Name: generation and multi-phenotypic high-content screening of coxiella burnetii transposon mutants
Uploaded: 2015-05-13T15:00:00.000+00:00
Duration: 11 min 44 s
Description: Watch this Scientific Journal Video about Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants at JoVE.com

This work was supported by an Avenir/ATIP grant to Matteo Bonazzi and a Marie Curie CIG (N&#176; 293731) to Eric Martinez. The authors would like to thank Dr. Robert Heinzen for sharing C. burnetii strains, antibodies and tools, Virginie Georget and Sylvain DeRossi (Montpellier Rio Imaging-MRI, 1919 route de Mende, 34293 Montpellier) for their technical assistance and data analysis.

<ol>
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Dot/Icm type IV secretion system requires endocytic maturation of the pathogen-occupied vacuole.</a> PloS one. 8 (1), e54566 (2013).</li><li>Vogel, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+a+tiger+into+a+house+cat:+using+Legionella+pneumophila.+to+study+Coxiella+burnetii..">Turning a tiger into a house cat: using Legionella pneumophila. to study Coxiella burnetii..</a> Trends in microbiology. 12 (3), 103-105 (2004).</li><li>Van Schaik, E. J., Chen, C., Mertens, K., Weber, M. M., Samuel, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathogenesis+of+the+obligate+intracellular+bacterium+Coxiella+burnetii.">Molecular pathogenesis of the obligate intracellular bacterium Coxiella burnetii.</a> Nature reviews. Microbiology. 11, 561-573 (2013).</li><li>Beare, P. 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=Dot/Icm+type+IVB+secretion+system+requirements+for+Coxiella+burnetii+growth+in+human+macrophages.">Dot/Icm type IVB secretion system requirements for Coxiella burnetii growth in human macrophages.</a> mBio. 2, (2011).</li><li>Omsland, 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=Isolation+from+animal+tissue+and+genetic+transformation+of+Coxiella+burnetii.+are+facilitated+by+an+improved+axenic+growth+medium.">Isolation from animal tissue and genetic transformation of Coxiella burnetii. are facilitated by an improved axenic growth medium.</a> Applied and environmental microbiology. 77 (11), 3720-3725 (2011).</li><li>Beare, P. a., Sandoz, K. M., Omsland, A., Rockey, D. D., Heinzen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Advances+in+genetic+manipulation+of+obligate+intracellular+bacterial+pathogens.">a Advances in genetic manipulation of obligate intracellular bacterial pathogens.</a> Frontiers in microbiology. 2 (May), 97 (2011).</li><li>Brodin, P., Christophe, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-content+screening+in+infectious+diseases.">High-content screening in infectious diseases.</a> Current opinion in chemical biology. 15 (4), 534-539 (2011).</li></ol>

The authors declare that they have no competing financial interests.

The study of host/pathogen interactions has proven to be a remarkable method to understand bacterial infections and develop alternative strategies to counter infectious diseases. However, due to the diversity of strategies elaborated by different bacterial pathogens, the identification and characterization of bacterial virulence factors and of the host signaling pathways that are targeted during infections represent a real challenge. This calls for the development of new approaches for the large-scale identification of key host/pathogen interaction hubs. The recent development of innovative, high-throughput and high-content screening techniques represents an invaluable resource that can be adapted to the study of intracellular bacterial pathogens15. Here, we have used the zoonotic bacterial pathogen Coxiella burnetii as a model to develop screening approaches that combine transposon mutagenesis and fluorescence-based assays. Importantly, this screening method allows the simultaneous monitoring of multiple steps of the Coxiella intracellular cycle, providing a global overview of the strategies developed by this bacterium to invade, replicate and persist within infected cells.
The approach described here is based on two well-established techniques, transposon mutagenesis and fluorescence-based assays, which have been successfully applied to the study of bacterial pathogens. Combining these techniques in the context of high-throughput/high-content screens allows us to evaluate the effects of a high number of bacterial mutations by analyzing a very high number of events (typically 15,000 infected cells per bacterial mutations are imaged and analyzed). This provides an important statistical analysis of events such as bacterial invasion of host cells and intracellular replication, which are, by nature, subject to high variability. It is important to note that cell lines other than epithelial can be used for this type of screening. However, flat and large epithelial cells are optimal for image analysis as host cell organelles are easier to detect. Because the majority of automated microscopes can automatically handle a large number of plates, there are virtually no limits to the number of mutants that can be screened simultaneously. Depending on the pathogen, the user can privilege the use of an epifluorescence or a confocal microscope. The time of image acquisition will mostly depend on the sensitivity of the microscope camera, on the number of fields acquired per well and on the number of channels acquired per field of view. The user can decide how to adjust these factors to optimize the screening protocol. As an example, we imaged one 96-well plate/hr using the conditions indicated at point 5.1. Image analysis largely depends on the machine (or cluster of machines) used. We use a 12-core (2 x 3.06 GHz 6-Core), 48 GB RAM workstation. This machine requires approximately 40 min to analyze images acquired from one plate.
An important aspect to be taken into account when developing these assays is the set up of new (or the optimization of existing) protocols to allow the manipulation and processing of a large number of samples. A typical example is the development of the single primer colony PCR approach, which allowed us to rapidly amplify and sequence Coxiella DNA fragments containing the site of insertion of each transposon, from very small samples. Based on our experience, the high-fidelity polymerase has to be carefully selected and tested in order to obtain reproducible results. The only limitation of this approach may hide in the observation that, in the majority of cases, about 30% of the processed samples are not exploitable, either due to the PCR or the sequencing steps. However, considering that the isolation of new Coxiella transposon mutants is not a rate-limiting step, this does not represent a major issue. Similarly, the development of a reliable assay to quantify the bacterial concentration of mutant stocks has been key for this approach. Due to the tendency of Coxiella to aggregate when in suspension, the use of optical density readings is not applicable to calculate the concentration of Coxiella cultures and the only existing alternative was quantitative PCR (qPCR). Here, the use of a fluorescently tagged DNA intercalating agent significantly speeded up bacteria quantitation.
This approach can also take advantage of the use of stable cell lines expressing fluorescent markers for several intracellular compartments depending on the pathogen used. Another important aspect is the use of cell culture media devoid of phenol red. We observed that this pH indicator has a natural fluorescence spanning the red and green spectrum that saturates the signal recorded on the automated fluorescence reader.
The strategy presented here relies on random transposon mutagenesis. For the mutants of interest, we recommend validating unique transpositions (and clonality) using Southern blot and PCR amplifications of the transposon insertion site.
Besides the equipment described in the protocol section, teams interested in using the screening approach presented here, will take great advantage in the set up of a relational database for data collection, a server for data storage and a workstation for rapid image analysis.
Importantly, the method here described is suited for the study of other intracellular bacterial pathogens provided a random mutagenesis method exists for the pathogen, cell lines can be infected by the pathogen and this one displays a specific phenotype during infection.

The emerging, endemic bacterium Coxiella burnetii is responsible for large outbreaks of Q fever, a debilitating flu-like zoonosis with severe health and economic impact1. The main reservoirs of Coxiella are domestic and farm animals, and it is estimated that more than 90% of dairy cattle in the US carry C. burnetii2. Humans are accidental hosts that are infected by inhalation of contaminated aerosols. Human Q fever manifests either as an acute or chronic disease, which may have fatal complications with a mortality rate reaching 65%1,3. With an infectious dose of 1 - 10 organisms, Coxiella is the most infectious pathogen known and it has been investigated as a potential bio weapon4. The recent explosive outbreak of Q fever in the Netherlands (2007 - 2010), with cases escalating from 182 to more than 2,000 per year, stands as an example of the severe virulence of this pathogen5.
The remarkable efficiency of Coxiella infections is likely associated with its resistance to environmental stress, combined with its unique adaptation to host cells. Indeed, Coxiella is present in the environment in the form of metabolically inactive small cell variants (SCV), which are remarkably resistant to several harsh conditions (desiccation, temperature, etc.). SCVs are up taken by phagocytic cells via &#945;V&#946;3 integrins6 while invasion of non-phagocytic cells is mediated by the Coxiella adhesion/invasion OmpA7 and a yet unidentified receptor. Following uptake, Coxiella resides in tight-fitting vacuoles, positive for the early endosomal markers Rab5 and EEA18. Bacteria respond to endosomal acidification by converting to metabolically active large cell variants (LCVs) and activating a Dot/Icm type 4 secretion system (T4SS)9, highly homologous to that of Legionella pneumophila10. The secretion of Dot/Icm effectors allow Coxiella to generate a large, LAMP1-positive acidic compartment containing active lysosomal enzymes where bacteria can thrive and actively protect infected cells from apoptosis11. Hence, the intracellular cycle of Coxiella is controlled by the Dot/Icm-mediated translocation of bacterial effectors12, however, the microbial factors involved in host cell invasion, bacterial replication and dissemination of the infection remain largely unknown.
Combining transposon mutagenesis and fluorescence-based assays, we are developing unbiased approaches for the simultaneous identification of bacterial factors involved in the main steps of Coxiella infections: 1) internalization within host cells, 2) intracellular replication, 3) cell-to-cell spread, and 4) persistence. To date, we have screened over 1,000 mutations in 500 Coxiella coding sequences, which provided us with unprecedented insights into the host-pathogen interactions that regulate Coxiella pathogenesis7. Of note, this approach can be applied to the study of other intracellular pathogens that share cell biology features with Coxiella.

<table><tbody><tr><td>Citric acid</td><td>Sigma</td><td>C0759-500G</td><td>ACCM-2 medium component</td></tr><tr><td>Sodium citrate</td><td>Sigma</td><td>S4641-500G</td><td>ACCM-2 medium component</td></tr><tr><td>Potassium phosphate</td><td>Sigma</td><td>60218-100G</td><td>ACCM-2 medium component</td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>M2670-100G</td><td>ACCM-2 medium component</td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C5080-500G</td><td>ACCM-2 medium component</td></tr><tr><td>Iron sulfate</td><td>Sigma</td><td>F8633-250G</td><td>ACCM-2 medium component</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9625-500G</td><td>ACCM-2 medium component</td></tr><tr><td>L-cysteine</td><td>Sigma</td><td>C6852-25G</td><td>ACCM-2 medium component</td></tr><tr><td>Bacto Neopeptone</td><td>BD (Beckton-Dickinson)</td><td>211681</td><td>ACCM-2 medium component</td></tr><tr><td>Casamino acids</td><td>BD (Beckton-Dickinson)</td><td>223050</td><td>ACCM-2 medium component</td></tr><tr><td>Methyl-B-cyclodextrin</td><td>Sigma</td><td>C4555-10G</td><td>ACCM-2 medium component</td></tr><tr><td>RPMI w/glutamax</td><td>Gibco</td><td>61870-010</td><td>ACCM-2 medium component</td></tr><tr><td>RPMI w/glutamax, without phenol red</td><td>Gibco</td><td>32404-014</td><td>For cell culture and infection</td></tr><tr><td>Fetal bovine serum</td><td>GE healthcare</td><td>SH30071</td><td>For cell culture</td></tr><tr><td>Trypsin EDTA (0.25%)</td><td>Life Technologies</td><td>25200-056</td><td>For cell culture</td></tr><tr><td>Saponin</td><td>Sigma</td><td>47036</td><td>For immunofluorescence staining</td></tr><tr><td>Ammonium chloride</td><td>Sigma</td><td>A9434</td><td>For immunofluorescence staining</td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A2153</td><td>For immunofluorescence staining</td></tr><tr><td>Electroporation cuvette 0.1 cm</td><td>Eurogentec</td><td>ce0001-50</td><td>For &lt;em&gt;Coxiella&lt;/em&gt; transformation</td></tr><tr><td>Trackmates screw top tubes with caps</td><td>Thermo Scientific</td><td>3741</td><td>2D barcoded screwcap</td></tr><tr><td>96-well microplate with black walls and bottom</td><td>Greiner</td><td>655076</td><td>Flat dark bottom, for dsDNA quantitation</td></tr><tr><td>96-well PCR microplate</td><td>Biorad</td><td>2239441</td><td>DNAse/RNAse free</td></tr><tr><td>96-well plate, deepwell</td><td>Labcon</td><td>949481</td><td>For &lt;em&gt;Coxiella&lt;/em&gt; mutants infections</td></tr><tr><td>PicoGreen</td><td>Life Technologies</td><td>P7581</td><td>For dsDNA quantitation</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T9284-500ML</td><td>For dsDNA quantitation</td></tr><tr><td>Phusion high fidelity DNA polymerase</td><td>New England Biolabs</td><td>M0530L</td><td>For single primer colony PCR</td></tr><tr><td>Celltracker Red CMTPX</td><td>Life Technologies</td><td>C34552</td><td>For imaging</td></tr><tr><td>Anti-LAMP1 antibody</td><td>Sigma</td><td>94403-1ML</td><td>For immunofluorescence staining</td></tr><tr><td>Hoechst 33258</td><td>Sigma</td><td>L1418</td><td>For imaging</td></tr><tr><td>Fluorescence microplate reader Infinite 200 Pro</td><td>Tecan</td><td></td><td></td></tr><tr><td>Epifluorescence automated microscope Cellomics</td><td>Thermo Scientific</td><td></td><td></td></tr></tbody></table>

generation and multi-phenotypic high-content screening of coxiella burnetii transposon mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Upon isolation of transposon mutants, single primer colony PCR is a robust, high-throughput method to identify the site of transposon insertion for each mutant. This approach derives from a typical nested PCR protocol but here a single primer hybridizes specifically and/or non-specifically to the template DNA depending on the stringency of the annealing temperature (Figure 1A). The typical PCR products consist of multiple DNA fragments, most of which are specific (Figure 1B). The use of a different sequencing primer that anneals right upstream of the transposon ITR, and downstream of the sequence recognized by the amplification primer provides specificity for the sequencing step (Figure 1C). Automated software for sequence analysis aligns the obtained sequences to the Coxiella genome providing the exact site of transposon insertions (Figure 1C). All transposon insertions can be then annotated on the Coxiella genome (Figure 1D).
Each Coxiella mutant is isolated and amplified axenically in ACCM-2 medium prior to either storage or screening. Figure 2 illustrates an example of 38 transposon mutants in 16 dot/icmCoxiella genes (Figure 2A). To assess the viability of Coxiella mutants, axenic growth curves are obtained by sampling bacterial cultures for 7 days post inoculation and applying the bacterial concentration assay described in 1.5 (Figurie 2B). Amplified mutants are then incubated with epithelial cells, in triplicate 96-well plates for 7 days. All Coxiella mutants generated being GFP-tagged, intracellular growth curves are obtained by measuring the GFP fluorescence intensity of every well, every 24 hr, and plotting the measured values as a function of time (Figure 2C).
Intracellular growth curves provide quantitative analysis of the phenotypes associated with every transposon insertion in the Coxiella genome. To add qualitative information about the same transposon mutants, we opted for automated image acquisition and analysis. Seven days post infection, plates are fixed, processed for immunofluorescence as described in 4 and analyzed using an automated, epifluorescence microscope as described in 5. Automated image analysis software such as CellProfiler (Broad Institute, <a href="http://www.cellprofiler.com/" target="_blank">www.cellprofiler.com</a>) processes the acquired channels independently and segments identified objects for comparative analysis (Figure 3). This allows the identification and morphological characterization of host cell nuclei, cell contours, lysosomes and Coxiella colonies (Figure 3 top panels). Correlating Coxiella colonies with cells and lysosomes allows the identification and specific morphological analysis of Coxiella-containing vacuoles (which are LAMP1 positive, Figure 3 bottom left panel). Correlating Coxiella colonies with host cell contours allows the identification and specific morphological analysis of infected cells (Figure 3 bottom center panel). Finally, the 4 channels are merged for illustration and quality control purposes (Figure 3 bottom right panel).
Data obtained from automated image analysis can be plotted against each other to obtain &#8220;multi-phenotypic scatter plots&#8221;. As an example, in Figure 4A the average area (in &#956;m2) of Coxiella colonies is plotted against the number of colonies per cell (Figure 4A), in order to identify mutations that affect intracellular replication of Coxiella (replication phenotype) and/or the capacity of bacteria to invade host cells (internalization phenotype). Statistical analysis was used to define regions in the resulting scatter plot corresponding to mild (-4 &#60; Z-score &#8804; -2) and severe (Z-score &#8804; -4) phenotypes. Mutants were observed in 3 main clusters: mutations that resulted in a defective intracellular growth of Coxiella were grouped in the left-most part of the plot (Figure 4A, pink and red dots); mutations that affected Coxiella internalization in cells were grouped in the bottom part of the plot (Figure 4A, light and dark blue dots) and finally, green dots in the right-most region of the plot correspond to mutations resulting in non-significant phenotypes (Z-score &#62; -2). Importantly, mutants that fail to replicate but are still able to invade host cells, are detected after 7 days of infection as single bacteria, or small colonies, adjacent to host cell nuclei (Figure 4C, second panel). Hence, the size of Coxiella &#8220;colonies&#8221; will be significantly affected but the number of infected cells will not vary as compared to WT Coxiella-infected cells. On the contrary, mutations that affect the capacity of Coxiella to invade host cells result in a decrease in the number of colonies/cell. When this number is significantly below 1, it indicates that, on average, there is a decrease in the overall number of infected cells. Alternatively, the average area (in &#956;m2) of Coxiella colonies can be plotted against the number of host cells surviving infection (Figure 4B), to identify mutations that confer cytotoxicity to Coxiella (cytotoxic phenotype). As above, statistical analysis was used to define regions in the resulting scatter plot corresponding to mild (-4 &#60; Z-score &#8804; -2) and severe (Z-score &#8804; -4) phenotypes. Most screened mutations did not significantly affect cell survival, regardless of bacterial replication within host cells (Figure 3B green dots). 37 mutations mildly affected host cell survival (Figure 3B, light red dots), and 7 mutations were particularly detrimental to host cell survival (Figure 3B, dark red dots). Please note that additional parameters obtained by the automated image analysis procedure can be used to derive other charts, according to experimental needs.
<img alt="Figure 1" src="/files/ftp_upload/52851/52851fig1highres.jpg" width="700" /> 
Figure 1: Sequencing and annotation of Coxiella transposon mutants. (A) Single primer colony PCR is used to amplify DNA fragments containing the site of transposon insertion. An amplification primer (Amp) is used both as a specific and non-specific primer depending on the stringency of annealing temperature. (B) Typical result of single primer colony PCR. Each reaction produces a number of fragments of variable size, some of which contain the transposon insertion site; some others are randomly amplified as by-products of the low stringency PCR cycle. (C) The use of a sequencing primer (Seq) that hybridizes to the transposon sequence allows the sequencing of the fragments of interest. (D) Sequence analysis software allows the automatic annotation of transposon insertions on the bacterial genome. <a href="https://www.jove.com/files/ftp_upload/52851/52851fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52851/52851fig2highres.jpg" width="700" /> 
Figure 2: Axenic and intracellular growth of Coxiella transposon mutants. (A) In the pilot screen, we have isolated, sequenced and screened 38 transposon mutants in 16 core genes of the Coxiella dot/icm secretion system (indicated in red). (B) In order to assess the viability of each transposon mutant, the growth of each isolate in axenic culture medium is monitored over 8 days using a fluorescently tagged DNA intercalating agent. (C) Each mutant is then used to infect epithelial cells. As the transposon possesses a GFP cassette, intracellular bacterial growth is monitored over 7 days of infection by following the variations of GFP fluorescence associated with Coxiella replication, using a microplate reader. <a href="https://www.jove.com/files/ftp_upload/52851/52851fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52851/52851fig3highres.jpg" width="700" /> 
Figure 3: Automated image analysis of Coxiella infections. An automated epifluorescence microscope is used to image 21 positions per well of triplicate 96-well plates. The image analysis software segments objects in each acquired channels for quantification and analysis. In all cases, objects touching the border of the images are excluded. (A) The Hoechst channel is used to identify host cell nuclei (circled in green). (B) These are used as seeds to identify host cell contours in the Cy3 channel (the position of nuclei is circled in blue, cell contours are in green). (C) The Cy5 channel is used to identify LAMP1-positive compartments (circled in green); only the objects included in the previously identified cell contours (in red) are retained for image analysis. (D) The GFP channel is used to identify Coxiella colonies (circled in green). (E) Correlating Coxiella colonies with LAMP1-positive compartments allows the identification of Coxiella-containing vacuoles (CCVs); only the objects included in the previously identified cell contours (in green) are retained for image analysis. (F) Correlating Coxiella colonies with cell contours allows the identification of infected cells (pseudocolored). (G) Images acquired in the 4 fluorescence channels (corresponding to Coxiella colonies (green), host cell nuclei (blue), host cell plasma membrane (grey), LAMP1-positive compartments (red)) are merged and used for illustration and quality control. Scale bars 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52851/52851fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52851/52851fig4highres.jpg" width="700" /> 
Figure 4: Large-scale identification of Coxiella factors involved in host/pathogen interactions. (A) The average area (in &#956;m2) of Coxiella colonies is plotted against the relative number of colonies per cell, to identify replication and internalization phenotypes of interest. Green dots represent phenotypes that deviate from WT Coxiella by a Z-score &#62; -2 (not significant). Pink and light blue dots represent replication and internalization phenotypes respectively, with a Z-score between -2 and -4 (mild phenotypes). Red and dark blue dots represent phenotypes with a Z-score &#8804; -4 (strong phenotypes). (B) The average area (in &#956;m2) of Coxiella colonies was plotted against the total number of cells (infected and not infected) that survived 7 days of infection to estimate the cytotoxic effect resulting from transposon insertions. Green dots represent phenotypes that deviate from WT Coxiella by a Z-score &#62; -2 (not significant). Pink dots represent cytotoxic phenotypes with a Z-score between -2 and -4 (mild phenotypes). Red dots represent cytotoxic phenotypes with a Z-score &#8804; -4 (strong phenotypes). Arrows indicate mutants illustrated by the corresponding lowercase letter in C. (C) Representative images of replication, internalization and cytotoxic phenotypes. In all cases, host cell nuclei are in red, Coxiella colonies are in green. Scale bars 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/52851/52851fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

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Immunology and Infection

9.8K Views.  Université Montpellier. Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Video: Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

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

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN). Both are effective in newborns and during the acute phase of the disease but not in the chronic phase, and their use is associated with important side effects. These facts underscore the urgent need to intensify the search for new drugs against T. cruzi.
T. cruzi is transmitted through hematophagous insect vectors of the Reduviidae and Hemiptera families. Once in the mammalian host, it multiplies intracellularly as the non-flagellated amastigote form and differentiates into the trypomastigote, the bloodstream non-replicative infective form. Inside the insect vector, trypomastigotes transform into the epimastigote stage and multiply through binary fission.
This paper describes an assay based on measuring the activity of the cytoplasmic &#946;-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red &#946;-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a &#946;-galactosidase-overexpressing plasmid and used for in vitro pharmacological screening in epimastigote, trypomastigote, and amastigote stages. This paper also describes how to measure the enzymatic activity in cultured epimastigotes, infected Vero cells with amastigotes, and trypomastigotes released from the cultured cells using the reference drug, benznidazole, as an example. This colorimetric assay is easily performed and can be scaled to a high-throughput format and applied to other T. cruzi strains.

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing &#946;-galactosidase

We describe a high-throughput colorimetric assay measuring &#946;-galactosidase activity in three life cycle stages of Trypanosoma cruzi, the causative agent of Chagas disease. This assay can be used to identify trypanocidal compounds in an easy, fast, and reproducible manner.

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many ...

Biochemistry

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

Biology

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of antibiotics and other secondary metabolites. Streptomyces coelicolor is a well-characterized, non-pathogenic species that is amenable to a variety of analyses in the lab. The phenotyping methods described here use S. coelicolor as a model streptomycete; however, the methods are applicable to all members of this large genus as well as some closely related actinomycetes. Phenotyping is necessary to characterize new species of Streptomyces identified in the environment, and it is also a vital first step in characterizing newly isolated mutant strains of Streptomyces. Proficiency in phenotyping is important for the many new researchers who are entering the field of Streptomyces research, which includes the study of bacterial development, cell division, chromosome segregation, and second messenger signaling. The recent crowdsourcing of antibiotic discovery through the isolation of new soil microbes has resulted in an increased need for training in phenotyping for instructors new to the field of Streptomyces research and their college or high school students. This manuscript describes methods for bacterial strain propagation, storage, and characterization through visual and microscopic examination. After reading this article, new researchers (microbiology education laboratories and citizen scientists) should be able to manipulate Streptomyces strains and begin visual characterization experiments.

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus Streptomyces.

Streptomycetes are filamentous soil bacteria belonging to the phylum Actinobacteria that are found throughout the world and produce a wide array of ...

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. The non-replicative elementary body (EB) initiates infection of the host. Once inside, the EB differentiates into the reticulate body (RB). The RB then undergoes multiple rounds of replication, before differentiating back to the infectious EB form. This cycle is essential for chlamydial survival as failure to switch between cell types prevents either host invasion or replication.
Limitations in genetic techniques due to the obligate intracellular nature of Chlamydia have hampered identification of the molecular mechanisms involved in the cell-type development. We designed a novel dual promoter-reporter plasmid system that, in conjunction with live-cell microscopy, allows for the visualization of cell type switching in real time. To identify genes involved in the regulation of cell-type development, the live-cell promoter-reporter system was leveraged for the development of a forward genetic approach by combining chemical mutagenesis of the dual reporter strain, imaging and tracking of Chlamydia with altered developmental kinetics, followed by clonal isolation of mutants. This forward genetic workflow is a flexible tool that can be modified for directed interrogation into a wide range of genetic pathways.

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

This protocol utilizes fluorescent promoter-reporters, live-cell microscopy, and individual inclusion extraction in a directed forward genetic approach to identify and isolate developmental mutants of Chlamydia trachomatis.

The intracellular bacterial pathogen Chlamydia trachomatis undergoes a developmental cycle consisting of two morphologically discrete developmental forms. ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The &#8220;last-line&#8221; antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Summary

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Chapters in this video

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

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

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing β-galactosidase

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing