and freeware software, and thus will be a valuable research and teaching resource.

Quantification of Orofacial Phenotypes in Xenopus

A method to quantify the orofacial size and shape of Xenopus laevis embryos has been developed. In this protocol, traditional size measurements are combined with geometric morphometrics to allow for more sophisticated analyses of orofacial development and defects.

Xenopus has become an important tool for dissecting the mechanisms governing craniofacial development and defects. A method to quantify orofacial ...

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...