Name: a microscopic phenotypic assay for the quantification of intracellular mycobacteria adapted for high-throughput/high-content screening
Uploaded: 2014-01-17T13:45:00
Description: 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

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 ...

<ol>
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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. 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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.

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			<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>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:287px;">sealing tape, breathable, sterile</td>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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		<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>
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			<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>
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			<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>
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		<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>
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		<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>
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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 ...

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