This work was in part supported by grants from the Bill and Melinda Gates Foundation (OPP1109789), the National Natural Science Foundation of China (31570129), and start-up funds from the Tsinghua University School of Medicine to B.J. B.J. is a Wellcome Trust Investigator (207487/Z/17/Z).

1. Renilla-Firefly Dual-luciferase Reporter
NOTE: For a visual representation of this method, see Figure 2.
<ol>
	<li>Inoculate mycobacterial reporter strains from -80 &#176;C stock with 2 mL of 7H9 medium. Wild-type dual-luciferase, as well as mutated Renilla (reporter) strains, needs to be used to allow the calculation of mistranslation rates (see step 1.7). Shake at 37 &#176;C for 1 to 2 days till OD600&#160;reaches the stationary phase (OD &#62; 3).</li>
	<li>Aliquot and dilute to OD600nm around 0.1 - 0.5. For typical experiments, use three independent biological replicate cultures. Anhydrotetracycline (ATC), a tetracycline analog inducer of reporter expression (which is controlled by a tetracycline-inducible promoter) to a final concentration of 50 ng/mL.</li>
	<li>To measure effects of kasugamycin on mistranslation rates21, add different doses of kasugamycin to the culture (see Figure 4 for the indicated doses) at the same time (at the tested doses, kasugamycin has no antimicrobial activity). Note that it is important to include at least one non-induced control for each reporter. Culture-induce cultures for 4-6 h at 37 &#176;C with shaking.</li>
	<li>Transfer the bacterial cultures to a 2 mL tube, and centrifuge at 3,220 x g for 5 min at room temperature to pellet down the bacteria. Discard the supernatant.</li>
	<li>Disrupt the bacteria by adding 40 &#956;L of 1x passive lysis buffer (supplied by a dual-luciferase kit), which has been diluted (1:1) in double-distilled water. Transfer the resuspended bacterial lysate to a white 96-well plate, one well per sample, and shake at room temperature for 20 min. 
	CAUTION: Do not over incubate the bacteria in lysis buffer (for more than 30 min).</li>
	<li>Add 80 &#956;L of firefly substrate to each well, shake for 15 s, and measure the luminescence by luminometer with 1,000 ms as integration time. Use either an automated injector or a multichannel pipette to avoid pipetting errors.</li>
	<li>Add 80 &#956;L of Renilla substrate to each well, shake for 15 s, and measure the luminescence by luminometer with 1,000 ms as integration time.</li>
	<li>Subtract the background luminescence - measured using either wild-type M. smegmatis (i.e., not containing reporters) or uninduced reporter lysate - from the measured values. Use the corrected values to calculate the mistranslation rates of each condition by using the following equation11,25, where DN refers to the activity in the mutated reporter strain: 
	<img alt="Equation 1" src="/files/ftp_upload/59453/59453eq1.jpg" /></li>
</ol>
2. Nluc/GFP Reporter
NOTE: For a visual representation of this method, see Figure 3.
<ol>
	<li>Inoculate the bacterial reporter strain from -80 &#176;C stock with 2 mL of 7H9 medium. Shake at 37 &#176;C for 1 to 2 days till OD600&#160;reaches the stationary phase (OD &#62;3).</li>
	<li>Subculture to 50 mL of 7H9 medium and grow till OD600&#160;reaches the late stationary phase (&#62;4).</li>
	<li>Before aliquoting the bacteria to a 96-well plate, add ATC at a final concentration of 50 ng/mL and mix well. Induction of the bulk culture ensures that all wells contain the same amount of inducer and that the induction of the reporter is synchronized. Aliquot the bacteria to a clear, round-bottomed 96-well plate, with 100 &#956;L of volume in each well.</li>
	<li>To screen for small molecules affecting mistranslation rates, add the compound at the indicated concentration to select wells. For the purposes of this protocol, use kasugamycin (at doses indicated in Figure 5) as an illustration. Add different doses of kasugamycin to select wells (each experimental group should contain at least two biological replicates).</li>
	<li>Shake and induce the samples at 37 &#176;C for 16-20 h. 
	NOTE: It is necessary to seal the plate with film. Also, all the edge wells need to be filled with at least 200 &#956;L of sterile water to limit evaporation from the test wells.</li>
	<li>Take 80 &#956;L from each well, using a multichannel pipette, and transfer the samples to a black 96-well plate (which maximizes the fluorescence signal measurement). Measure the GFP signal by luminometer with 20 ms as integration time.</li>
	<li>After measuring the GFP signal, centrifuge the plate at 3,220 x g for 10 min. Transfer 50 &#956;L of the supernatant to a white-bottomed 96-well plate (which maximizes the luminescence signal measurement), add 50 &#956;L of Nluc substrate to each well, mix them well, and measure the luminescence by luminometer with 1,000 ms as integration time.</li>
	<li>Determine the Nluc/GFP ratio by dividing the corrected Nluc luminescence values by GFP fluorescence: this measure (in arbitrary units) is a relative measure of aspartate for asparagine mistranslation.</li>
</ol>

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The authors have nothing to disclose.

The protocol described here can be adapted for the measurement of mistranslation rates in a wide variety of organisms. There are a number of considerations that should be kept in mind when adapting the protocol to other systems. First, the purpose of the measurement should be considered. For the accurate measurement of mistranslation rates using gain-of-function reporters, what is needed is (a) a robust readout assay (e.g., enzymatic function [in this case, luciferase activity] with a broad linear range). Also, look for (b) a loss of function mutation in a critical residue of the reporter protein that results in a very significant loss of function, below the expected rate of mistranslation. For example, if the expected mistranslation rate to be measured is approximately 10-3/codon, the loss of function mutation needs to be greater than 1,000-fold; otherwise, the lower range of mistranslation events will not be sensitively detected. Lastly, for the accurate measurement of mistranslation rates using gain-of-function reporters, (c) a robust benchmark reporter is needed &#8212; in this case, firefly luciferase for section 1 of the protocol. Ideally, the benchmark reporter should be fused to the mutated enzymatic reporter, guaranteeing (for all intents and purposes) that they are both expressed in equimolar ratios. The benchmark (and primary reporter) readout should be robust to perturbations to exposed environments. For example, the fluorescence of wild-type GFP is highly susceptible to perturbation by changes in pH28, making it unsuitable as a benchmark for the measurement of mistranslation rates in phagocytosed mycobacteria in macrophages, which reside in phagosomes that are below pH 729. On the other hand, for applications such as small molecule screening, the most important considerations for a primary screen will be the reproducibility of the measurements (i.e., precision, as opposed to accuracy), the minimization of manual handling, and small culture volumes. The accurate measurements of mistranslation rates are less important and can be performed as secondary assays. Finally, it should be noted that the reporters described in this protocol, based on the enzymatic function of luciferase enzymes, will only measure mean mistranslation rates of a bacterial population but cannot give information about single-cell heterogeneity variation in mistranslation rates. Given the importance of single-cell variability in adaptive phenotypes30,31,32, fluorescent reporters for the measurement of mistranslation events have been developed12,33, including for the measurement of mistranslation in mycobacteria5.
Having considered the requirements for the measurement of mistranslation, it should be noted that the genetic gain-of-function reporters such as described in this protocol can only measure one type of mistranslation (i.e., one amino acid substitution for one codon) per reporter. Therefore, for the measurement of different mistranslation events, multiple reporters are required. For example, for the measurement of mistranslation by ribosomal decoding errors of near-cognate codons by lysyl-tRNA at one position, at least 16 reporters are required2,3,11. High-precision mass spectrometry and bioinformatics allow for the potential identification of many different types of mistranslation events simultaneously18; however, these methods also come with caveats. In general, they are less accurate than gain-of-function reporters. Furthermore, as stated earlier, they are less suitable for the detection of certain types of mistranslation, namely those that could be conflated with nonenzymatic deamidation23. Finally, mass spectrometry is unsuitable as a read-out for medium- or high-throughput screening.
For the adaptation of these reporters and protocols for the measurement of mistranslation in other systems, further considerations include a robust expression of the reporter in the desired model system. We initially attempted to use the dual-luciferase system developed by Farabaugh and colleagues in our mycobacterial system without modifications but had no success, due to a lack of reporter expression. Extensive troubleshooting identified that both codon-optimization and a minor sequence modification of the reporters were required to allow for robust expression25 (and see above). It is conceivable that similar adaptations of the reporters would be required for use in other systems.
Finally, consideration should be given to the type of mistranslation event being measured. For example, if there is a need to measure stop-codon readthrough (nonsense suppression), the stop codon&#8217;s loss of function mutation needs to be introduced within a noncritical region of the primary reporter protein34. Otherwise, only specific nonsense suppression events that regain the critical residue (and, hence, reporter function) will be measured by the assay, which would potentially lead to a substantial underestimation of the true mistranslation rates. There is increasing evidence that translational error plays a possible adaptive role in a large number of organisms1,12,13,35. However, the measurement of mistranslation events is still limited to a small, albeit growing number of model species. The adaptation of sensitive methods to measure mistranslation has the potential to further increase scientists&#8217; understanding of the role of translation error in physiology and pathology.

The flow of information in molecular biology requires the translation of genetic information to functional proteins. As with all biological systems, gene translation also involves measurable errors. Estimates of the rates of error in translation are typically quoted as approximately 1/10,000 per codon (reviewed by Ribas de Pouplana et al.1). However, error rates vary widely, from fewer than 10-5 to more than 0.05/codon1,2,3,4,5. The wide range of error rates, spanning more than three orders of magnitude, is due to the fact that errors can arise from multiple steps in the translation pathway: from stochastic, mutational, or stress-induced errors in aminoacylation6,7,8,9,10, physiological misacylation of asparaginyl- and glutaminyl-tRNAs5, or ribosomal decoding errors2,3,11. Measurably high error rates, representing over 0.01/codon, suggested that translational errors may perform physiological functions1,12 and that mistranslation may be context-specific13.
We and others have shown that naturally occurring errors in gene translation may be adaptive, especially during environmental stress1,5,12,14,15,16,17,18. In mycobacteria, errors generated by the two-step indirect glutamine/asparagine tRNA aminoacylation pathway19,20 result in a remarkably increased tolerance for the first-line antituberculosis antibiotic rifampicin5. Therefore, we speculated that decreasing mycobacterial mistranslation with a small molecule may potentiate killing by rifampicin. We screened for and identified the naturally occurring aminoglycoside kasugamycin as a compound that could decrease mycobacterial mistranslation, potentiate rifampicin-mediated killing of mycobacteria both in vitro and in vivo21, and limit the emergence of rifampicin resistance21, which threatens the global control of tuberculosis22 &#8212; the world&#39;s most deadly pathogen.
To study translational error, methods for the measurement of mistranslation have to be employed. There are multiple methods that have been developed for the measurement of mistranslation, each with advantages and disadvantages. Briefly, precision mass spectrometry-based methods have several advantages, the most important of which is that with newer algorithms for the detection of multiple types of translational error, a relatively unbiased measurement of mistranslation can be performed18. However, mass spectrometry is not very suitable for the measurement of deamidation mistranslation events &#8212; precisely the type of mistranslation that occurs in mycobacteria due to the error-prone indirect tRNA aminoacylation pathway. This is because of high-frequency nonenzymatic deamidation that occurs in the processing of samples for mass spectrometry23, resulting in an extremely high background signal. Therefore, for the detection of errors in this pathway, genetic gain-of-function reporters offer distinct advantages. Specifically, suitable gain-of-function reporters can have extremely low background rates, allowing the measurement of very low error rates11.
Since performing experiments with pathogenic Mycobacterium tuberculosis requires specialized facilities and extra precautions, we perform most experiments in the nonpathogenic mycobacterium M. smegmatis &#8212;&#160;and have shown earlier that results between the two species are broadly comparable5,21. To measure mistranslation rates in mycobacteria generated by the indirect tRNA aminoacylation pathway, we modified a Renilla-Firefly dual-luciferase system that had been previously developed to measure ribosomal decoding errors in E. coli11 for use in mycobacteria. We made three specific modifications: the original reporter did not express efficiently in mycobacteria, and therefore, the sequence was codon-optimized, and the C-terminal three amino acids of the firefly luciferase, serine-lysine-leucine, which has been annotated as a trafficking signal in some systems24, was modified to isoleucine-alanine-valine25. The original reporter had a critical lysine residue in the firefly luciferase mutated. Instead, we mutated either a critically conserved aspartate (D120) or glutamate (E144) residue in the Renilla luciferase to asparagine and glutamine, respectively25 (Figure 1). The reporter was subcloned into the episomal tetracycline-inducible plasmid pUV-tetOR (see the Table of Materials). Gain-of-function reporters mutate critically conserved functional residues in enzymes/fluorescent proteins that renders them nonfunctional11,26. Translational (or theoretically, transcriptional) errors that synthesize the functional variant of the protein would result in measurable enzyme activity in a subset of translated proteins. To correct for variation in protein abundance, the mutated reporter is coexpressed with a functional protein that acts as a benchmark and allows for accurate quantification of gain-of-function11. Whilst the Renilla-Firefly dual-luciferase reporter allowed for the accurate measurement of specific mycobacterial mistranslation rates5,25 (Figure 2 and section 1 of the protocol), we quickly realized that it is not suitable for medium/high-throughput screening of molecules that would alter the mistranslation rate. This is due mainly to two reasons, namely, a) the relative lack of potency of Renilla luciferase meant that a minimum of 1 mL of mycobacterial culture/sample was required to measure mistranslation rates, and b) the requirement for lysis of the cells prior to enzyme activity measurement required excessive manual handling: mycobacterial cells have a thick and multi-layered cell wall and envelope that is relatively resistant to lysis. Therefore, we looked to develop a new gain-of-function reporter system that could be used with small volumes (e.g., in a 96-well plate system) and did not require cell-lysis for measurements. We used the highly potent Nluc luciferase and identified a critical aspartate residue that, when mutated to asparagine, resulted in 2 logs loss of function (Figure 1). Furthermore, the small size of Nluc allowed it to have an N-terminal secretion signal tag &#8212; from antigen 85A, a major secreted antigen in mycobacteria27 &#8212; that would allow Nluc to be secreted into culture supernatant and circumvent the requirement for cell lysis. The benchmark protein, GFP, was expressed from the same promoter as the mutated Nluc, but from an integrated vector (see the Table of Materials), and could be measured in intact cells21 (Figure 3). Despite these advantages, the Nluc/GFP reporter (section 2 of the protocol) also has disadvantages: the relatively modest reduction in Nluc activity (100-fold) with the D140N mutation would not permit the measurement of extremely low mistranslation rates, making the reporter more suitable as a screening tool than for the accurate measurements of translational error. Furthermore, Nluc has no critical glutamate residues; therefore, only asparagine-to-aspartate error rates could be measured. The general principles described in this work should allow researchers to either use these reporters as we have done or modify reporters as appropriate for an accurate and/or facile measurement of other specific translational error rates in their model system of choice.

<table><tbody><tr><td>Middlebrook 7H9</td><td>BD Difco</td><td>271310</td><td></td></tr><tr><td>Anhydrotetracycline</td><td>Cayman Chemical&amp;nbsp;</td><td>10009542</td><td></td></tr><tr><td>Kasugamycin</td><td>sigma</td><td>K4013</td><td></td></tr><tr><td>Dual-luciferase reporter assay system</td><td>promega</td><td>E1960</td><td></td></tr><tr><td>Nano-Glo luciferase assay</td><td>promega</td><td>N1120</td><td></td></tr><tr><td>Fluoroskan Ascent FL luminometer</td><td>Thermo</td><td>/</td><td></td></tr><tr><td>Assay Plate, 96 Well White, Flat Bottom High Binding, No Lid</td><td>Costar</td><td>3922</td><td></td></tr><tr><td>Assay Plate, 96 Well Black, Flat Bottom High Biding, No Lid</td><td>Costar</td><td>3925</td><td></td></tr><tr><td>96 Well Cell Culture Cluster, Flat Bottom with Lid</td><td>Costar</td><td>3599</td><td></td></tr><tr><td>Non-commercial reagents (plasmids)</td><td></td><td></td><td></td></tr><tr><td>pUV-TetOR-RenFF</td><td>NA</td><td>NA</td><td>episomal shuttle plasmid that allows tetracycline-inducible expression of the wild-type dual luciferase reporter</td></tr><tr><td>pUV-TetOR-Ren-D120N-FF</td><td></td><td></td><td>dual-luciferase reporter with mutated Renilla</td></tr><tr><td>pUV-TetOR-Ag85ASec-Nluc-D140N</td><td>NA</td><td>NA</td><td>episomal shuttle plasmid with a tetracyclin-inducible secretable version of mutated Nluc luciferase</td></tr><tr><td>pMC1S-GFP</td><td>NA</td><td>NA</td><td>Mycobacterial integrated (L5 site) vector for tetracycline-inducible expression of GFP</td></tr></tbody></table>

measurement of specific mycobacterial mistranslation rates with gain-of-function reporter systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

A cartoon illustrating the general outline of the two reporter systems used in this work are shown in Figure 1. An overview of section 1 of the protocol is shown in Figure 2 and an overview of section 2 in Figure 3. The effect of kasugamycin on mycobacterial mistranslation is shown in Figure 4, as measured by the Renilla-Firefly reporter system. To show the specificity of the kasugamycin action, the reporter was also expressed in a strain of M. smegmatis in which ksgA was deleted (&#8710;ksgA). KsgA is an rRNA dimethyl transferase, and ksgA&#173;-deleted strains are relatively resistant to kasugamycin. Kasugamycin action on mistranslation was also measured using the Nluc/GFP reporter, as shown in Figure 5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59453/59453fig1.jpg" /> 
Figure 1: Cartoon illustrating the two gain-of-function reporter systems. (A) The Renilla-Firefly reporter system is comprised of two luciferase enzymes expressed as a fusion protein and under the control of a tetracycline-inducible promoter. Expression of the wild-type dual enzyme results in high measurable activity of both Renilla and firefly luciferases (top). Mutation of a critical aspartate residue, D120, in Renilla to asparagine renders the Renilla enzyme (D120N) inactive, but firefly luciferase is still active. Mistranslation of asparagine to aspartate (in this case, from physiologically misacylated Asp-tRNAAsn tRNA5,21) results in a small proportion of the translated Renilla proteins gaining activity, which can be measured. Comparison of the Renilla/Firefly activity of the mutated reporter compared with the wild-type dual enzyme allows the calculation of the asparagine to an aspartate mistranslation rate. (B) The Nluc/GFP reporter system operates on a similar principle. The mutated Nluc (D140N) gene has an N-terminal secretion signal derived from antigen 85A, a major secreted mycobacterial protein. Both the nluc and gfp genes are expressed from identical tetracycline-inducible promoters, to allow the use of GFP as a relative expression benchmark. Note the lack of wild-type Nluc control strain: this reporter is primarily used in screening, where measurements of relative mistranslation rates are sufficient for the primary screen. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59453/59453fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59453/59453fig2.jpg" /> 
Figure 2: Outline of measuring the mistranslation rate using Renilla-Firefly reporter (section 1 of the protocol). Fresh cultures of M. smegmatis, transformed with a plasmid expressing the dual-luciferase reporters, are grown. The reporter expression is induced, and investigational compounds or conditions are tested. Following 4-6 h of reporter expression, the cultures are pelleted and lysed and the lysates are tested for dual-luciferase activity. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59453/59453fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59453/59453fig3.jpg" /> 
Figure 3: Outline of measuring the mistranslation rate Nluc-GFP reporter (section 2 of the protocol). A fresh culture of M. smegmatis, transformed with the Nluc and GFP reporters, is grown to a sufficient volume for all the plates to be tested (assuming ~10 mL/96-well plate). Immediately prior to pipetting the bacterial culture into each well, induce the expression of the reporters with ATC added to the bulk culture. Incubate the wells by shaking them overnight. To measure the relative mistranslation rates, transfer the cultures to a black plate (to increase the sensitivity of fluorescence detection), measure the GFP fluorescence, and then, spin the plates down to pellet the bacteria. Transfer the supernatant to a white plate for Nluc activity measurement. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59453/59453fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59453/59453fig4.jpg" /> 
Figure 4: Representative result of using Renilla-Firefly reporter measuring the mistranslation rate in the presence of kasugamycin in different strains. Mistranslation rates of aspartate for asparagine in (A) wild-type M. smegmatis and (B) a strain deleted for ksgA (&#8710;ksgA) following treatment with kasugamycin (ksg) as measured by the Renilla-Firefly dual reporter. The cultures were treated with kasugamycin at indicated doses (in micrograms/milliliter) for 6 h prior to cell lysis and measurements. The corrected Ren/FF ratios (y-axes) are indicative of the aspartate for asparagine mistranslation rates. The deletion of ksgA results in a higher baseline mistranslation rate, which is more resistant to modulation by kasugamycin. The bars indicate the mean values, with standard deviation as error. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59453/59453fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59453/59453fig5.jpg" /> 
Figure 5: Representative result of using Nluc-GFP reporter measuring the mistranslation rate in the presence of kasugamycin. Relative mistranslation rates of aspartate for asparagine in wild-type M. smegmatis as measured by the Nluc/GFP reporter, following treatment with kasugamycin (ksg) overnight (16 h). The bars indicate the mean values, with standard deviation as error. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59453/59453fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems at JoVE.com

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

measurement-specific-mycobacterial-mistranslation-rates-with-gain

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

5.8K Views.  Tsinghua University School of Medicine. In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

Video: Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biochemistry

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 Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Genetics

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.

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 ...

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 ...

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

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 ...

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers&#8217; safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 &#176;C, 85 &#176;C, or 95 &#176;C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 &#181;L of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of &#60;1 log10eCFU/mL (i.e., &#60;10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

A Tuberculosis Molecular Bacterial Load Assay TB-MBLA

We describe a tuberculosis molecular bacterial load assay test performed after heat inactivation of sputum. Heat inactivation renders sputum samples noninfectious and obviates the need for containment level 3 laboratories for tuberculosis molecular tests.

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Biology

High-Throughput Screening Assay for Mycobacterium abscessus Using Dual Reporters

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

Author Spotlight: Scalable Drug Screening Protocol for Efficient Discovery of M. abscessus Treatments

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

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