This work was supported by BC Lung Association and Mitacs.

1. Bacterial Strain and Growth Medium
<ol>
	<li>Make albumin dextrose and salt stock solution (ADS) by solubilizing 25 g of bovine serum albumin, 10.0 g of dextrose, and 4.05 g of sodium chloride in 460 mL of deionized water. Filter-sterilize the ADS and store at 4 &#176;C.</li>
	<li>Make 7H9 broth by adding 4.7 g of 7H9 powder and 2 mL of glycerol to 900 mL of purified water. Autoclave the 7H9 broth at 121 &#176;C for 10 min and allow it to cool to room temperature before proceeding. Make 7H9ADST by adding 100 mL of ADS and 0.5 mL of Tween80 to 900 mL of 7H9 broth. Store at 4 &#176;C.</li>
	<li>Weigh 50 mg of kanamycin sulfate and dissolve in 1 mL of deionized water; the final concentration is 50 mg/mL. Filter-sterilize and store at -20 &#176;C. Add 0.5 mL of kanamycin stock solution per 1 L of 7H9ADST. 
	NOTE: This medium should be made fresh, so scale the volumes appropriately according to the culture size.</li>
	<li>Grow M. tuberculosis in 7H9ADST supplemented with kanamycin in standing culture. Shake the culture daily and dilute it before the OD600 reaches 1.0 to avoid clumping. 
	&#8203;NOTE: The M. tuberculosis strain used for the development of this method was H37Rv transformed with pJAK2.A plasmid12. pJAK2.A is an integrative plasmid based on the pMV361 vector, which allows high-level expression of the firefly luciferase gene from the hsp60 promoter and can be selected using kanamycin.</li>
</ol>
2. THP-1 Medium and Maintenance
<ol>
	<li>Add 50 mL of heat-inactivated fetal bovine serum (FBS) and 5 mL of 200 mM L-glutamine to 500 mL of RPMI 1640 to make RPMI incomplete medium (approximately 10% FBS and 2 mM glutamine).</li>
	<li>Maintain an THP-1 cell culture according to standard protocol13. Briefly, grow THP-1 cells in RPMI incomplete medium while maintaining a cell density of 0.2 to 1 million per mL of medium between passages.</li>
</ol>
3. High-throughput Intracellular Screening Using Luciferase-expressing M. tuberculosis H37Rv
<ol>
	<li>Measure the optical density of an actively growing bacterial suspension in a spectrophotometer at a wavelength of 600 nm. Calculate the bacterial density using the conversion factor of 0.1 OD600 = 3 x 107 bacteria per mL.</li>
	<li>Pipette out sufficient bacteria for a multiplicity of infection (MOI) of 10:1 into a new centrifuge tube. Pellet at 3,000 x g for 10 min and aspirate the liquid. Add 50 &#181;L of human serum to 450 &#181;L of RPMI1640. Scale the volume to appropriate values for the experiment.</li>
	<li>To opsonize the bacteria, resuspend the pellet at a density of 1 x 108 bacteria per 500 &#181;L of RPMI1640 containing 10% human serum. Allow the mixture to incubate at 37 &#176;C for 30 min. Determine the THP-1 cell culture density by counting with a hemocytometer and an inverted microscope.</li>
	<li>Pellet the cells in sterile centrifuge tubes at 100 x g and 37 &#176;C for 10 min. Aspirate the supernatant and resuspend the cells in RPMI incomplete at a density of 1 million cells per mL. Add phorbol-12-myristate-13-acetate (PMA) to a 40 ng/mL final concentration. 
	NOTE: This will be referred to as the differentiation mix.</li>
	<li>Combine opsonized M. tuberculosis with THP-1 differentiation mix at a MOI of 10:1 and aliquot the final mix at 100 &#181;L per well in a 96-well flat-bottom white plate. Regularly stir the mixture to ensure uniformity. Allow the differentiation and infection to proceed overnight at 37 &#176;C in a humidified incubator containing 5% CO2.</li>
	<li>Wash the wells twice with 100 &#181;L of RPMI each. Add compounds diluted to the desired concentrations in RPMI incomplete and incubate for 3 days.</li>
	<li>Aspirate the medium from the wells. Add 50 &#181;L of luciferase assay reagent to each well. Seal the plates with transparent adhesive plate sealers. Allow 5 min of incubation at 22 &#176;C and then obtain a readout in a luminometer at 1 s per well.</li>
</ol>
4. Cytotoxicity Analysis Using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay14
<ol>
	<li>Differentiate THP-1 cells in RPMI incomplete supplemented with 40 ng/mL of PMA in clear 96-well plates. Maintain a cell density of 1 million per mL and aliquot 100 &#181;L per well. Allow differentiation to proceed overnight at 37 &#176;C in a humidified incubator containing 5% CO2 .</li>
	<li>Aspirate the medium from the wells and wash them twice with RPMI 1640. Add compounds diluted in RPMI incomplete to the wells. Incubate for 3 days.</li>
	<li>Dissolve 0.5 g of MTT in 100 mL of phosphate-buffered saline (PBS) to make a stock solution of 5 mg/mL. Sterile filter and store at -20 &#176;C, away from light; it is best to make this solution fresh.</li>
	<li>2.5 h before the end of the 3-day incubation period, add 25 &#181;L of MTT solution to each well and complete the incubation period.</li>
	<li>Prepare 50% N,N-dimethyl formamide (DMF) by mixing 250 mL of DMF with 250 mL of deionized water.</li>
	<li>Prepare MTT extraction buffer as follows: Weigh 100 g of SDS in a 500-mL bottle and add 300 mL of 50% DMF. Apply low heat to allow the SDS to dissolve. Add 10 mL of pure acetic acid and 12.5 mL of 1 M HCl. Fill up to the 500-mL mark with 50% DMF; the final composition of the extraction buffer is 50% DMF, 20% SDS, 2.5% acetic acid, and 2.5% 1 M hydrochloric acid.</li>
	<li>At the end of the treatment period, add 100 &#181;L of extraction buffer (warmed to 45 &#176;C to dissolve any crystals) to each well. Allow the mixture to incubate overnight at 37 &#176;C in a humidified incubator containing 5% CO2. Read the absorbance at 570 nm. 
	&#8203;NOTE: The cytotoxicity assay is best performed in parallel with an intracellular screen using same-batch and age of THP-1 cells.</li>
</ol>
5. In-broth Activity Analysis Using a Resazurin Assay3
<ol>
	<li>Grow M. tuberculosis in 7H9ADST to the mid-log phase (~ 0.5 - 0.8 OD600). Dilute the culture with the 7H9ADST to 0.01 OD600. Dilute the compounds in 7H9ADST to 2x the testing concentrations and aliquot 100 &#181;L of each diluted compound into each well.</li>
	<li>Transfer 100 &#181;L of the diluted bacterial suspension into each well. Allow the plates to incubate at 37 &#176;C in a humidified incubator for 5 days. Dissolve 10 mg of resazurin in 100 mL of deionized water and sterile filter.</li>
	<li>Add 30 &#181;L of resazurin solution and monitor the color change after 48 h; bacterial growth is indicated by a color conversion from blue to pink. 
	NOTE: A quantitative analysis can also be performed by measuring either the fluorescence at 590 nm with excitation at 530 - 560 nm or the absorbance at 570 nm and 600 nm15.</li>
</ol>

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The authors declare no competing financial interests for this work.

The goal of this study was to create a simple and cost-effective HTS method using a human intracellular infection model for M. tuberculosis. Tuberculosis is a human disease characterized by the infection of alveolar macrophages by M. tuberculosis. Due to biosafety issues, research involving biological models of both the bacterium and the host cells has been used in the past. However, it has been shown that the usage of surrogate bacteria and non-human models are poor predictors of hit-to-lead success in drug development, indicating that drug screening is best done with human cells infected with M. tuberculosis19,20,21,22.
In this method, we have advanced and adapted current state-of-the-art screening protocols for the human macrophage-like THP-1 cell-line. In order to achieve high-throughput, we introduced several technical improvements to the infection protocol. First and foremost, we replaced all steps involved in CFU determination and substituted them with a luciferase-based reporter system. Firefly luciferase was chosen due to the simple end-point assay, the rapid degradation by host cell lysosome enzymes, and the minimal equipment requirements. This substitution effectively eliminated a 30-day incubation period, as well as the labor and consumables costs associated with plating and colony-counting steps.
A second major improvement we introduced was batch processing and infection, which further improves throughput and consistency between the wells with a simpler infection protocol. By combining the differentiation and infection steps into a single step, we were able to shorten the protocol by one day. At the same time, we were able to reduce the three rounds of washing that would normally occur between differentiation and infection, which is a source of possible THP-1 cell loss due to detachment.
This protocol was developed for screening inside the THP-1 cell line, which confers several advantages. THP-1, as an immortalized cell line, can be reliably cultured in vitro for over 20 passages23. This is especially important for large screening campaigns, where it can be challenging to maintain enough cells to supply a high-throughput setup. Additionally, testing in THP-1 provides a homogenous genetic background that minimizes variability in results. This is highly beneficial for testing compounds that influence host cell responses10. As an added bonus, gene expression in the THP-1 cell line can be down-regulated by small interfering RNAs (siRNAs)24. This provides a valuable tool for downstream investigations into the mode of action of hit compounds. Although this method was designed using THP-1 cell line, it can be easily adapted for human primary cells, such as peripheral blood mononuclear cells (PBMC), as was previously shown10.
To best mimic the actual interaction between alveolar macrophages and M. tuberculosis, the intracellular assay protocol includes a step to opsonize the bacteria. Opsonization with human serum coats M. tuberculosis and facilitates cell entry via complement receptor 3 (CR3)25. Naked bacteria are more likely to enter macrophages via the lectin receptor8. Given that the bronchoalveolar fluid is known to contain components of human serum25, opsonization, or the lack thereof, may have a fundamental impact on the screening outcome. However, some may choose to skip this step in order to further simplify the infection process16, or it may not be feasible to obtain enough human serum due to the size of the library being screened11.
The intracellular assay includes a step to remove all liquid containing unattached material from the wells prior to the addition of the luciferase reagent. This step is designed to increase the signal-to-noise ratio while reducing the amount reagent used per well. However, the inclusion of this step may generate false-positive data for compounds that kill THP-1 but not M. tuberculosis, since detached and lysed THP-1 and free-floating M. tuberculosis would be removed. Therefore, the manufacturer&#39;s suggested protocol of adding equal amounts of luciferase reagent (100 &#181;L) to each well should be followed in order to assay for M. tuberculosis survival in wells containing cytotoxic compounds.
In contrast to the firefly luciferase system, the bacterial lux system does not require external reagents for signal generation26. However, the firefly system is preferred over the lux system for the following reasons: First, the bacterial system may be toxic in mycobacteria26. Second, a more complex reporter system (5 genes for lux versus 1 gene for firefly) is more susceptible to signal inhibition by test compounds. Lastly, commercially available reagents for the firefly luciferase assay provide the necessary conditions for optimal signal generation. On the other hand, the bacterial luciferase system relies on ATP production and the cofactors present inside the bacteria for signal generation. These can vary between different treatments and are not as easy to control. Therefore, the addition of the luciferase reagent to the firefly system standardizes the reaction conditions and provides more reliable measurements of luciferase activity across all treatments.
CFU determination has long be the gold standard for quantifying bacterial density. In contract, bioluminescence, like most reporters, is not a direct measure of bacteria. Instead, RLU is a function of both CFU and the metabolic state of the bacteria. Others have demonstrated the mostly linear relationship between RLU and CFU for bioluminescent mycobacteria under specific conditions5. In any case, a significant reduction in the luciferase assay signal, no matter the underlying cause-other than the actual inhibition of luciferase enzyme activity-would indicate a reduction of the M. tuberculosis fitness inside the host cell. Therefore, these compounds would be of interest from a drug screening standpoint and should not be excluded in method development.
An alternative to the luciferase-based intracellular screening protocol is the automated fluorescent microscopy-based approach11,27,28,29. The luciferase assay output is measured by a luminometer, and the data obtained is quantitative, whereas fluorescent microscopy generates images that are qualitative. However, through clever computer programming, images can be analyzed to generate quantitative data. Furthermore, fluorescent microscopy allows multiple fluorophores to be used concurrently, which is very helpful for gathering valuable parameters such as cell viability, cell counts, and actual rate of infection. As one might predict, these benefits come with some setbacks. The initial investment on automated fluorescent microscopy equipment is many times greater than the cost of a luminometer or fluorometer and is therefore out of reach for many research labs. For those who have access to the equipment, the sample processing must be considered prior to image acquisition and data analysis. Those two steps affect the overall time investment with increases in library size. The inclusion of additional fluorescent labels in host cells requires fixing, staining, and washing steps, thus necessitating additional user intervention and time investments. Furthermore, fluorescent microscopy data collection and analysis, although automated, still requires significantly more time and resources than the simple luciferase assay readout. Therefore, the luciferase reporter-based intracellular screening method is simpler and capable of higher throughput.
The luciferase-based intracellular screening method has one significant limitation compared to fluorescent microscope-based screening methods. This is due to the fact that the luciferase assay provides no data regarding the health status of host macrophages. Cytotoxic compounds would cause the death of macrophages, and thus live bacteria may be released into the medium and would no longer contribute to the final luciferase assay signal. As a result, cytotoxic compounds would appear to cause the death of intracellular M. tuberculosis and thus generate a great number of false positives. To address this issue, we have complemented our method with an MTT assay to assess the cytotoxicity of compounds on host macrophages. This module of the screening method gives us additional information about the drugability of compounds of interest and allows the early elimination of less-than-ideal drug candidates.
Alternatively, one may also use the luciferase-based intracellular assay prior to performing automated fluorescent microscopy. In screens of large compound libraries, this allows for the quick and efficient assessment of compound effectiveness in the macrophage infection model. As a result, automated fluorescent microscopy can be reserved for detailed studies on better candidates, as illustrated by a previously published study11.
The low cost and simple nature of the luciferase-based intracellular assay also greatly benefits researchers who wish to test smaller chemical libraries. Overall, the luciferase-based intracellular assay has proven to be an extremely flexible tool for research labs of all calibers and for screening projects of various sizes.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. Drug-sensitive TB is a treatable disease that requires multiple antibiotics for a period of 6 months. Despite being a treatable disease, TB mortality was estimated to be 1.5 million in 20151. In the past 10 years, there have been increasing concerns over the prevalence of drug-resistant M. tuberculosis. Multidrug-resistant TB (MDR-TB) is defined as TB that is resistant to at least Isoniazid (INH) and Rifampicin (RMP), and most MDR-TB strains are also resistant to select second-line TB drugs, thus limiting treatment options. The effects of drug resistance create a greater challenge for treating patients co-infected with Human Immunodeficiency Virus (HIV); 400,000 patients worldwide died of HIV-associated TB in 20151. Disappointingly, the United States Food and Drug Administration has approved only one new TB drug against MDR-TB, bedaquiline, in the past 40 years2. Advances in finding better and shorter TB therapies are urgently needed in order to stay ahead in the fight against TB and MDR-TB.
Traditionally, TB drug screens are performed under in vitro growth conditions in growth medium, whereby compounds are added to a growing culture and their effectiveness in eradicating the microorganisms are determined by counting colony forming units (CFU) on solid medium. As CFU counts are labor intensive, time consuming, and costly, various indirect methods have been developed to alleviate this problem. Such methods include the Alamar Blue viability assay3, the determination of fluorescence4 from green fluorescent protein (GFP) or luminescence5 from luciferase-expressing bacteria, and the estimation of total adenosine triphosphate (ATP)6,7.
Typical TB is characterized by an M. tuberculosis infection of the lung, where the bacteria reside and replicate inside the phagosomes of alveolar macrophages8. The simple in-broth phenotypic screen may suit extracellular pathogens; however, in the historical perspective, hit compounds against M. tuberculosis identified using this method often fail to live up to expectations during downstream validation steps in infection models. We propose that TB drug is best performed in an intracellular host cell infection model. Nevertheless, intracellular models possess many technological and biological barriers to high-throughput screening (HTS) development. A big hurdle is the complexity of the infection process, exemplified by numerous steps and the elaborate removal of extracellular bacteria by in-between washing. A second major hurdle is the lengthy time requirements, as growth detection, normally done by CFU counting on culture plates, is a process that takes over 3 weeks to complete. One solution to replace CFU counts has been provided by automated fluorescent microscopy in combination with fluorescent bacteria. However, this solution requires an initial equipment investment that is out of reach for many research labs. A simple, low-cost, and disease-relevant HTS method would greatly enhance the drug discovery process.
In this study, we report a new, modular HTS system that is aimed at providing a rapid, and highly scalable, yet economical, assay suitable for determining the activity of compounds against intracellular M. tuberculosis. This system is composed of three modules: (i) intracellular, (ii) cytotoxicity, and (iii) in-broth assays. The combined final result provides a comprehensive description of the compound properties, with additional information as to the potential mode of action. This screening system has been used in several projects with various compound libraries that target mode of action, including the analysis of drug synergy9, the stimulation of autophagy10, and the inhibition of M. tuberculosis-secreted virulence factor (unpublished). Compounds of unknown mode of action have also been studied11. A modified version of this method was also adopted by our industrial partner as the primary screening method to identify new compounds against intracellular M. tuberculosis11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>RPMI 1640</td>
			<td>Sigma-Aldrich</td>
			<td>R5886</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>12483020</td>
			<td></td>
		</tr>
		<tr>
			<td>Middlebrook 7H9</td>
			<td>Becton, Dickinson and Company</td>
			<td>271210</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween80</td>
			<td>Fisher Scientific</td>
			<td>T164</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin, Bovine pH7</td>
			<td>Affymetrix</td>
			<td>10857</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Scientific</td>
			<td>BP350</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>kanamycin sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP906</td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma-Aldrich</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>MTT</td>
			<td>Sigma-Aldrich</td>
			<td>M2128</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide (DMF)</td>
			<td>Fisher Scientific</td>
			<td>D131</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Hydrocholoric acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>351279212</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Fisher Scientific</td>
			<td>351269</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Fisher Scientific</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin</td>
			<td>Alfa Aesar</td>
			<td>B21187</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1</td>
			<td>American Type Culture Collection</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>M. tuberculosis H37Rv</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well flat bottom white plate</td>
			<td>Corning</td>
			<td>3917</td>
			<td></td>
		</tr>
		<tr>
			<td>95-well flat bottom clear plate</td>
			<td>Corning</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent plate sealer</td>
			<td>Thermo Fisher Scientific</td>
			<td>AB-0580</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Biomate 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate spectrophotometer</td>
			<td>Biotek</td>
			<td>Epoch</td>
			<td></td>
		</tr>
		<tr>
			<td>luminometer</td>
			<td>Applied Biosystems</td>
			<td>Tropix TR717</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

High-throughput intracellular screening using M. tuberculosis expressing the luciferase gene
Figure 2A and Table 1 contain the raw data collected by the luminometer, expressed in relative luminescent units (RLU), showing the effect of an increasing concentration of the TB drug rifampicin on M. tuberculosis inside THP-1 cells. Figure 2A is a scatter plot of the raw luminescence measured in RLU for various concentrations of rifampicin. The error bars indicate the standard error of the mean (SEM). Figure 2B shows the percent reduction in luminescence in treated wells compared to the untreated wells. The data shows that rifampicin is capable of reducing &#62; 99.9% of RLU from intracellular M. tuberculosis at a concentration of 0.1 &#181;g/mL. This is in accordance with the previously published MIC between 0.1 and 0.4 &#181;g/mL16. Luminescence produced in each well is an indication of the total luciferase expressed by M. tuberculosis and thus is an indicator of the metabolic status of M. tuberculosis inside the well. It is normal for raw luminescent levels to vary between experiments. As such, a comparison of raw data would generate unreliable conclusions. Thus, the data should be normalized against a defined negative control, which would be the samples treated with DMSO only. The resulting values can be expressed as the percent reduction in M. tuberculosis in the wells (Figure 2B).
Cytotoxicity analysis using the MTT assay
The MTT assay is a well-established assay for eukaryotic cytotoxicity. This colorimetric assay indicates live cells through the conversion of MTT (yellow) to purple-colored formazan. This assay is performed without an M. tuberculosis infection because the bacteria are capable of metabolizing MTT, which reduces the observed toxicity of the compounds. A common suggestion for the MTT assay is to use media without the pH indicator phenol red due to the absorption at 570 nm17. However, the acidified extraction buffer described in this method is able to minimize interference caused by phenol red17.
Figure 3 illustrates the cytotoxicity caused by an increasing concentration of a test compound coded G1-1H. Normally, a 50% inhibitory concentration (IC50) is employed to indicate the level of cytotoxicity. In the case of G1-1H, concentrations of 10 &#181;M and 3 &#181;M are clearly below the IC50 concentration.
In-broth activity analysis using the resazurin assay
The resazurin assay is commonly used for the analysis of cytotoxicity in eukaryotic cells, but it can also be used to monitor live bacteria in broth3. The resazurin assay is a redox-based assay similar to the MTT assay. It measures NADPH levels and NADPH dehydrogenase activity through the conversion of resazurin into resorufin, a red fluorophore. The easiest and quickest way to determine drug efficacy is to look for the lowest treatment concentration where the wells remain blue in color. Figure 4 shows part of a 96-well plate showing the effects various concentrations of the antibiotic apramycin have on resazurin conversion by M. tuberculosis. The in-broth MIC was determined to be between 2.5 and 5 &#181;g/mL. This is slightly higher than the previously published value of 1.5 &#181;g/mL18, but it is still well within the acceptable range. In many cases, the color change is rather gradual, so it is difficult to make a confident determination. Under these conditions, it is better to quantify the actual amount of resazurin conversion using either absorbance or fluorescence. Due to the similar absorbance characteristics of resazurin and resorufin, MIC determination using absorbance requires measurements using two different wavelengths and complex calculations15. Therefore, the best method is to measure fluorescence using 530- to 560-nm excitation wavelengths and a 590-nm emission wavelength, as described in the manufacturer&#39;s literature15.
Figure 4 illustrates a potential source of inconsistency associated with multiday incubations that could drastically alter screening results. Due to improper humidifying of the 37 &#176;C incubator used for this experiment, wells on the edges of the plates suffered significantly more evaporation. All 15 DMSO-treated wells are supposed to be identical, but the wells along the left and top edges had reduced volumes. These wells also appear to have different colors than the other DMSO-treated wells in the middle of the plate. The same inconsistency can also be observed in 2.5 &#181;g/mL apramycin-treated wells. In this case, the reduced volume would also lead to a quantitative reading by a spectrophotometer and fluorometer. Evaporation becomes significant for all experiments with prolonged incubation times, so care must be taken to keep incubators well-humidified to avoid this source of inconsistency.
<img alt="Figure 1" src="/files/ftp_upload/55273/55273fig1.jpg" /> 
Figure 1: Method Schematics. Diagram depicting assay schematics for 3 separate modules of the high-throughput screening system. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55273/55273fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55273/55273fig2.jpg" /> 
Figure 2: Representative Data from an Intracellular to Examine the Effectiveness of Rifampicin in Eliminating M. tuberculosis inside THP-1 Cells. (A) Graph of the mean luminescence reading (in relative luminescence units) for each treatment (rifampicin) concentration. The error bars denote the standard error of the mean for each triplicate. (B) Graph of the calculated percent reduction in luminescence caused by a treatment of rifampicin, where higher values indicate greater effectiveness. The 90% inhibitory concentration (IC90) in this case is somewhere between 0.01 and 0.1 &#181;g/mL. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55273/55273fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55273/55273fig3.jpg" /> 
Figure 3: Representative Data from a Cytotoxicity (MTT) Assay to Examine the Toxic Effects of Compound G1-1H on Differentiated THP-1 Cells. Graph of the calculated &#34;percent of control&#34; values for each concentration of the treatment compound G1-1H, where higher values indicate healthier THP-1 cells. The IC50 in this case is somewhere between 10 and 30 &#181;M. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55273/55273fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55273/55273fig4.jpg" /> 
Figure 4: Representative Data from a Resazurin Assay to Examine the Effectiveness of Apramycin at Inhibiting M. tuberculosis In-broth. Only qualitative data is collected in this case due to equipment limitations in the biosafety level 3 (BSL3) lab. The photo shows part of a 96-well plate containing M. tuberculosis treated with either DMSO or varying amounts of apramycin. The DMSO-treated wells exhibited a conversion of resazurin to resorufin, as indicated by the color conversion from blue to pink. The apramycin-treated samples clearly underwent the color conversion below 5 &#181;g/mL. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55273/55273fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[Rifampicin]</td>
			<td>Rep1</td>
			<td>Rep2</td>
			<td>Rep3</td>
			<td>Average</td>
			<td>% Reduction</td>
		</tr>
		<tr>
			<td>4 &#181;g/mL</td>
			<td>184</td>
			<td>190</td>
			<td>210</td>
			<td>195</td>
			<td>100</td>
		</tr>
		<tr>
			<td>1 &#181;g/mL</td>
			<td>244</td>
			<td>215</td>
			<td>159</td>
			<td>206</td>
			<td>100</td>
		</tr>
		<tr>
			<td>0.1 &#181;g/mL</td>
			<td>1037</td>
			<td>731</td>
			<td>976</td>
			<td>915</td>
			<td>98</td>
		</tr>
		<tr>
			<td>0.01 &#181;g/mL</td>
			<td>19200</td>
			<td>24400</td>
			<td>23919</td>
			<td>22506</td>
			<td>54</td>
		</tr>
		<tr>
			<td>0 &#181;g/mL</td>
			<td>39877</td>
			<td>49655</td>
			<td>57728</td>
			<td>49087</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1: Raw data from the luciferase assay for the determination of the intracellular IC90 of rifampicin.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>[G1-1H]</td>
			<td>Rep 1 A570</td>
			<td>Rep 2 A570</td>
			<td>Rep 3 A570</td>
			<td>Average A570</td>
			<td>% of untreated</td>
		</tr>
		<tr>
			<td>30 &#181;M</td>
			<td>0.056</td>
			<td>0.056</td>
			<td>0.055</td>
			<td>0.056</td>
			<td>7</td>
		</tr>
		<tr>
			<td>10 &#181;M</td>
			<td>0.518</td>
			<td>0.488</td>
			<td>0.492</td>
			<td>0.499</td>
			<td>62</td>
		</tr>
		<tr>
			<td>3 &#181;M</td>
			<td>0.652</td>
			<td>0.638</td>
			<td>0.656</td>
			<td>0.649</td>
			<td>80</td>
		</tr>
		<tr>
			<td>0 &#181;M</td>
			<td>0.822</td>
			<td>0.782</td>
			<td>0.815</td>
			<td>0.806</td>
			<td>100</td>
		</tr>
	</tbody>
</table>
Table 2: Raw Data from the MTT Assay for the Determination of the Cytotoxicity of Test Compound G1-1H at Different Concentrations.

Watch this Scientific Journal Video about System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis at JoVE.com

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.

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 ...

system-for-efficacy-cytotoxicity-screening-inhibitors-targeting

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

8.5K Views.  University of British Columbia. 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.

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

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

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.

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.

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

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...