Financial support for this work was provided by the European Community (ERC-STG INTRACELLTB Grant n&#176; 260901, MM4TB Grant n&#176; 260872), the Agence Nationale de Recherche, the Feder (12001407 (D-AL) Equipex Imaginex BioMed), and the Region Nord Pas de Calais. We gratefully acknowledge the technical assistance of Gaspard Deloison, Elizabeth Werkmeister, Antonino Bongiovanni and Frank Lafont from the platform BICeL.

1. High-throughput Genome-wide siRNA Screening
Screening performed in a human Type-II pneumocytes model A549 cell line upon infection with Mtb H37Rv expressing Green Fluorescent Protein (GFP). This procedure is outlined in Figure 1A.
<ol>
	<li>Resuspend the dried siRNA library that is stored in mother plates (96-well plates) with 1x&#160;siRNA buffer to reach a concentration of 4 &#956;M, then transfer 10 &#956;l of the mixture into a 384-well daughter plate (daughter plate 1).</li>
	<li>Add 10 &#956;l of 1x siRNA buffer in daughter plate 1 to dilute siRNA by 2-fold. After siRNA resuspension, plates are sealed with peelable aluminum seal and can be stored at -20 &#176;C at least 6 months and up to 2-3 years, but storage time may vary depending on siRNA library manufacturer&#39;s recommendations.</li>
	<li>Dilute siRNAs in daughter plate 1 into daughter plate 2 to reach a concentration of 500 nM. After siRNA resuspension, plates are sealed with peelable aluminum seal and can be stored at -20 &#176;C at least 6 months and up to 2-3 years, but storage time may vary depending on siRNA library manufacturer&#39;s recommendations.</li>
	<li>Before use, thaw daughter plate 2 at room temperature.</li>
	<li>Take 2.5 &#956;l of siRNA from daughter plate 2 and place into a 384-well assay plate.</li>
	<li>In the same 384-well assay plate in step 1.5, add 2.5 &#956;l negative and positive control siRNA to their respective wells.</li>
	<li>Dilute the transfection reagent in 1x D-PBS to yield enough solution to provide 0.1 &#956;l transfection reagent in each well and preincubate the diluted transfection solution at room temperature for 5 min.</li>
	<li>Add 7.5 &#956;l of the transfection reagent/PBS solution mixture to each well in the assay plate and incubate for 30 min at room temperature.</li>
	<li>Add 40 &#956;l of A549 cells (1,500 cells/well) suspended in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). Maintain cells for a 3 day-incubation period at 37 &#176;C in an atmosphere containing 5% CO2. These cells divide every 24 hr, thus about 12,000 cells are in the wells three days after transfection.</li>
	<li>Wash a two-week old GFP-expressing Mtb H37Rv culture out with D-PBS (free from MgCl2 and CaCl2) by centrifugation at 4,000 x g for 5 min and discard the washes. Repeat this step 3x. (For GFP-Mtb H37Rv culture conditions see Protocol 3).</li>
	<li>Suspend the bacterial pellet in 10 ml of RPMI 1640 medium supplemented with 10% FBS and decant for 1 hr at room temperature to allow bacterial aggregates to sediment.</li>
	<li>Collect the bacterial supernatant and measure OD600 (OD600 should be between 0.6-0.8) and GFP-fluorescence (RFU value) using a microplate reader to determine the bacterial concentration. Calculate the titer of the suspension using a reference regression line displaying RFU value = f (CFU value) that had been generated prior to the experiment on another culture that had been prepared in the same conditions. Prepare bacterial suspension containing 2.4 x 106 bacteria/ml, which corresponds to a multiplicity of infection (MOI) of 5.</li>
	<li>Remove the medium in the 384-well assay plate and add 25 &#956;l of freshly prepared bacterial suspension.</li>
	<li>Incubate the 384-well assay plate at 37 &#176;C for 5 hr in an atmosphere containing 5% CO2.</li>
	<li>Remove the medium and gently wash the cells with RPMI medium supplemented with 10% FBS 3x.</li>
	<li>To kill the remaining extracellular bacteria, treat the cells with 50 &#956;l of fresh RPMI-FBS medium containing 50 &#956;g/ml of amikacin at 37 &#176;C for 1 hr in an atmosphere containing 5% CO2.</li>
	<li>Remove the medium-containing amikacin and add 50 &#956;l of fresh RPMI medium supplemented with 10% FBS. Incubate the 384-well assay plate at 37 &#176;C for 5 days in an atmosphere containing 5% CO2.</li>
	<li>Prior to image acquisition, add 10 &#956;l of freshly prepared 30 &#956;g/ml of DAPI in PBS (final concentration 5 &#956;g/ml) and incubate for 10 min at 37 &#176;C.</li>
	<li>Load the plate into automated confocal microscope. 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_1highres.jpg" src="/files/ftp_upload/51114/screenshot_1.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_1highres.jpg" target="_blank">Click here to view larger image.</a></li>
	<li>Set the exposure parameters. Record DAPI fluorescence using excitation laser 405 nm with emission filter 450 nm and GFP fluorescence using excitation laser 488 nm with emission filter 520 nm. 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-1highres.jpg" src="/files/ftp_upload/51114/screenshot_2-1.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-1highres.jpg" target="_blank">Click here to view larger image.</a> 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-2highres.jpg" src="/files/ftp_upload/51114/screenshot_2-2.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-2highres.jpg" target="_blank">Click here to view larger image.</a></li>
	<li>Select the wells and the fields in each well to be acquired, which is then referred as layout and sublayout parameters. 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-1highres.jpg" src="/files/ftp_upload/51114/screenshot_3-1.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-1highres.jpg" target="_blank">Click here to view larger image.</a> 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-2highres.jpg" src="/files/ftp_upload/51114/screenshot_3-2.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-2highres.jpg" target="_blank">Click here to view larger image.</a></li>
	<li>Generate the experiment file using the parameters from steps 1.20 and 1.21, and run the automatic acquisition. 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-1highres.jpg" src="/files/ftp_upload/51114/screenshot_4-1.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-1highres.jpg" target="_blank">Click here to view larger image.</a> 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-2highres.jpg" src="/files/ftp_upload/51114/screenshot_4-2.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-2highres.jpg" target="_blank">Click here to view larger image.</a></li>
	<li>Transfer images to remote server. 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_5highres.jpg" src="/files/ftp_upload/51114/screenshot_5.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_5highres.jpg" target="_blank">Click here to view larger image.</a></li>
	<li>Evaluate images using image analysis software. Detect cell nuclei from DAPI channel using nuclei detection algorithm and the bacterial area from GFP channel using pixel intensity properties algorithm (Figure 4A).</li>
</ol>
Note: This protocol is optimized to study the effect of gene silencing on the intracellular Mtb growth. Mtb is a slow-growth bacterium which divides every 20 hr in optimal conditions. After 5 days post-infection the amount of extracellular Mtb is still low in absence of cell lysis and didn&#39;t affect the quality of the analysis. This protocol must be optimized in terms of length of antibiotic treatment and incubation time to be adapted for siRNA screens using fast-growth bacteria like Mycobacteria smegmatis&#160;and&#160;Escherichia coli that are extensively released and can infect new cells.
2. High-throughput Compound Screening
Screening performed on Mtb H37Rv infected host cells. This procedure is outlined in Figure 1B.
<ol>
	<li>Thaw the 384-well mother plates containing the compound library solubilized in DMSO 100%. Transfer 0.5 &#956;l of the compounds in 384-well daughter plates containing 10 &#956;l of RPMI 1640 medium supplemented with 10% FBS.</li>
	<li>Wash a two-week old GFP-expressing Mtb H37Rv culture out with D-PBS (free from MgCl2 and CaCl2) by centrifugation at 4,000 x g for 5 min and discard the washes. Repeat this step 3x (For GFP-Mtb H37Rv culture conditions see Protocol 3).</li>
	<li>Suspend the bacterial pellet in 10 ml&#160;of RPMI 1640 medium supplemented with 10% FBS and decant for 1 hr at room temperature to allow bacterial aggregates to sediment.</li>
	<li>Collect the bacterial supernatant and measure OD600 (OD600 should be between 0.6-0.8) and GFP-fluorescence (RFU value) using a microplate reader. Calculate the titer of the suspension using a reference regression line displaying RFU value = f (CFU value) that had been generated prior to the experiment. Typical concentration is 1 x 108 bacteria/ml.</li>
	<li>Harvest 6 day old primary human macrophages at 4 x 105 cells/ml in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 50 ng/ml recombinant human M-CSF (For human Peripheral Blood Monocyte Cells purification and macrophages differentiation see Protocol 4).</li>
	<li>Incubate the diluted primary cells with bacilli at different MOI, ranging from 1-5, in suspension with mild shaking at 90 rpm for 2 hr at 37 &#176;C.</li>
	<li>Wash the infected cells by centrifugation at 350 x g to remove the extracellular bacteria. After each centrifugation step, resuspend the pellet in RPMI 1640 medium supplemented with 10% FBS. Repeat this step 2x.</li>
	<li>Suspend the infected cells in RPMI 1640 medium supplemented with 10% FBS and amikacin at 50 &#956;g/ml and incubate the suspension with mild shaking for 1 hr at 37 &#176;C.</li>
	<li>Remove the cell-culture medium containing amikacin by centrifugation at 350 x g and wash the infected cells with complete RPMI 1640 medium supplemented with 10% FBS and 50 ng/ml recombinant human M-CSF. Repeat once.</li>
	<li>Add 40 &#956;l of the infected macrophages suspension in the same assay plate in step 2.1, which already contains 10 &#956;l of the compound dilutions. The final concentration of DMSO in each well has now reached 1%.</li>
	<li>Incubate the assay plates for 5 days at 37 &#176;C in an atmosphere containing 5% CO2.</li>
	<li>Stain the live cells with cell-permeant far-red fluorescent dye.</li>
	<li>Load the plate into automated confocal microscope. 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_1highres.jpg" src="/files/ftp_upload/51114/screenshot_1.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_1highres.jpg" target="_blank">Click here to view larger image.</a></li>
	<li>Set the exposure parameters. Record far-red fluorescence using excitation laser 640 nm with emission filter 690 nm and GFP fluorescence using excitation laser 488 nm with emission filter 520 nm. 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-1highres.jpg" src="/files/ftp_upload/51114/screenshot_2-1.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-1highres.jpg" target="_blank">Click here to view larger image.</a> 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_2-2highres.jpg" src="/files/ftp_upload/51114/screenshot_2-2.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_2-2highres.jpg" target="_blank">Click here to view larger image.</a></li>
	<li>Select the wells and the fields in each well to be acquired, which is then referred as layout and sublayout parameters. 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-1highres.jpg" src="/files/ftp_upload/51114/screenshot_3-1.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-1highres.jpg" target="_blank">Click here to view larger image.</a> 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_3-2highres.jpg" src="/files/ftp_upload/51114/screenshot_3-2.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_3-2highres.jpg" target="_blank">Click here to view larger image.</a></li>
	<li>Generate the experiment file using the parameters from steps 2.14 and 2.15 and run the automatic acquisition. 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-1highres.jpg" src="/files/ftp_upload/51114/screenshot_4-1.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-1highres.jpg" target="_blank">Click here to view larger image.</a> 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_4-2highres.jpg" src="/files/ftp_upload/51114/screenshot_4-2.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_4-2highres.jpg" target="_blank">Click here to view larger image.</a></li>
	<li>Transfer images to remote server. 
	<img fo:content-width="5in" fo:src="/files/ftp_upload/51114/screenshot_5highres.jpg" src="/files/ftp_upload/51114/screenshot_5.jpg" /> 
	<a href="https://www.jove.com/files/ftp_upload/51114/screenshot_5highres.jpg" target="_blank">Click here to view larger image.</a></li>
	<li>Evaluate images using image analysis software. Detect cell area from far-red channel using pixel intensity properties algorithm and bacterial area from GFP channel using pixel intensity properties algorithm (Figure 4B).</li>
</ol>
Note: This protocol can be adapted for Mtb mutant library screening by replacing compounds by mutants expressing a fluorescent protein (One well/One mutant) (Figure 1C, see also Brodin&#160;et al.4). Fluorescent mutants are first seeded in wells (20 &#956;l of bacterial suspension per well). Bacteria are then recovered by 30 &#956;l of cell suspension. After centrifugation at 350 x g for 1 min, the plate is incubated at 37 &#176;C in an atmosphere containing 5% CO2. Incubation time and MOI depend on the assay. As an example, for visualization of early cellular events such as phagosome acidification, the cells can be infected for 2 hr with MOI ranging from 1-20. Lysosomes are stained using Lysotracker dye at 2 &#956;M for 1.5 hr at 37 &#176;C in an atmosphere containing 5% CO2 and then fixed with either 10% formalin or 4% paraformaldehyde (PFA). Confocal images are acquired and finally analyzed using image analysis scripts featuring appropriate algorithms for lysosomes detection and subcellular localization4.
3. Green Fluorescent Protein Expressing Mycobacterium tuberculosis H37Rv (GFP-H37Rv) Culture Conditions
For long term storage, GFP-H37Rv were frozen in D-PBS (around 1 x 108 mycobacteria per vial).
<ol>
	<li>Resuspend one frozen-stock vial of GFP-H37Rv in an Erlenmeyer flask containing 50 ml of 7H9 broth medium supplemented with Middlebrook OADC enrichment 10%, Glycerol 0.5%, Tween-80 0.05%, and Hygromycin B (50 &#956;g/ml).</li>
	<li>Incubate 8 days at 37 &#176;C.</li>
	<li>Measure OD600 of the GFP-H37Rv culture.</li>
	<li>Dilute the GFP-H37Rv culture to obtain OD600 = 0.1 in fresh 7H9 broth medium supplemented with Middlebrook OADC enrichment 10%, Glycerol 0.5%, Tween-80 0.05% and Hygromycin B (50 &#956;g/ml).</li>
	<li>Incubate the GFP-H37Rv at 37 &#176;C for 8 days more before use for the assay.</li>
</ol>
4. Human Peripheral Blood Monocyte Cells Purification from Whole-blood or Buffy-coat Preparation
<ol>
	<li>Dilute the blood pouch 2x in 1x&#160;D-PBS&#160;(free from MgCl2 and CaCl2) containing 1% FBS.</li>
	<li>Isolate the monocytes by Ficoll density gradient centrifugation at 400 x g for 20 min.</li>
	<li>Collect the isolated monocytes.</li>
	<li>Wash the monocytes 3x with 1x&#160;D-PBS (free from MgCl2 and CaCl2) containing 1% FBS by centrifugation at 400 x g for 10 min at room temperature.</li>
	<li>Concentrate the cells up to 1 x 107 cell/ml.</li>
	<li>Purify monocytes using CD14-magnetic beads according to the manufacturer&#39;s protocol (see Materials).</li>
	<li>After CD14-monocytes purification, seed the cells at 1.5 x 106 cell/ml in RPMI 1640 complemented with 10% FBS and 40 ng/ml of human Macrophages Colony Stimulating Factor (hM-CSF) and incubated for 4 days at 37 &#176;C in an atmosphere containing 5% CO2.</li>
	<li>After 4 days replace the medium with fresh RPMI 1640 complemented with 10% FBS and 40 ng/ml&#160;of hM-CSF and incubated for 2 days at 37 &#176;C in an atmosphere containing 5% CO2.</li>
	<li>After 6 days, the cells can be used for the assay.</li>
</ol>

<ol>
	<li>Gandhi, N. 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=Multidrug-resistant+and+extensively+drug-resistant+tuberculosis:+a+threat+to+global+control+of+tuberculosis.">Multidrug-resistant and extensively drug-resistant tuberculosis: a threat to global control of tuberculosis.</a> Lancet. 375, 1830-1843 (2010).</li><li>Barry, C. E., 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=The+spectrum+of+latent+tuberculosis:+rethinking+the+biology+and+intervention+strategies.">The spectrum of latent tuberculosis: rethinking the biology and intervention strategies.</a> Nat. Rev. Microbiol. 7, 845-855 (2009).</li><li>Christophe, T., Ewann, F., Jeon, H. K., Cechetto, J., Brodin, P. <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+imaging+of+Mycobacterium+tuberculosis-infected+macrophages:+an+in+vitro+model+for+tuberculosis+drug+discovery.">High-content imaging of Mycobacterium tuberculosis-infected macrophages: an in vitro model for tuberculosis drug discovery.</a> Future Med. Chem. 2, 1283-1293 (2010).</li><li>Brodin, 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=High+content+phenotypic+cell-based+visual+screen+identifies+Mycobacterium+tuberculosis+acyltrehalose-containing+glycolipids+involved+in+phagosome+remodeling.">High content phenotypic cell-based visual screen identifies Mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling.</a> PLoS Pathog. 6, (2010).</li><li>Bermudez, L. E., Goodman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+invades+and+replicates+within+type+II+alveolar+cells.">Mycobacterium tuberculosis invades and replicates within type II alveolar cells.</a> Infect. Immun. 64, 1400-1406 (1996).</li><li>Dobos, K. M., Spotts, E. A., Quinn, F. D., King, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Necrosis+of+lung+epithelial+cells+during+infection+with+Mycobacterium+tuberculosis+is+preceded+by+cell+permeation.">Necrosis of lung epithelial cells during infection with Mycobacterium tuberculosis is preceded by cell permeation.</a> Infect. Immun. 68, 6300-6310 (2000).</li><li>McDonough, K. A., Kress, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity+for+lung+epithelial+cells+is+a+virulence-associated+phenotype+of+Mycobacterium+tuberculosis.">Cytotoxicity for lung epithelial cells is a virulence-associated phenotype of Mycobacterium tuberculosis.</a> Infect. Immun. 63, 4802-4811 (1995).</li><li>Lee, H. M., Yuk, J. M., Shin, D. M., Jo, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dectin-1+is+inducible+and+plays+an+essential+role+for+mycobacteria-induced+innate+immune+responses+in+airway+epithelial+cells.">Dectin-1 is inducible and plays an essential role for mycobacteria-induced innate immune responses in airway epithelial cells.</a> J. Clin. Immunol. 29, 795-805 (2009).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. 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+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> J. Biomol. Screen. 4, 67-73 (1999).</li><li>Birmingham, 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=Statistical+methods+for+analysis+of+high-throughput+RNA+interference+screens.">Statistical methods for analysis of high-throughput RNA interference screens.</a> Nat. Methods. 6, 569-575 (2009).</li><li>Carralot, 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=Automated+high-throughput+siRNA+transfection+in+raw+264.7+macrophages:+a+case+study+for+optimization+procedure.">Automated high-throughput siRNA transfection in raw 264.7 macrophages: a case study for optimization procedure.</a> J. Biomol. Screen. 14, 151-160 (2009).</li><li>Zhang, X. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illustration+of+SSMD,+z+score,+SSMD*,+z*+score,+and+t+statistic+for+hit+selection+in+RNAi+high-throughput+screens.">Illustration of SSMD, z score, SSMD*, z* score, and t statistic for hit selection in RNAi high-throughput screens.</a> J. Biomol. Screen. 16, 775-785 (2011).</li><li>Song, O. 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=Confocal-based+method+for+quantification+of+diffusion+kinetics+in+microwell+plates+and+its+application+for+identifying+a+rapid+mixing+method+for+high-content/throughput+screening.">Confocal-based method for quantification of diffusion kinetics in microwell plates and its application for identifying a rapid mixing method for high-content/throughput screening.</a> J. Biomol. Screen. 15, 138-147 (2010).</li></ol>

No conflicts of interest declared.

We describe here the methods required for a phenotypic assay using a GFP-expressing Mtb H37Rv strain to infect fluorescently labeled host cells, which makes it appropriate for High-content/High-throughput screens. This protocol could be applied to a broad range of compounds, fluorescent probes and Mtb mutants. For each protocol described above, fixation and immunolabeling steps could be performed prior to image acquisition. We use an automated fluorescent confocal microscope equipped with a 20X (NA 0.70...

Among the emerging and re-emerging infectious pathogens reported during the last years, Mycobacterium tuberculosis (Mtb) holds a prominent place being responsible for 1.4 million deaths and 8.7 million new infections in 2011 (Global tuberculosis report 2012, www.who.int/topics/tuberculosis/en/). Despite the availability of multidrug therapies, the number of infected people is still on the rise and multidrug resistant (MDR) as well as extensively drug resistant (XDR) Mtb are quickly spreading all over the world1. Moreover, when taking into consideration the presence of Mtb antigens, it is evident that one third of the global population is considered as being latently infected by Mtb. Statistically, in one case out of ten, there is evolution towards the active form of the disease with subsequent clinical symptoms2. Therefore, new means to fight Mtb are urgently needed. In this context, we developed an in vitro visual phenotypic assay relying on monitoring Mtb invasion and multiplication into host&#160;cells by automated confocal fluorescence microscopy3. The adaptation of the assay in 384-well microtiter plates in combination with automated image acquisition and analysis, allowed High-content/High-throughput Screening (HC/HTS) of medium scale libraries of compounds, siRNAs and bacterial mutants. The screening of a genome wide RNAi library on this phenotypic assay thus enabled the identification of the key host-factors involved in Mtb trafficking and intracellular replication but also the elucidation of host-pathways exploited by the tubercle bacillus. Another adaptation of this particular phenotypic assay was for the identification of bacterial factors essential to Mtb intra-phagosomal persistence. For instance, the arrest of phagosome maturation is considered as one of the major mechanisms that facilitates the survival and replication of Mtb in macrophage. The monitoring of the subcellular localization of Mtb knock-out mutants in fluorescently labeled-acidic compartments allowed for the identification of bacterial genes involved in the survival process4. Finally, the high-content imaging of Mtb also offers an excellent method to quantify drug efficiency for inhibiting various phenomena like intracellular bacterial growth3. Altogether, this type of high throughput phenotypic assay allows accelerating drug discovery against TB and the data collected by these different approaches contribute to a better understanding of the host manipulation exerted by Mtb.

<table border="0" cellpadding="0" cellspacing="0" width="1010">
	<colgroup>
		<col />
		<col /> 
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">&#181;clear-plate black, 384 well</td>
			<td style="width:160px;">Greiner bio-one</td>
			<td style="width:175px;">781091</td>
			<td style="width:388px;">127.8/86/15 MM with Lid, TC treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CellCarrier 384 well plate</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;">6007550</td>
			<td>Black, Clear Bottom, with Lid, TC treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">V-bottom white , 384 well-plate</td>
			<td style="width:160px;">Greiner bio-one</td>
			<td style="width:175px;">781280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">sealing tape, breathable, sterile</td>
			<td style="width:160px;">Corning</td>
			<td style="width:175px;">3345</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Lipofectamine RNAiMax</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">13778150</td>
			<td>Transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Dimethyl sulfoxide</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">34943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">RPMI 1640 + GlutaMAX-I</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">61870-010</td>
			<td>Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">D-PBS 1X [-]MgCl2/[-]CaCl2</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">14190-094</td>
			<td>Dulbecco&#39;s Phosphate Salin Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">D-PBS 1X [+]MgCl2/[+]CaCl2</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">14190-091</td>
			<td>Dulbecco&#39;s Phosphate Salin Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Fetal bovine serum</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">2610040-79</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">FICOLL PAQUE PLUS</td>
			<td style="width:160px;">DUTSCHER</td>
			<td style="width:175px;">17-1440-03</td>
			<td>Ficoll for Peripherical Blood Monocyte Cells purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">CD14 MicroBeads, human</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-050-201</td>
			<td>Purification of CD14+ Monocytes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Human M-CSF, premium gr. (1000 &#956;g)</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-096-493</td>
			<td>Macrophage Colony Stimulating Factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">LS Columns</td>
			<td style="width:160px;">Miltenyi</td>
			<td style="width:175px;">130-042-401</td>
			<td>Columns for CD14+ Monocytes isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Tween 80</td>
			<td style="width:160px;">Euromedex</td>
			<td style="width:175px;">2002-A</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Glycerol high purity </td>
			<td style="width:160px;">Euromedex</td>
			<td style="width:175px;">50405-EX</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;"> Middlebrook OADC enrichment</td>
			<td style="width:160px;">Becton-Dickinson</td>
			<td style="width:175px;">211886</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">7H9</td>
			<td style="width:160px;">Becton-Dickinson</td>
			<td style="width:175px;">W1701P</td>
			<td>Mycobacteria culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Versene 1X</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">15040033</td>
			<td>Non enzymatic cell dissociation solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">DAPI</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">D1306</td>
			<td>Nuclei dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hoechst 33342</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">H3570</td>
			<td>Nuclei dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Syto60</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">S11342</td>
			<td>Nuclei/cytoplasm dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Formalin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">HT5014</td>
			<td>Cell fixation solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">siRNA targeting Dectin-1</td>
			<td style="width:160px;">Santa-Cruz</td>
			<td style="width:175px;">sc-63276</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">*siGenome* Non targeted siRNA pool </td>
			<td style="width:160px;">Dharmacon</td>
			<td style="width:175px;">D-001206-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Rifampicin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">R3501</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Isoniazid (INH)</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">I3377-50G</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Hygromycin B</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:175px;">10687-010</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Amikacin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:175px;">A1774</td>
			<td>antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Automated Confocal Microscope OPERA</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Image acquisition</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Columbus 2.3.1 Server Database</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Data transfert, storage and analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Acapella 2.6 software</td>
			<td style="width:160px;">PerkinElmer</td>
			<td style="width:175px;"> </td>
			<td>Image-based analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">GraphPad Prism5 software</td>
			<td style="width:160px;">GraphPad </td>
			<td style="width:175px;"> </td>
			<td>Statistical analysis</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:287px;">Excel 2010</td>
			<td style="width:160px;">Microsoft</td>
			<td style="width:175px;"> </td>
			<td>Statistical analysis</td>
		</tr>
	</tbody>
</table>

a microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

High-throughput genome-wide siRNA screening
Mtb is able to colonize immune cells in vitro as well as several other lung epithelial cells. For instance, Mtbis able to infect and damage A549 epithelial cells that are commonly used as a model for human type II pneumocytes5-7. Dectin-1 was reported as a host cell receptor involved in Mtb uptake, proinflammatory response and antibacterial effect on intracellular mycobacterial growth in A549 cells8</s...

Watch this Scientific Journal Video about A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening at JoVE.com

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

a-microscopic-phenotypic-assay-for-quantification-intracellular

Research

JoVE Journal

Immunology and Infection

7.7K 次观看.  Université de Lille. 在这里，我们描述了一种表型检测，适用于小干扰合成RNA（siRNA）、化合物和 结核 分枝杆菌突变库的高通量/高含量屏幕。该方法依靠使用自动共聚焦显微镜检测荧光标记的荧光结 核分枝杆菌 。

视频: A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.
Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.
Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.
Note: Definitions/Abbreviations
BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. 
CFU = Colony Forming Unit (a measure of mycobacterial viability).

A Functional Whole Blood Assay to Measure Viability of Mycobacteria, using Reporter-Gene Tagged BCG or M.Tb BCG lux/M.Tb lux

We describe an alternative approach to the enumeration of mycobacteria in vitro, which uses reporter-gene tagged mycobacteria instead of colony-forming units (CFU). &#x201C;Survival&#x201D; of organisms as well as host response-markers are measured simultaneously, providing a low-cost, versatile and functional system for studies of host/pathogen interactions in the context of tuberculosis.

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

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.

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.

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

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up.
Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans&#8212;Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches.
We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans&#8212;E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.

A High-throughput, High-content, Liquid-based C. elegans Pathosystem

Here we describe a protocol that is an adaptable, whole host, high-content screening tool that can be utilized to study host-pathogen interactions and be used for drug discovery.

The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to ...