Name: a high-throughput compatible assay to evaluate drug efficacy against macrophage passaged mycobacterium tuberculosis
Uploaded: 2017-03-24T15:00:00
Description: Watch this Scientific Journal Video about A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis at JoVE.com

We thank Dr. Frank Wolschendorf for access to the Cytation 3 automated imaging plate reader. This work was funded in part by NIH grant R01-AI104499 to OK. Parts of the work were performed in the UAB CFAR facilities and by the UAB CFAR Flow Cytometry Core/Joint UAB Flow Cytometry Core, which are funded by NIH/NIAID P30 AI027767 and by NIH 5P30 AR048311.

NOTE: As M. tuberculosis mc26206 is an avirulent strain17,18, all work in this protocol can be performed in a Biosafety Level 2 facility (BSL-2).
1. Culture Conditions for Green Fluorescent Protein Expressing M. tuberculosis mc26206 (Mtb-GFP)
NOTE: The M. tuberculosis H37Rv derived auxotroph strain mc26206 (&#916;panCD, &#916;leu...

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Here, we have described in detail an alternative Mtb infection model suitable for drug efficacy testing. This model takes into account two key factors that should be given more consideration during the early TB drug development process: the presence of physiologically relevant barriers to drug penetration and metabolic changes of Mtb during infection. While we have previously shown the benefits of our infection model and proposed the possibility of scaling up the infection for throughput compatibility<s...

Tuberculosis (TB) remains a serious global health threat despite the availability of anti-TB chemotherapy regimens for over 40 years1. This is due in part to the requirement for long treatment periods of over 6 months using multiple drug combinations, which leads to patient non-compliance2. The emergence of drug-resistant TB in recent years has further compounded problems in a field where successful development of clinically approved drugs is virtually non-existent3. Indeed, despite exhaustive anti-TB drug development, only a single drug has been FDA approved for clinical use in the past 40 years4. Thus, new generations of anti-TB drugs are urgently needed to address this problem.
A key problem in TB drug discovery is the lack of successful transfer from compounds with in vitro activity to efficacy in the clinical setting5,6,7. Initially, target based approaches were used to screen for anti-Mtb drugs5, which failed to translate into whole bacterial cells. Even when Mtb cells are used, it is often performed using broth grown cultures, which do not accurately predict drug efficacy in vivo8,9. These problems have been recognized and drug screening assays against macrophages containing Mtb or latent Mtb have been successfully established8,10,11,12. However, even these more advanced assays do not give sufficient consideration to the penetration barriers that drugs encounter in the non-vascularized pulmonary lesions, and in the necrotic foci at the site of infection. Indeed, even for the first-line TB drug rifampicin, sub-optimal dosing has been questioned due to inadequate in vivo tissue and cerebral spinal fluid (CSF) penetration13,14,15 as well as decreased efficacy against intracellular Mtb8,9. As such, new models and assays that would take into account these parameters during the early lead development process would undoubtedly improve TB drug discovery efforts.
To address this need, we recently established an inexpensive, rapid, and BSL-2 compatible alternative infection model for Mtb drug efficacy testing16. This infection model produced densely packed Mtb within large macrophage aggregate structures, which recapitulated physiologically relevant cellular penetration barriers and generated macrophage-passaged Mtb with an altered physiological status resembling latent Mtb. Mtb derived from this infection model was combined with the resazurin microtiter assay (REMA) to evaluate drug efficacy, which produced results consistent with other intracellular infection models and correlated well with the reported ability of common TB drugs to achieve high CSF concentrations relative to serum concentrations16.
Here we describe in detail the generation of Mtb/macrophage aggregate structures to produce macrophage-passaged Mtb suitable for drug susceptibility testing using REMA. In particular, we show how this infection system could be adapted to a 96-well format for compatibility with throughput screening of candidate anti-TB drugs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7H9</td>
			<td>BD Difco</td>
			<td>271310</td>
			<td>Follow manufacturer&#39;s recommendations</td>
		</tr>
		<tr>
			<td>Middlebrook OADC</td>
			<td>BD Biosciences</td>
			<td>212351</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyloxapol</td>
			<td>Sigma</td>
			<td>T8761</td>
			<td>Prepare 20% stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>D-Pantothenic acid hemicalcium salt</td>
			<td>Sigma</td>
			<td>P5710</td>
			<td>Prepare 24 mg/ml&#160; stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>L-leucine</td>
			<td>MP Biomedicals</td>
			<td>194694</td>
			<td>Prepare 50 mg/ml&#160; stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>Hygromycin B</td>
			<td>EMD Millipore</td>
			<td>400051</td>
			<td>Prepare 200 mg/ml&#160; stock solution in H2O</td>
		</tr>
		<tr>
			<td>Nalgene Square PETG media bottle</td>
			<td>Thermo Fisher</td>
			<td>2019-0030</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Hyclone</td>
			<td>SH30027.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S12450H</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning</td>
			<td>MT25005CI</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Hyclone</td>
			<td>SH30237.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 3 plate reader</td>
			<td>Biotek</td>
			<td></td>
			<td>Interchangable with any fluorescent plate reader and microscope</td>
		</tr>
		<tr>
			<td>Gen5 Software</td>
			<td>Biotek</td>
			<td></td>
			<td>Recording and analysis of rezasurin coversion</td>
		</tr>
		<tr>
			<td>Rifampicin&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP2679250</td>
			<td>Prepare 10 mg/ml stock solution in H2O</td>
		</tr>
		<tr>
			<td>Moxifloxacin Hydrochloride</td>
			<td>Acros Organics</td>
			<td>457960010</td>
			<td>Prepare 10 mg/ml stock solution in H2O</td>
		</tr>
		<tr>
			<td>Resazurin Sodium Salt</td>
			<td>Sigma</td>
			<td>R7017</td>
			<td>Prepare 800 &#956;g/mL stock solution in H2O; filter sterilize</td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td>Fisher Scientific</td>
			<td>T164500</td>
			<td>Prepare 20% stock solution in H2O; filter sterilize</td>
		</tr>
	</tbody>
</table>

a high-throughput compatible assay to evaluate drug efficacy against macrophage passaged mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

To confirm the robustness of adapting this infection model to 96-well plate format, we here examined the drug susceptibility of Mtb derived from our 96-well adapted infection model to rifampicin (RIF) and moxifloxacin (MOXI) according to the template given in Figure 1A. We demonstrate that the generation of Mtb/macrophage aggregate structures key to this assay can be reliably produced in a 96-well plate format (Figure 2), thereby enablin...

Watch this Scientific Journal Video about A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis at JoVE.com

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

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