Research in the Keestra-Gounder lab is supported by grants from the NIAID of the NIH under Award Number R21AI122092 and from the American Diabetes Association under Award Number 1-18-JDF-035.

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J., Phalipon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+Cell+Targeting+by+Enteropathogenic+Bacteria+T3SS+Effectors.">Host Cell Targeting by Enteropathogenic Bacteria T3SS Effectors.</a> Trends in Microbiology. 26 (4), 266-283 (2018).</li><li>Karin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+NF-kappaB+is+activated:+the+role+of+the+IkappaB+kinase+(IKK)+complex.">How NF-kappaB is activated: the role of the IkappaB kinase (IKK) complex.</a> Oncogene. , (1999).</li><li>Berti, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+real-time+RT-PCR+analysis+of+inflammatory+gene+expression+associated+with+ischemia-reperfusion+brain+injury.">Quantitative real-time RT-PCR analysis of inflammatory gene expression associated with ischemia-reperfusion brain injury.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 22 (9), 1068-1079 (2002).</li><li>Fried, M., Crothers, D. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-step+cross-linking+method+for+identification+of+NF-kappaB+gene+network+by+chromatin+immunoprecipitation.">Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation.</a> Biotechniques. 39 (5), 715-725 (2005).</li><li>Ernst, O., Vayttaden, S. J., Fraser, I. D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+NF-kappaB+Activation+in+TLR-Activated+Macrophages.">Measurement of NF-kappaB Activation in TLR-Activated Macrophages.</a> Methods in Molecular Biology. 1714, 67-78 (2018).</li><li>Rodriguez, M. S., Thompson, J., Hay, R. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+small+Rho+GTPases+is+a+pathogen-induced+process+detected+by+NOD1.">Manipulation of small Rho GTPases is a pathogen-induced process detected by NOD1.</a> Nature. 496 (7444), 233-237 (2013).</li><li>Wong, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+characterization+of+NF-kappaB+binding+uncovers+non-canonical+motifs+and+advances+the+interpretation+of+genetic+functional+traits.">Extensive characterization of NF-kappaB binding uncovers non-canonical motifs and advances the interpretation of genetic functional traits.</a> Genome Biology. 12 (7), R70 (2011).</li><li>Gupta, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firefly+luciferase+mutants+as+sensors+of+proteome+stress.">Firefly luciferase mutants as sensors of proteome stress.</a> Nature Methods. 8 (10), 879-884 (2011).</li><li>Cogswell, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NF-kappa+B+regulates+IL-1+beta+transcription+through+a+consensus+NF-kappa+B+binding+site+and+a+nonconsensus+CRE-like+site.">NF-kappa B regulates IL-1 beta transcription through a consensus NF-kappa B binding site and a nonconsensus CRE-like site.</a> Journal of Immunology. 153 (2), 712-723 (1994).</li><li>Thornberry, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Heterodimeric+Cysteine+Protease+Is+Required+for+Interleukin-1-Beta+Processing+in+Monocytes.">A Novel Heterodimeric Cysteine Protease Is Required for Interleukin-1-Beta Processing in Monocytes.</a> Nature. 356 (6372), 768-774 (1992).</li></ol>

The authors have nothing to disclose.

The major contribution of the protocol described is that it provides a fast and easy method to detect NF-&#954;B activation in cells, which allows for high throughput analysis of multiple stimulatory conditions or drugs affecting NF-&#954;B activation. Here, we describe a protocol for NF-&#954;B activation in Salmonella-infected HeLa cells. These cells can be used for infection with other pathogens as well to study the impact of bacterial infection on NF-&#954;B activation. In addition, NF-&#954;B-dependent luci...

The nuclear factor-&#954;B (NF-&#954;B) family of proteins are important transcription activators that regulate gene expression in various biological pathways. Activation of NF-&#954;B induces transcription of target genes, many of which are important for immune and inflammatory responses, cell proliferation, stress responses and cancer progression1,2. NF-&#954;B plays an integral role in mediating early inflammatory outcomes for pathogen clearance. Given the many biological processes mediated by NF-&#954;B activation, disruptions in its signaling can have serious consequences for health and disease. Loss of function mutations in NF-&#954;B signaling are associated in several immune deficiency phenotypes, while gain of function mutations are associated with several types of cancers, including B-cell lymphomas and breast cancers3. Additionally, many pathogens have been shown to directly modulate the activation state of NF-&#954;B through expression of virulence factors4,5,6,7.
Activation of NF-&#954;B is known to be a consequence of many variable stimuli including bacterial products such as lipopolysaccharides (LPS), flagellin and peptidoglycans known as pathogen-associated molecular patterns (PAMPs). These PAMPs are detected by pattern recognition receptors (PRRs) such as the Toll-like receptors (TLRs) and Nod-like receptors (NLRs) leading to the activation of NF-&#954;B and the subsequent expression of an array of NF-&#954;B-dependent inflammatory genes8. In addition to PRR activation by PAMPs, other bacterial products, such as bacterial effector proteins, can induce the activation of NF-&#954;B. Interestingly, bacteria also express effector proteins that actively attenuate the NF-&#954;B pathway and enhance their pathogenicity, underscoring the importance of NF-&#954;B as an essential mediator of immunity9.
There are five different subunits that form the NF-&#954;B dimers; p50, p52, RelA (p65), RelB and cRel. The two main NF-&#954;B heterodimers are the p50:RelA and the p52:RelB dimers. The activated NF-&#954;B dimers bind to DNA sites, known as &#954;B sites, in the promoter and enhancer regions of various target genes. Under normal homeostatic conditions, NF-&#954;B interacts with a family of inhibitor proteins known as I&#954;B proteins to remain inactive. Upon stimulation, I&#954;B is phosphorylated by I&#954;B Kinase (IKK), which allows it to be targeted for ubiquitination, and subsequently degradation. Degradation of I&#954;B activates NF-&#954;B by revealing a nuclear localization signal. NF-&#954;B then translocates to the nucleus, where it binds &#954;B sites in the promoter region of target genes and promote transcription10. Thus, activation of NF-&#954;B upregulates mRNA expression of NF-&#954;B target genes, and this change can be measured through RNA quantification assays such as RT-qPCR11.
Several methods exist and are commonly used for the measurement of NF-&#954;B activation, including electrophoretic mobility shift assays (EMSA), nuclear translocation, and gene reporter assays. EMSA is used to detect protein complexes with nucleic acids. Stimulated cells are fractionated to isolate nuclear proteins, including the translocated NF-&#954;B, which is then incubated with radiolabeled nucleotides containing the NF-&#954;B binding domain. The samples are run on a gel and imaged by autoradiography of 32P-labeled nucleic acid. If NF-&#954;B is present in the protein fraction, it will bind the nucleotides, which will migrate slower through the gel and present as discrete bands. Nuclear fractions of cells lacking activated NF-&#954;B (e.g., unstimulated control cells) will produce no bands as the nucleotides migrate faster to the end of the gel. A major drawback of this method is that it is largely quantitative in the binary sense (i.e., on or off) and does not adequately capture meaningful differences in NF-&#954;B binding capacity. Additionally, this method does not consider chromatin structures that are functionally important for NF-&#954;B target genes12,13.
Similar to the previous method, there is a &#34;non-shift&#34; assay in which multi-well plates are coated with nucleotides containing the NF-&#954;B binding sequence. Following treatment of cells with nuclear fractions of protein, NF-&#954;B will bind to the nucleotides bound to the well. Anti-NF-&#954;B antibodies are then added, which will interact with the bound NF-&#954;B and produce a colorimetric signal proportional to the amount of NF-&#954;B, indicating the degree of NF-&#954;B activation. This method is advantageous over the EMSA in that it does not require radiolabeled nucleic acids and is quantitative, in comparison. However, a caveat of this method is that it again does not differentiate between chromatin states of NF-&#954;B target genes14.
Another method by which NF-&#954;B activation may be detected is by chromatin immunoprecipitation (ChIP), whereby DNA and interacting proteins are cross-linked with formaldehyde and immunoprecipitated with specific anti-NF-&#954;B antibodies. The specific nucleotide fragments are then purified and identified through PCR amplification or direct high throughput sequencing. Results generated from this method provide semi-quantitative results of NF-&#954;B binding activity with target genes. However, the results are highly dependent on the fixation conditions and purification processes at each step15.
In nuclear translocation assays, cells are stimulated to induce NF-&#954;B activation and then fixed. Anti-p65 antibodies are added to fixed cells. Alternatively, the p65 subunit itself can be tagged with a fluorescent peptide such as green fluorescent green (GFP). In either case, immunofluorescence will allow imaging of localization of p65 to determine cellular distribution. By measuring the proportion of cytosolic and nuclear localized protein, investigators can determine the relative activation state of NF-&#954;B. A drawback of this method is that the immunofluorescence is comparatively time consuming, requires expensive antibodies, and needs relatively greater technical expertise16.
Reporter genes are commonly used tools to study the regulatory and expression patterns of a gene of interest. Typically, reporter genes are constructed from the promoter sequence of a gene of interest fused to a gene coding for an easily detectable protein. Proteins with enzymatic activities, fluorescence, or luminescence properties are commonly chosen for their ability to be assayed and quantified. Thus, the read-out (e.g., luminescence, fluorescence) serves as a signal for detection of the gene expression. These reporter constructs can then be introduced into different cell types, such as epithelial cells or macrophages.
Described in the protocol is the use of a cloned HeLa cell line (HeLa 57A) that is stably transfected with a luciferase reporter containing three copies of the &#954;B consensus of the immunoglobulin &#954;-chain promoter region17. Expression of luciferase is dependent on the activation of NF-&#954;B, which occurs following cell stimulation. Stimulated cells are easily lysed using cell lysis buffer provided in the luciferase assay kit. A portion of the cell lysate is then mixed with luciferase assay buffer that contains luciferin. Luciferin is the substrate of luciferase and is required for the generation of light in the presence of luciferase. After combining the assay buffer with the lysate, the solution will emit light in a process known as luminescence. The amount of light produced, given in lumens, is proportional to the amount of luciferase present in the lysate and serves as a measure of NF-&#954;B activation. The lumen readings are interpreted in comparison to an unstimulated standard to account for baseline NF-&#954;B activity and the signal itself is stable for several minutes to allow for reliable measurement. In addition, the HeLa 57A cell line is stably transfected with a NF-&#954;B-independent &#946;-galactosidase reporter. The &#946;-galactosidase reporter is constitutively expressed, and &#946;-galactosidase activity can be measured to control for cell viability or variation in cell numbers17. The luciferase values can then be adjusted to the &#946;-galactosidase values and reported as fold increase over the unstimulated control cells.
Since NF-&#954;B is a transcription factor responsible for the increased expression of NF-&#954;B-dependent target genes, a follow up experiment to control for NF-&#954;B-dependent increased gene expression is quantitative reverse transcription polymerase chain reaction (RT-qPCR). RT-qPCR is a highly sensitive method by which changes in the gene expression can be quantified over several orders of magnitude. Stimulated and control cells are harvested for RNA via phenol-chloroform extraction. Following phase separation, RNA is extracted as the major component of the aqueous layer. RNA is then precipitated and washed to produce a pure pellet. This pellet is then reconstituted and further cleaned of contaminant DNA via DNase treatment. The pure RNA is then reverse transcribed to create complementary DNA (cDNA). This cDNA can then be analyzed through quantitative PCR techniques, where the abundance of a specific mRNA sequence is quantified to determine gene expression. This technique does not elucidate translational control, post translational modification, protein abundance, or protein activity. However, many genes, particularly those involved in pro-inflammatory processes, are regulated via NF-&#954;B and their mRNA abundance is indicative of their expression.
The method proposed here utilizes a fast and simple way by which NF-&#954;B activation can be detected via luminescence assays of cellular lysate. RT-qPCR of NF-&#954;B target gene expression can be used to quantify expression of particular genes, as well as validate functional activity of NF-&#954;B activation. The major advantages of such a system are its simplicity and speed, which allows for high throughput screening of a range of conditions that modulate NF-&#954;B activation. This protocol is suitable for other cell lines expressing an NF-&#954;B::luciferase reporter, and has been demonstrated in stably transfected RAW264.7 cells18. The amount of time required to handle samples, starting from cell lysis to generating a luminescence signal, is minimal and takes the span of about an hour. Measurement of NF-&#954;B requires only basic laboratory equipment such as opaque plates, a plate reader capable of measuring luminescence, and simple data analysis software such as a spreadsheet program.

<table><tbody><tr><td>Adhesive film</td><td>VWR International</td><td>60941-070</td><td></td></tr><tr><td>Chloroform</td><td>Fisher Bioreagents</td><td>C298-500</td><td></td></tr><tr><td>DMEM</td><td>Thermo Fisher</td><td>11665092</td><td></td></tr><tr><td>DNAse treatment kit</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>dNTPs</td><td>Promega</td><td>U1511</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Bioreagents</td><td>BP2818100</td><td>molecular grade</td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F0926</td><td></td></tr><tr><td>HeLa 57A cells</td><td></td><td></td><td>Ref # 15</td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Applied Biosystems</td><td>4368814</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Bioreagents</td><td>BP26181</td><td></td></tr><tr><td>Kanamycin</td><td>Fisher Bioreagents</td><td>BP906-5</td><td></td></tr><tr><td>LB agar</td><td>Fisher Bioreagents</td><td>BP1425-500</td><td></td></tr><tr><td>Lysogeny broth</td><td>Fisher Bioreagents</td><td>BP1426-500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Chemical</td><td></td><td></td></tr><tr><td>NanoDrop ND-1000</td><td>Thermo Scientific</td><td></td><td>spectrophotometer</td></tr><tr><td>Promega luciferase assay system</td><td>Promega</td><td>E1501</td><td>Cell lysis buffer &amp;amp; luciferin substrate</td></tr><tr><td>Random Hexamers</td><td>Thermo Scientific</td><td>SO142</td><td></td></tr><tr><td>Real-time GAPDH forward primer</td><td></td><td></td><td>5&#039;-CCAGGAAATGAGCTTGAC&lt;br/&gt; AAAGT-3&#039;</td></tr><tr><td>Real-time GAPDH reverse primer</td><td></td><td></td><td>5-&#039;CCCACTCCTCCACCT&lt;br/&gt; TTGAC-3&#039;</td></tr><tr><td>Real-time IL-23 forward primer</td><td></td><td></td><td>5-&#039;GAGCCTTCTCTGCTCCC&lt;br/&gt; TGAT-3&#039;</td></tr><tr><td>Real-time IL-23 reverse primer</td><td></td><td></td><td>5&#039;-AGTTGGCTGAGGCCCAGTAG-3&#039;</td></tr><tr><td>Real-time IL-6 forward primer</td><td></td><td></td><td>5&#039;-GTAGCCGCCCCACACAGA-3&#039;</td></tr><tr><td>Real-time IL-6 reverse primer</td><td></td><td></td><td>5&#039;-CATGTCTCCTTTCTCAGG&lt;br/&gt; GCTG-3&#039;</td></tr><tr><td>Reverse Transcriptase</td><td>Applied Biosystems</td><td>4308228</td><td></td></tr><tr><td>RNAse inhibitor</td><td>Thermo Scientific</td><td>EO0381</td><td></td></tr><tr><td>RT buffer</td><td>Promega</td><td>A3561</td><td></td></tr><tr><td>SL1344</td><td></td><td></td><td>Ref # 17</td></tr><tr><td>SL1344 &lt;em&gt;&amp;Delta;sipA sopB&lt;/em&gt;::MudJ &lt;em&gt;sopE2&lt;/em&gt;::pSB1039</td><td></td><td></td><td>Ref # 18</td></tr><tr><td>SL1344 &lt;em&gt;&amp;Delta;sopE &amp;Delta;sipA sopB&lt;/em&gt;::MudJ &lt;em&gt;sopE2&lt;/em&gt;::pSB1039</td><td></td><td></td><td>Ref # 18</td></tr><tr><td>SYBR green</td><td>Applied Biosystems</td><td>4309155</td><td>2x mastermix</td></tr><tr><td>Tri-reagent</td><td>Molecular Research Center</td><td>TR 118</td><td>guanidine thiocyanate</td></tr><tr><td>Trypsin -EDTA</td><td>Thermo Fisher</td><td>25300054</td><td>0.05% Trypsin-EDTA</td></tr><tr><td>Ultrapure water</td><td>Fisher Bioreagents</td><td>BP248450</td><td></td></tr><tr><td>Well plate for PCR</td><td>VWR International</td><td>89218-294</td><td>384-well plate</td></tr></tbody></table>

nf-&#954;b-dependent luciferase activation and quantification of gene expression in salmonella infected tissue culture cells

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-&#954;B is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (I&#954;B&#945;). Activation of NF-&#954;B requires the degradation of I&#954;B&#945;, which then exposes a nuclear localization signal on NF-&#954;B and promotes its trafficking to the nucleus. Once in the nucleus, NF-&#954;B binds to the promotor region of NF-&#954;B target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.
The activation of NF-&#954;B occurs independently of transcription or translation. Therefore, the activation state of NF-&#954;B must be measured either by quantifying NF-&#954;B specifically in the nucleus, or by quantifying expression of NF-&#954;B target genes. In this protocol, cells stably transfected with an NF-&#954;B::luciferase reporter construct are assayed for NF-&#954;B activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-&#954;B, which traffics to the nucleus and binds to &#954;B sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-&#954;B activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

The assay described here focuses on activation of the transcription factor NF-&#954;B using a NF-&#954;B-dependent luciferase reporter that is stably transfected into a line of HeLa cells. Activated NF-&#954;B translocates to the nucleus where it binds &#954;B binding sites of target genes, including the pro-inflammatory cytokines IL6 and IL23. A general overview of NF-&#954;B activation is depicted in Figure 1. The gram-negative bacterium <...

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NF-&#954;B-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-&#954;B) activation in cell lines expressing NF-&#954;B::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

The dimeric transcription factor NF-&#954;B regulates many cellular response pathways, including inflammatory pathways by inducing the expression of ...

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10.4K Views.  University of Colorado Anschutz Medical Campus. Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation in cell lines expressing NF-κB::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

Video: NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Genetics

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

Biochemistry

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Developmental Biology

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Biology

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

Summary

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Chapters in this video

Using Luciferase to Image Bacterial Infections in Mice

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)