1. Microbiology and Infectious Disease
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Kasugamycin potentiates rifampicin and limits emergence of resistance in Mycobacterium tuberculosis by specifically decreasing mycobacterial mistranslation

  1. Swarnava Chaudhuri
  2. Liping Li
  3. Matthew Zimmerman
  4. Yuemeng Chen
  5. Yu-Xiang Chen
  6. Melody N Toosky
  7. Michelle Gardner
  8. Miaomiao Pan
  9. Yang-Yang Li
  10. Qingwen Kawaji
  11. Jun-Hao Zhu
  12. Hong-Wei Su
  13. Amanda J Martinot
  14. Eric J Rubin
  15. Veronique Anne Dartois  Is a corresponding author
  16. Babak Javid  Is a corresponding author
  1. Tsinghua University School of Medicine, China
  2. The State University of New Jersey, United States
  3. Harvard TH Chan School of Public Health, United States
  4. Beth Israel Deaconess Medical Center, Harvard Medical School, United States
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Cite this article as: eLife 2018;7:e36782 doi: 10.7554/eLife.36782

Abstract

Most bacteria use an indirect pathway to generate aminoacylated glutamine and/or asparagine tRNAs. Clinical isolates of Mycobacterium tuberculosis with increased rates of error in gene translation (mistranslation) involving the indirect tRNA-aminoacylation pathway have increased tolerance to the first-line antibiotic rifampicin. Here, we identify that the aminoglycoside kasugamycin can specifically decrease mistranslation due to the indirect tRNA pathway. Kasugamycin but not the aminoglycoside streptomycin, can limit emergence of rifampicin resistance in vitro and increases mycobacterial susceptibility to rifampicin both in vitro and in a murine model of infection. Moreover, despite parenteral administration of kasugamycin being unable to achieve the in vitro minimum inhibitory concentration, kasugamycin alone was able to significantly restrict growth of Mycobacterium tuberculosis in mice. These data suggest that pharmacologically reducing mistranslation may be a novel mechanism for targeting bacterial adaptation.

https://doi.org/10.7554/eLife.36782.001

eLife digest

A bacterium called Mycobacterium tuberculosis is responsible for nearly 98% of cases of tuberculosis, which kills more people worldwide than any other infectious disease. This is due, in part, to the time it takes to cure individuals of the disease: patients have to take antibiotics continuously for at least six months to eradicate M. tuberculosis in the body.

Bacteria, like all cells, make proteins using instructions contained within their genetic code. Cell components called ribosomes are responsible for translating these instructions and assembling the new proteins. Sometimes the ribosomes produce proteins that are slightly different to what the cell’s genetic code specified. These ‘incorrect proteins’ may not work properly so it is generally thought that cells try to prevent the mistakes from happening.

However, scientists have recently found that the ribosomes in M. tuberculosis often assemble incorrect proteins. The more mistakes the ribosomes let happen, the more likely the bacteria are to survive when they are exposed to rifampicin, an antibiotic which is often used to treat tuberculosis infections. This suggests that it may be possible to make antibiotics more effective against M. tuberculosis by using them alongside a second drug that decreases the number of ribosome mistakes.

Chaudhuri, Li et al. investigated the effect of a drug called kasugamycin on M. tuberculosis when the bacterium is cultured in the lab, and when it infects mice. The experiments found that Kasugamycin decreased the number of incorrect proteins assembled by the M. tuberculosis bacterium. When the drug was present, rifampicin also killed M. tuberculosis cells more efficiently. Furthermore, in the mice but not the cell cultures, kasugamycin alone was able to restrict the growth of the bacteria. This implies that M. tuberculosis cells may use ribosome mistakes as a strategy to survive in humans and other hosts.

When it was given with rifampicin, kasugamycin caused several unwanted side effects in the mice, including weight loss; this may mean that the drug is currently not suitable to use in humans. Further studies may be able to find safer ways to decrease ribosome mistakes in M. tuberculosis, which could speed up the treatment of tuberculosis.

https://doi.org/10.7554/eLife.36782.002

Introduction

The long treatment duration of regimens for tuberculosis are thought in part to be due to phenotypic resistance (tolerance) of a subpopulation of genetically susceptible bacteria to antibiotic-mediated killing (Gold and Nathan, 2017; Maisonneuve and Gerdes, 2014; Lewis, 2010; Wakamoto et al., 2013). A better mechanistic understanding of antibiotic tolerance is required to rationally devise regimens that may reduce tuberculosis regimen duration. Multiple mechanisms have been proposed for how mycobacteria tolerate antibiotics, including non-replicating persistence (Saito et al., 2017; Gold and Nathan, 2017), antibiotic efflux (Adams et al., 2011) and phenotypic variation in cell-size (Rego et al., 2017; Richardson et al., 2016). We proposed that in mycobacteria, increased specific errors in gene translation – mistranslation – led to intracellular protein variants of the drug target of rifampicin, RpoB, which resulted in phenotypic resistance to rifampicin (Javid et al., 2014; Su et al., 2016). Clinical isolates with mutations in the essential amidase gatCAB that mediates variation in cellular mistranslation rates had both increased mistranslation and rifampicin tolerance, suggesting that this is a clinically relevant mode of antibiotic tolerance (Su et al., 2016).

The indirect aminoacylation pathway is present in the majority of bacterial species (with the exception of some proteobacteria such as Escherichia coli), all archaea, some mitochondria and other organelles (Sheppard and Söll, 2008). Bacteria lacking specific glutamine and/or asparagine tRNA synthetases instead utilize a non-discriminatory glutamyl- (asparaginyl) synthetase that forms misacylated Glu-tRNAGln and Asp-tRNAAsn aminoacyl complexes, respectively (Curnow et al., 1997; Rathnayake et al., 2017). These misacylated complexes are specifically recognized by GatCAB and amidated to the cognate Gln-tRNAGln and Asn-tRNAAsn aminoacyl tRNAs, thereby preserving the fidelity of the genetic code. We recently identified that in mycobacteria, strains with mutations in gatA causing partial loss of function are not only viable, but can be isolated from patient samples (Su et al., 2016). These strains have much higher rates of specific mistranslation – of glutamine to glutamate, and asparagine to aspartate – since a proportion of misacylated Glu-tRNAGln and Asp-tRNAAsn complexes are not fully converted to the cognate aminoacyl forms before taking part in translation at the ribosome. Importantly, wild-type GatCAB could also be limiting. Wild-type mycobacteria flow-sorted for lower GatCAB expression had both higher mistranslation rates and rifampicin tolerance (Su et al., 2016), suggesting that targeting the indirect tRNA aminoacylation pathway may present a novel and attractive means for increasing mycobacterial rifampicin susceptibility.

Here, we identify the natural product kasugamycin as a small molecule that can specifically decrease mistranslation due to the indirect tRNA aminoacylation pathway. At sub-inhibitory concentrations, kasugamycin, but not another aminoglycoside streptomycin can increase mycobacterial rifampicin susceptibility both in vitro and in animal infection.

Results

Kasugamycin increases mycobacterial discrimination against misacylated tRNAs

We hypothesized that a small molecule that could specifically decrease mycobacterial mistranslation would result in increased susceptibility to rifampicin. GatCAB-mediated mistranslation is not due to ribosomal decoding errors – but rather due to misacylated Glu-tRNAGln and Asp-tRNAAsn complexes taking part in translation (Su et al., 2016). In addition to other reported activities in E. coli (Lange et al., 2017; Müller et al., 2016; Kaberdina et al., 2009; Moll and Bläsi, 2002), the aminoglycoside kasugamycin decreased ribosomal misreading of mRNA (van Buul et al., 1984), but it was not known if it could also decrease errors due to translation of misacylated tRNAs, as the indirect tRNA aminoacylation pathway is not present in E. coli.

We tested whether kasugamycin could increase fidelity of misacylated tRNA-mediated mycobacterial mistranslation using a gain-of-function genetic reporter (Figure 1A). Kasugamycin at sub-inhibitory doses (Supplementary file 1) increased translational fidelity in both the model organism Mycobacterium smegmatis (Msm) and pathogenic Mycobacterium tuberculosis (Mtb) (Figure 1B,C and Figure 1—figure supplement 1). Importantly, kasugamycin decreased mistranslation in mycobacterial strains with mutated gatA that have extremely high misacylated-tRNA-mediated mistranslation due to partial loss of GatCAB function (Su et al., 2016) – Figure 1D, verifying that it was effective in increasing discrimination against translation of misacylated Glu-tRNAGln and Asp-tRNAAsn aminoacyl-tRNAs.

Figure 1 with 3 supplements see all
Kasugamycin decreases mycobacterial mistranslation due to the indirect tRNA aminoacylation pathway.

(A) Schematic of the Nluc-luciferase/GFP gain-of-function reporter to measure mistranslation. Kasugamycin (Ksg) decreases asparagine-to-aspartate mistranslation in both Msm (B) and Mtb (C) in a dose-dependent manner (see Materials and methods). (D) Kasugamycin (50 µg/ml) reduces asparagine-to-aspartate and glutamine-to-glutamate mistranslation – measured using a Renilla-FF luciferase dual reporter (see Materials and methods and Su et al., 2016) in a strain (HWS.4 – M. smegmatis-gatA-V405D) with a specific defect in the indirect tRNA aminoacylation pathway. (E) An E. coli cell-free translation system spiked with a non-discriminatory aspartyl synthetase used in conjunction with the Nluc-GFP mistranslation reporter shows that kasugamycin can specifically decrease translational error from misacylated Asp-tRNAAsn in a dose-dependent manner. *p<0.05, **p<0.01, ***p<0.001 by Student’s t-test.

https://doi.org/10.7554/eLife.36782.003

To further test whether kasugamycin was able to decrease Asp-tRNAAsn misacylation-mediated mistranslation, we developed a hybrid cell-free translation system. A commercially available E. coli coupled transcription-translation system was supplemented with a non-discriminatory aspartyl-synthetase (Ruan et al., 2008) that was able to misacylate E. coli tRNAAsn (see Materials and methods). Addition of the non-discriminatory synthetase markedly increased mistranslation, as measured by the Nano-luciferase-GFP gain-of-function reporter. Kasugamycin, at concentrations that did not decrease GFP signal, was able to reduce mistranslation-induced Nluc signal (Figure 1E), confirming that kasugamycin could increase ribosomal discrimination of misacylated tRNA.

One of the activities of kasugamycin in E. coli is the inhibition of the translation of canonical mRNA transcripts (i.e. those with a 5’ UTR including a Shine-Dalgarno sequence), but not leaderless transcripts lacking a 5’ UTR (Kaberdina et al., 2009; Moll and Bläsi, 2002), although permissive translation of leaderless transcripts was not universal (Schuwirth et al., 2006). We wanted to test whether selective inhibition of translation of canonical but not leaderless transcripts with kasugamycin was evident in mycobacteria, especially since mycobacteria have many annotated leaderless transcripts (Shell et al., 2015; Cortes et al., 2013). We constructed a reporter strain of M. smegmatis that expressed two fluorescent proteins, GFP and mCherry from the same basic promoter (Psmyc), but the promoter driving mCherry resulted in a leaderless transcript (Figure 1—figure supplement 2A and Materials and methods). The translation inhibitor chloramphenicol inhibited translation of both fluorescent proteins, but kasugamycin at 1500 µg/ml, more than 10 times higher concentrations than required to decrease mistranslation, did not significantly attenuate translation of either fluorescent protein (Figure 1—figure supplement 2B). Kasugamycin has also been reported to generate novel 61S ribosomes in E. coli (Kaberdina et al., 2009), but our attempts to isolate such structures from M. smegmatis were unsuccessful (not shown).

Kasugamycin acts on the ribosome as an inhibitor of 30S initiation (Wilson, 2014). Could other translation inhibitors – either targeting 30S initiation or other steps of translation also decrease mistranslation of misacylated tRNAs? We tested edeine, another 30S initiation inhibitor, and chloramphenicol, an inhibitor of peptide-bond formation (Wilson, 2014), both at sub-inhibitory concentrations with our gain-of-function mistranslation reporter. Intriguingly, edeine and kasugamycin, but not chloramphenicol could decrease mistranslation rates (Figure 1—figure supplement 3), suggesting that inhibition of 30S initiation might interfere with ribosomal discrimination of misacylated tRNAs by a yet to be characterized mechanism.

Kasugamycin increases mycobacterial susceptibility to rifampicin in vitro

We had previously showed that mistranslation generated via the indirect tRNA aminoacylation pathway in mycobacteria played an important role in rifampicin phenotypic resistance (Su et al., 2016; Javid et al., 2014). We therefore hypothesized that since kasugamycin could reduce mistranslation generated by this pathway, it would be able to reduce rifampicin tolerance. In keeping with our hypothesis, kasugamycin reduced mycobacterial rifampicin phenotypic resistance in wild-type (Figure 2A,B) and high mistranslating gatA mutant mycobacterial strains (Figure 2C). We then tested the ability of kasugamycin to enhance rifampicin-mediated killing of mycobacteria in axenic culture. Kasugamycin had no effect on mycobacterial growth, but addition to rifampicin significantly enhanced killing and sterilization of mycobacterial cultures (Figure 2D). Kasugamycin-mediated decrease in rifampicin tolerance was not due to its activity as an aminoglycoside – known protein translation inhibitors. The aminoglycoside streptomycin increases mistranslation (Leng et al., 2015). Plating of Msm on rifampicin-agar in the presence of sub-inhibitory concentrations of streptomycin increased the number of phenotypically-resistant colonies (Figure 2—figure supplement 1). Furthermore, in keeping with our observations with regard to 30S initiation inhibition and mistranslation, plating Msm on rifampicin-agar with sub-inhibitory concentrations of edeine but not chloramphenicol, decreased the number of phenotypically resistant colonies (Figure 2—figure supplement 2), confirming the link between reducing mistranslation rates and decreasing rifampicin tolerance.

Figure 2 with 2 supplements see all
Kasugamycin enhances in vitro killing of mycobacteria by rifampicin by specifically reducing mistranslation generated by the indirect tRNA aminoacylation pathway.

Ksg decreases plating rifampicin tolerance (Su et al., 2016) in both Msm (A) and Mtb (B) and an Msm strain with a specific defect in the indirect tRNA aminoacylation pathway (C) as well as (D) increases killing of Msm by rifampicin in axenic culture. (E) A strain (M. smegmatis-RpoB-N434T) with a single point mutation in rpoB is less tolerant to rifampicin because it is unable to mistranslate via the indirect pathway a single asparagine residue critical for rifampicin binding (Su et al., 2016). This strain is, however, relatively resistant to kasugamycin’s effects on rifampicin tolerance. *p<0.05, **p<0.01, ***p<0.001 by Student’s t-test. (F) Pre-treatment of Msm cultures with low-dose rifampicin (1 µg/ml) increases likelihood of rifampicin resistance upon subsequent challenge with high-dose rifampicin (100 µg/ml). Addition of Ksg but not streptomycin in pre-treatment abolishes the increased likelihood of rifampicin resistance. *p<0.05, ***p<0.001, ns = not significant by Fisher’s exact test.

https://doi.org/10.7554/eLife.36782.007

To further verify that kasugamycin’s effects on rifampicin susceptibility are due to its specific activity in decreasing mycobacterial mistranslation, we used a mycobacterial strain with a single point mutation in RpoB – strain Msm-RpoB-N434T. This strain has lower tolerance to rifampicin since a critical rifampicin-binding residue could no longer be mistranslated via the indirect pathway (Su et al., 2016). Msm-RpoB-N434T was less rifampicin tolerant than its parent strain, but kasugamycin was less potent at decreasing rifampicin tolerance further (Figure 2E). Since other reported activities of kasugamycin (Lange et al., 2017; Müller et al., 2016; Kaberdina et al., 2009) would not be affected by a single point mutation in the rpoB gene, we conclude that the major mechanism by which kasugamycin increased rifampicin susceptibility is by decreasing mistranslation-induced protein variants.

In E. coli, antibiotic resistance is preceded by tolerance (Levin-Reisman et al., 2017). Kasugamycin but not streptomycin given alongside rifampicin pre-treatment significantly reduced the likelihood of resistance following high-dose rifampicin challenge (Figure 2F), suggesting that kasugamycin may limit development of de novo resistance.

Kasugamycin increases rifampicin susceptibility in vivo

To test activity in vivo, we needed to establish whether kasugamycin had favorable tolerability and pharmacokinetics in an animal model, and if so, was kasugamycin administration with rifampicin able to boost killing of M. tuberculosis. We characterized the pharmacokinetics of parenterally administered kasugamycin and streptomycin in mice. Both agents showed dose-dependent concentration-time profiles in plasma, with rapid clearance (Figure 3—figure supplement 1). Administration of the maximum tolerated daily dose of kasugamycin – 400 mg/kg – resulted in a Cmax/EC(mistranslation)50 (peak plasma concentration divided by the half-maximal effective in vitro dose of kasugamycin in reducing mistranslation rates – Figure 1C) of ~2.5 and time over EC50 of 1 hr or only 4% of the dosing interval. Nevertheless, co-administration of kasugamycin resulted in an astonishing 30-fold boosting of rifampicin killing of Mtb in mouse lungs (Figure 3A). However, concomitant dosing of rifampicin and kasugamycin was poorly tolerated, even in the absence of tuberculosis (Figure 3—figure supplement 2). Histopathological examination of organs did not reveal a specific cause (not shown). Lower doses of kasugamycin co-administered with rifampicin were ineffective at enhancing rifampicin activity in vivo (not shown). In vitro, pre-treatment of axenic mycobacterial cultures with kasugamycin but not streptomycin decreased rifampicin tolerance, to a lesser degree (Figure 3—figure supplement 3). We thus opted for successive administration of high-dose kasugamycin intermittently with rifampicin in mice, which was well-tolerated. To specifically exclude the observed activity as being due to aminoglycoside-mediated inhibition of protein synthesis or post-antibiotic effects, we also included streptomycin-treated arms (Figure 3B). Since the driver of aminoglycoside efficacy is Cmax/MIC (peak plasma concentration divided by in vitro minimum inhibitory concentration) (Scaglione and Paraboni, 2006), we selected a streptomycin dose of 3 mg/kg, achieving a Cmax/MIC of 7, while the Cmax/MIC of kasugamycin was 0.8 (Supplementary file 1), thus avoiding underestimating streptomycin’s activity. Sequential treatment with rifampicin and kasugamycin, but not streptomycin led to significantly enhanced killing of Mtb in mouse spleens but not lungs (Figure 3C, Figure 3—figure supplement 4), which was not explained by differences in bulk-tissue PK (Figure 3—figure supplement 5). Thus, despite limited drug exposure resulting in effective concentrations achieved for only a small fraction of the treatment period, the in vitro effects on rifampicin tolerance were recapitulated in mice.

Figure 3 with 5 supplements see all
Kasugamycin enhances in vivo killing of M.tuberculosis by rifampicin.

(A) Lung burden of Mtb-infected mice treated for 2 weeks with rifampicin (10 mg/kg), Ksg (400 mg/kg) or combination of the two. (B) Schematic of the sequential Ksg (400 mg/kg)/rifampicin (10 mg/kg) or Streptomycin (3 mg/kg)/rifampicin (10 mg/kg) dosing schedule. (C) Lung (left panel) or spleen (right panel) burden of Mtb-infected mice treated for 4 weeks as per the schedule in (B). Each data point represents bacterial organ burden from a single mouse. *p<0.05, **p<0.01, ***p<0.001, ns = not significant by one-way ANOVA followed by Tukey’s post-hoc multi-comparison correction. Only key comparators are shown for clarity, complete statistical analysis is shown in Supplementary file 2.

https://doi.org/10.7554/eLife.36782.010

Discussion

There are multiple non-redundant models proposed for antibiotic tolerance (Abel Zur Wiesch et al., 2015; Aldridge et al., 2014; Balaban et al., 2013). In all these models, tolerance is mediated by generation of phenotypic heterogeneity within bacterial populations. Generation of stochastic errors during gene translation – mistranslation – is more prevalent, and occurs at higher rates, than previously appreciated (Mohler and Ibba, 2017; Ribas de Pouplana et al., 2014) and is one mechanism by which bacteria may generate considerable phenotypic heterogeneity. We show here that it is possible to identify a small molecule that can specifically decrease mistranslation rates, and hence increase antibiotic susceptibility.

There has been great interest in identifying small molecules that specifically target antibiotic-tolerant mycobacteria (Darby and Nathan, 2010; Zheng et al., 2014; Grant et al., 2013; Zheng et al., 2017; Sukheja et al., 2017; Alumasa et al., 2017; Wang et al., 2013; Vilchèze et al., 2017). These molecules have for the most part been identified via in vitro phenotypic screens that are specific for non-replicating persistence, although novel ‘target-specific’, whole-cell screens are proving highly useful in identifying compounds with activity against mycobacteria in vitro that are specific for certain stress-adaptation pathways (Zheng et al., 2017). With one exception (Wang et al., 2013), however, none of these candidates, and the pathways that they target, have been validated within an animal model of infection.

Kasugamycin is unique among aminoglycosides in its ability to decrease translational error – all other aminoglycosides increase mistranslation (Leng et al., 2015; Ribas de Pouplana et al., 2014; van Buul et al., 1984). In addition to its known effects in reducing ribosomal decoding errors, we have shown that kasugamycin can increase discrimination against physiologically misacylated tRNAs. It had been previously demonstrated that the ribosome has some proof-reading functionality beyond Watson-Crick base-pairing. This was limited to rejection of peptides formed from incorrect codon·anti-codon base-pairs (Zaher and Green, 2009). Misacylated tRNAs would still form cognate codon·anti-codon pairs at the ribosome, and would therefore not be rejected by such a mechanism. Rejection of misacylated aminoacyl tRNA formation had previously been described at the aminoacyl synthetase stage (Ibba and Söll, 1999), or by discrimination by EF-Tu (LaRiviere et al., 2001). Kasugamycin’s binding to the E. coli ribosome is close to the A794 and G926 residues (E. coli numbering) of 16S rRNA (Schuwirth et al., 2006). Given that these residues are universally conserved, it is likely that kasugamycin’s binding to mycobacterial and other bacterial ribosomes is in a similar location. Therefore, our data suggest that kasugamycin-bound ribosomes also possess a hitherto unknown ability to discriminate against misacylated EF-Tu·aminoacyl-tRNA complexes. Since edeine, another 30S initiation inhibitor, but not inhibitors of elongation (streptomycin) or peptide-bond formation (chloramphenicol) could also decrease mistranslation from misacylated tRNAs, this suggests a conserved mechanism, potentially involving translation initiation, directly or indirectly, in discrimination of physiologically misacylated tRNAs.

When kasugamycin and rifampicin were given daily, there was significant potentiation in mouse lungs after 2 weeks of treatment (Figure 3A), but with significant toxicity. However, in the better-tolerated alternate dosing schedule (Figure 3B), potentiation was seen only in mouse spleens, not lungs (Figure 3C). These differences could not be explained by differences in kasugamycin bulk tissue distribution (Figure 3—figure supplement 5). Even within a single organ/tissue, there can be significant heterogeneity in drug penetration and distribution (Prideaux et al., 2015), which we did not measure, and such heterogeneity of distribution in either rifampicin or kasugamycin or both may explain the differences observed. Furthermore, a greater proportion of M. tuberculosis may be resident in macrophages in lungs compared with spleens, and aminoglycosides have poor intra-cellular penetration (Brezden et al., 2016). As such, under this dosing schedule kasugamycin and rifampicin may be targeting the same mycobacterial subpopulation within spleens but different subpopulations within lungs, potentially explaining the observations.

The maximum dose of kasugamycin that could be administered to mice was limited due to toxicity and pharmacokinetics. At the maximum tolerated dose (400 mg/kg, once daily), the peak plasma concentration of 300 µg/ml failed to reach the in vitro minimum inhibitory concentration of 400 µg/ml. Intriguingly, these limitations allowed us to identify the relative in vivo potency of kasugamycin compared with conventional anti-microbials. Most anti-tuberculous drugs require peak plasma concentrations orders of magnitude greater than in vitro MIC for measurable efficacy (Mitchison, 2012; Pasipanodya and Gumbo, 2011; Pasipanodya et al., 2013). By contrast, the bacteriostatic kasugamycin given alone, administered intermittently (10 doses in 30 days) to infected mice, and with a Cmax/MIC <1, and at plasma concentrations <25% of MIC for 99% of the dosing interval was able to restrict Mtb growth in vivo. Streptomycin, a bactericidal aminoglycoside, given at far greater equivalent doses, had no effect. Mycobacteria increase specific mistranslation rates under conditions such as nutrient limitation and low pH (Javid et al., 2014), which mimic potential in vivo environments. These data suggest the efficacy of kasugamycin may not be as a conventional anti-microbial, but possibly by targeting bacterial adaptation to the host via reducing mistranslation.

There are several alternative mechanisms by which kasugamycin alone may have led to bacterial growth restriction in vivo. Kasugamycin’s biological activities differ with other aminoglycosides in additional ways than its contrasting effects on translational fidelity. Most aminoglycosides inhibit translocation during protein synthesis, whereas kasugamycin inhibits translation initiation by blocking the mRNA channel in the small ribosomal subunit (Schluenzen et al., 2006; Schuwirth et al., 2006). The reported structures of kasugamycin bound to the ribosome suggest that during 70S (leaderless) initiation, there is less steric hindrance of mRNA passage than in canonical initiation (Schluenzen et al., 2006), potentially explaining why kasugamycin is permissive for translation of some, but not all (Schuwirth et al., 2006) leaderless transcripts (Kaberdina et al., 2009; Moll and Bläsi, 2002). With regard to potentiation of rifampicin in vitro, our data strongly suggest that kasugamycin is acting by reducing mistranslation generated by the indirect tRNA aminoacylation pathway, and hence protein variants that mediate rifampicin tolerance (Figure 2E). However, although we did not find evidence that in mycobacteria kasugamycin selectively blocks translation of canonical but not leaderless mRNA transcripts (Figure 1—figure supplement 2), or form alternate ribosomes (Kaberdina et al., 2009) (not shown), we cannot formally exclude the possibility that these mechanisms play a role in kasugamycin’s activity in restricting M. tuberculosis growth in mice in the absence of rifampicin. Other potential mechanisms may be that kasugamycin is concentrated in macrophages, such that the intracellular concentration exceeded the MIC. Although we did not measure the intra-macrophage concentration of kasugamycin, almost all aminoglycosides enter cells poorly due to their polar structure (Brezden et al., 2016).

Synergistic drug combinations have the potential to radically improve treatment for tuberculosis. New methods for modelling synergy from purely empirical in vitro measurements can identify novel combinations (Cokol et al., 2017). More rational approaches can rescue current drugs that have limitations due to emergence of resistance or toxicity (Blondiaux et al., 2017). Our data suggest that targeting mycobacterial mistranslation may be a generally effective strategy, and not only in the context of potentiating rifampicin activity. Since most bacteria, with the notable exception of E. coli and a few other proteobacteria utilize the indirect tRNA pathway for synthesis of aminoacylated glutamine and/or asparagine tRNAs (Curnow et al., 1997), targeting adaptive mistranslation (Ribas de Pouplana et al., 2014) may be a useful strategy in the treatment of diverse bacterial infections.

Materials and methods

Bacterial strains, culture and antibiotics

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Mycobacterium smegmatis mc2-155 and its derivatives were cultured in Middlebrook 7H9 (BD DifcoTM) broth supplemented with 0.2% glycerol, 0.05% Tween-80, 10% Albumin-dextrose-salt (ADS), and on Luria-Bertani agar (LB agar) for plate assays. The high mistranslating strain HWS.4 with a mutation, gatA-V405D and the M. smegmatis strain with a point mutation in rpoB Msm-rpoB-N434T are previously described (Su et al., 2016). Mycobacterium tuberculosis-H37Rv was cultured in Middlebrook 7H9 broth with 0.2% glycerol, 0.05% Tween-80, 10% OADC (oleic acid, albumin, dextrose and catalase), and in Middlebrook 7H11 agar (BD Difco) supplemented with OADC for plate assays. Rifampicin, kasugamycin and streptomycin were purchased from Sigma. Rifampicin was dissolved in dimethyl sulphoxide (DMSO); kasugamycin and streptomycin were dissolved in water, and filter sterilized.

Antibiotic dose selection

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The in vitro MICs of kasugamycin, streptomycin and rifampicin for M. smegmatis and M. tuberculosis are given in Supplementary file 1. For in vitro experiments, kasugamycin and streptomycin doses were selected that had no effects on growth either in axenic culture or plating. Doses are given in the Figure legends. The only exception is Figure 1—figure supplement 2 – testing whether kasugamycin could inhibit translation of canonical/leaderless transcripts, when a dose of 1500 µg/ml was chose: this was 50x higher than a dose that could decrease mistranslation, and was close to the MIC. For the in vivo experiments, the maximum tolerated dose (400 mg/kg daily) of kasugamycin was given to mice. The streptomycin dose was calculated to represent 9x higher equivalent dose than kasugamycin (by Cmax/MIC) so as to not underestimate its effects, but not so high that streptomycin’s known bactericidal activity might interfere with interpretation of the data. Rifampicin (10 mg/kg) is a standard dose used in most in vivo experiments.

Generation of mistranslation reporter

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The nano-luciferase gene (nluc) sequence was obtained from Promega and optimized to accommodate mycobacterial codon usage preference and synthesized (Genscript). The nano-luciferase gene was fused downstream of the secretion signal sequence (first forty amino acids) of the secreted mycobacterial antigen 85A/B. The fused product was PCR amplified and cloned into the pJet1.2 cloning vector (Thermo Fisher Scientific) and verified by Sanger sequencing. Site directed mutagenesis was used to create a D140N mutation in nano-luciferase, and the mutation was verified by Sanger sequencing. The mutated nluc gene containing the secretion signal was then cloned into the tetracycline-inducible pUVtetOR vector having a hygromycin-resistant gene cassette. After sequence verification, the recombinant plasmid was transformed into M. smegmatis mc2-155 and M. tuberculosis-H37Rv using standard methodology. Codon optimized green fluorescence protein (GFP) sequence was cloned into tetracycline inducible pMC1s vector, which has kanamycin-resistant gene cassette, and electroporated into M. smegmatis mc2-155 and M. tuberculosis-H37Rv containing the nano-luciferase (N-luc) reporter.

Mistranslation assays

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The basis of the N-luc/GFP assay is a gain of Nano-luciferase (N-luc) signal, similar to previously published methods (Javid et al., 2014; Kramer and Farabaugh, 2007). The D140N mutation in N-luc caused approximately 100-fold reduction in activity compared with the wild-type enzyme. Mistranslation specifically of aspartate (D) for asparagine (N) in a subset of newly translated polypeptides would result in regaining of wild-type N-luc activity. Both N-luc and GFP were induced by the same tetracycline-responsive promoter, therefore total N-luc activity was corrected by dividing by total GFP fluorescence to account for variation in gene expression between samples. The N-luc/GFP ratio gave only a relative, not absolute, indication of specific mistranslation rates.

M. smegmatis mc2-155 containing the N-luc and GFP reporter plasmids was grown in 7H9 broth containing hygromycin (50 µg/ml) and kanamycin (20 µg/ml) to late log phase (OD600 = 2.0) at 37˚C, after which anhydrotetracycline (ATc, 50 ng/ml) was added to induce N-luc expression. The bacterial culture was then immediately aliquoted into a 96-well plate (100 µl, that is approximately 6 × 107 cells per well), different concentrations of kasugamycin or water control were added, and the cultures incubated for 16 hr at 37˚C with shaking. The cultures were then transferred to a 96-well black plate and GFP fluorescence measured. The plate was then centrifuged (4000 rpm for 10 min), and supernatants transferred to a 96-well white luminescence plate. The nano-luciferase assay was performed using Nano-Glo luciferase assay kit (Promega), and luminescence measured by the same machine. Relative mistranslation rates for M. tuberculosis H37Rv was measured in the same way, with minor modification. The H37Rv strain containing the N-luc-D140N and GFP reporter plasmids was induced with ATc (100 ng/ml) for 2 days before measuring the N-luc and GFP signals.

Mistranslation measurement using the Renilla-Firefly dual luciferase was performed as described previously (Su et al., 2016). Briefly, M. smegmatis mc2-155 strains harboring the reporters were grown till stationary phase. The cultures were diluted 20 times in fresh 7H9 medium, and expression of the dual luciferase was induced with 50 ng/ml anhydrotetracycline (ATc). After 6 hr, the bacterial cells were lysed and luciferase activities measured by dual luciferase assay kit (Promega). Measurements of fluorescence/luminescence of M. smegmatis were performed on a Fluoroskan Ascent FL Fluorimeter and Luminometer (Thermoscientifc), and for M. tuberculosis (and the Edeine/Chloramphenicol experiments) on a Biotek Synergy H1 plate reader (Fisher Scientific). The different instruments used was largely responsible for the differences in arbitrary unit (AU) values for mistranslation rates between the two species. Mistranslation rates were calculated as previously (Su et al., 2016). Tests of difference of means were performed by two-tailed Student’s t-test.

Cloning, expression and purification of Deinococcus radiodurans AspRS2 (Dr AspRS2)

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The Deinococcus radiodurans non-discriminatory aspartyl synthetase Dr AspRS2 is able to misacylate E. coli asparagine tRNA with aspartate (Ruan et al., 2008) (i.e. form Asp-tRNAAsn – the mycobacterial non-discriminatory enzyme does not recognize E. coli tRNA, not shown), and was therefore used in the E. coli cell-free translation system. Codon optimized Dr AspRS2 gene containing 6xHis-tag at the 5´end was synthesized (Genewiz) and cloned into XbaI and EcoRI restriction sites of pET28a(+) vector and transformed into E. coli BL21(DE3). Expression of Dr AspRS2 was induced by adding 1 mM IPTG. After one hour of induction at 37°C, the cells were harvested and Dr AspRS2 was purified by Ni-NTA affinity chromatography using standard methods. The final protein concentration was determined by Bradford reagent (Bio-Rad). The activity of the enzyme was confirmed by 3H-Aspartate labeling of E. coli tRNAAsn (not shown).

Cell-free measurement of mistranslation

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For cell-free measurement of mistranslation, a reporter expressing mutated N-luc (D140N) linked with wild-type GFP by a GGSGGG linker was generated. The mutated n-luc was produced by site directed mutagenesis and linked to WT gfp by Gibson assembly. The nluc linked gfp was then cloned into pIVEX vector (5 Prime) for in vitro coupled transcription-translation (IVT). The reporter measured relative mistranslation of aspartate for asparagine by gain-of-function Nluc activity/GFP fluorescence, as above. The coupled transcription-translation IVT reaction was carried out using an E. coli T7 S30 Extract System for Circular DNA kit (Promega) following manufacturer’s instructions with the following modifications. Since E. coli lacks the indirect tRNA aminoacylation pathway, the reaction mix was spiked with the non-discriminatory Dr AspRS2 to form misacylated Asp-tRNAAsn complexes that could take part in translation. 2 µM Dr AspRS2 or reaction buffer was added to the IVT reaction mix. To determine if kasugamycin could increase ribosomal discrimination of misacylated Asp-tRNAAsn-mediated translational errors, different concentrations of kasugamycin or carrier were added to the reaction mix and incubated on ice for 10 min before addition of the reporter template. Once the DNA template was added, the tube was mixed thoroughly and incubated at 37°C for 2 hr before the reaction was quenched by placing on ice for 5 min. The Nluc activity and GFP fluorescence was measured as above.

Inhibition of leaderless and canonical translation assay

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A dual-fluorescent reporter was constructed to measure translation of leaderless and canonical transcripts. The Psmyc promoter was cloned from plasmid PML1357 (Huff et al., 2010) (Addgene) and its transcription start site mapped by 5’ RACE (not shown). The gene for mCherry was fused directly to the transcription start site, forming a leaderless expressed gene, and gfp was cloned 3’ to a canonical Shine-Dalgarno sequence (Figure 1—figure supplement 2). Both fluorescent proteins had a C-terminal tag, AAV, which decreases stability and half-life of expressed proteins by targeting them for protease-mediated degradation (Andersen et al., 1998), thereby allowing monitoring of translation in real time. The two expression cassettes were subcloned into plasmid PSE100 and transformed into wild-type M. smegmatis. The transcription start sites of the two fluorescent promoters was verified by 5’RACE and was as predicted (not shown). Biological triplicates of the strain were grown to log phase, and then back-diluted to lag phase. After 4 hr growth, antibiotics (or carrier) were added to cultures. OD600, green and red fluorescence were measured by a Varioskan FLASH (Thermo) instrument.

Rifampicin-specific phenotypic resistance (RSPR) assay

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Rifampicin tolerance (phenotypic resistance) on agar medium was measured as previously described (Su et al., 2016). Stationary phase M. smegmatis mc2-155 cultures were serially diluted, and spread on LB-agar plates containing rifampicin (50 µg/ml), with or without kasugamycin/streptomycin. The plates were incubated at 37˚C for 5 – 7 days after which the number of colony-forming units (cfu) were counted. The number of bacterial cells in the inoculum was calculated by plating serial dilutions of the culture on antibiotic-free LB-agar plates and counting total plated cfu. For RSPR analysis of M. tuberculosis H37Rv, bacteria were spread on 7H11 agar medium containing rifampicin (0.2 µg/ml) with or without kasugamycin (31 µg/ml), and the plates were incubated for 6 weeks. For antibiotic pre-treatment experiments, M. smegmatis mc2-155 was grown in 7H9 broth containing kasugamycin (50 µg/ml) or streptomycin (0.25 µg/ml) and then spread on LB agar containing rifampicin. In all cases, doses of kasugamycin or streptomycin or other antibiotics were selected that did not by themselves decrease plating efficiency. Tests of difference of means were performed by two-tailed Student’s t-test.

Minimum duration of killing assay

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M. smegmatis mc2-155 was grown overnight in 7H9 (OD600 = 0.6) broth, and approximately 5 × 106 cells were inoculated into fresh 7H9 broth containing different concentrations of rifampicin (0, 10, 20, 50 µg/ml), with or without kasugamycin (50 µg/ml). At different time points, aliquots were removed from each culture, cells were washed once and 10-fold dilutions spread onto LB agar medium with sterile glass beads. The number of viable bacteria at each time point was calculated from the resulting number of colonies.

Rifampicin resistance assay

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M. smegmatis mc2-155 was grown to early stationary phase in 7H9 medium containing DMSO, rifampicin (1 µg/ml), or rifampicin with kasugamycin (50 µg/ml) or streptomycin (0.2 µg/ml) for 3 hr. After pre-exposure, cells were washed twice in PBS and 100 µl aliquots (2.5 × 107 cells) were transferred to wells in 96-well plates (240 wells/group in total, experiments conducted with three independent cultures over three separate days and results pooled) containing 7H9 and rifampicin (100 µg/ml), which was selective for bona fide genetic resistance to the antibiotic. After 4 days of incubation, the number of rifampicin resistant cultures was observed by the presence of turbid growth. The ‘resistance ratio’ was calculated as the percentage of wells with turbid (resistant) cultures divided by the total number of wells per condition. Statistical analysis was performed by Fischer’s exact test (GraphPad Prism) to compare conditions.

Pharmacokinetic (PK) analysis of kasugamycin and streptomycin

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All mouse experiments were approved by the Institutional Animal Care and Use Committee of the New Jersey Medical School, Rutgers University, Newark, NJ, under protocol number 15114D1018. For PK analysis, 8- to 10-week-old female BALB/c mice were injected intramuscularly with 100 mg/kg, 200 mg/kg or 400 mg/kg kasugamycin dissolved in 0.9% saline. Separate analysis for single-dose injections confirmed that intra-peritoneal and intra-muscular injection had exactly the same PK profiles (not shown). Blood was collected by tail snip after 15 and 30 mins, 1, 3, 5 and 8 hr following kasugamycin injection. Plasma was separated by centrifuging the blood at 5000 rpm for 5 min. Of plasma sample, 10 µl was extracted by adding 10 µl of acetonitrile/water (1:1) and 100 µl of acetonitrile: methanol (1:1) containing 10 ng/ml of verapamil (used as internal standard to correct for differences in injection volume that may happen during HPLC run), and plasma kasugamycin levels determined by high-pressure liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). The LC/MS/MS was performed on a Sciex Applied Biosystems Qtrap 4000 triple quadrupole mass spectrometer coupled to an Agilent 1260 HPLC system. Chromatography for kasugamycin was performed on a Cogent Diamond Hydride column (2.1 × 50 mm, particle size 4 µm) using a normal phase gradient elution. The gradient used 0.1% formic acid in Milli-Q deionized water and 0.1% formic acid in acetonitrile. Kasugamycin and verapamil were ionized using ESI-positive mode ionization and monitored using masses 380.17/112.10 and 455.4/156.2, respectively. Standard curve and quality control solutions were created by diluting 1 mg/ml of DMSO stocks of kasugamycin in acetonitrile/water (1:1). 10 µl of each dilution was added to 10 µl of drug-free plasma (Bioreclamation) and 100 µl of acetonitrile: methanol (1:1) containing 10 ng/ml verapamil. These standards were extracted as mentioned above.

For PK analysis of streptomycin, (8-10) week-old female BALB/c mice were intraperitoneally injected with 10 mg/kg, 20 mg/kg and 50 mg/kg of streptomycin dissolved in 0.9% saline, and blood was collected by tail snip after 15 and 30 mins, 1, 3, 5 and 8 hr. To 10 µl of plasma samples, 15 µl of extraction solution (35% trichloroacetic acid in water) was added. Then 10 µl of internal standard (20 µg/ml amikacin in water) and 70 µl of water was added to the mixture, and centrifuged at 3000 rpm for 5 min at 10˚C. Of the extract, 70 µl was transferred to an analysis plate, and 3 µl was injected on LC/MS/MS for analysis. Agilent Zorbax SB-C8, 4.6 × 75 mm, 3.5 µm was used as liquid chromatography column and Sciex Applied Biosystems Qtrap 4000 mass spectrometer was used for analysis.

Antibiotic toxicity in mouse

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This experiment was approved by the Institutional Animal Care and Use Committee of Tsinghua University under protocol number 17-BJ2. Six-week-old female BALB/c mice were given 10 mg/kg rifampicin orally by gavage and/or different doses (50 mg/kg and 400 mg/kg) of kasugamycin intraperitoneally daily for a week. Bodyweights of the mice were monitored daily. Mice were euthanized if they showed signs of visible distress or if they lost >20% of initial bodyweight.

Mouse infection and treatment

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All mouse infection and treatment experiments were approved by the Institutional Animal Care and Use committee of Rutgers University. Initial sample size calculation was calculated to have 80% power to detect a 0.5 log CFU reduction based on inter-animal variability established through a large number of similar animal efficacy experiments. For the subsequent animal experiments, sample size was determined from preliminary data. Nine-week-old female BALB/c mice were infected with M. tuberculosis H37Rv with a Glas-Col inhalation system. An inoculum of 1 × 107 cfu/ml bacteria was added to the nebulizer, and the initial bacterial lung load was determined by sacrificing four mice after 3 hr of infection. After 1 week, four mice were sacrificed to determine the bacterial burden in the lungs before start of treatment. The mice were treated daily with 10 mg/kg rifampicin orally (po) alone, or with 400 mg/kg kasugamycin intra-peritoneally (ip) alone, or with a combination of rifampicin (10 mg/kg) and kasugamycin (400 mg/kg) or carrier controls for 2 weeks. The mice were then sacrificed, the lungs harvested and homogenized; and the homogenates spread on Middlebrook 7H11 agar supplemented with OADC.

For the intermittent rifampicin-kasugamycin treatment procedure (Figure 2B), 10- to 12-week-old female BALB/c mice were infected with M. tuberculosis H37Rv, with 1 × 107 cfu/ml added to the nebulizer, and the lung bacterial load was determined by sacrificing four mice after 3 hr of infection. After 2 weeks of infection, four mice were sacrificed to determine bacterial burden in the lungs before start of treatment. The mice were given the following antibiotic regimen: The control group received PBS ip for 4 days, then water po for 5 days. From there on, they received PBS for 2 days followed by water for 5 days; and this cycle was repeated until the end of the treatment. This treatment procedure was used for the kasugamycin and streptomycin groups as well, with PBS being replaced by 400 mg/kg kasugamycin and 3 mg/kg streptomycin, respectively. The same procedure was also applied for the rifampicin only group, with water being replaced by 10 mg/kg rifampicin. Mice that received two antibiotics were given 400 mg/kg kasugamycin or 3 mg/kg streptomycin for 4 days, and then 10 mg/kg rifampicin for 5 days. This was followed by kasugamycin or streptomycin for 2 days and rifampicin for 5 days; and the cycle repeated until the end of the experiment. After 44 days of infection, mice were euthanized, lungs and spleens of the mice were harvested and homogenized, and the homogenates were spread on Middlebrook 7H11 agar supplemented with OADC. Statistical analysis of differences in means between groups was performed by one-way ANOVA followed by Tukey’s post-hoc correction for multiple samples (GraphPad Prism).

Statistical analysis

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Tests of significance are stated in the relevant methods sections and figure legends. All experiments were performed at least three times independently except for the edeine/chloramphenicol experiments (performed twice independently) and animal experiments. Figure 2A is representative of an experiment performed twice. For Figure 2B, data from two independent experiments are presented except for the Streptomycin arm, which was performed once.

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Decision letter

  1. Madhukar Pai
    Reviewing Editor; McGill University, Canada
  2. Gisela Storz
    Senior Editor; National Institute of Child Health and Human Development, United States
  3. Madhukar Pai
    Reviewer; McGill University, Canada
  4. Andreas Diacon
    Reviewer; Stellenbosch University, South Africa

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Kasugamycin potentiates rifampicin in Mycobacterium tuberculosis by specifically decreasing mycobacterial mistranslation" for consideration by eLife. Your article has been reviewed by 4 reviewers including Madhukar Pai as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Gisela Storz as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Andreas Diacon (Reviewer #3).

As you can see below, we received divergent feedback from the peer reviewers (reviewer #4, in particular, has raised several major concerns), and our final decision was reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

Reviewer #1:

I am not a basic TB scientist, and my comments are focused more on the relevance of this study and its potential implications for TB treatment.

Current TB treatment is long, and therapy for drug-resistant TB is particularly long, toxic, and expensive. Success rates are only about 50% and identification of new TB drug targets is a key priority for the TB field.

In this novel study, the authors have identified kasugamycin as a small molecule that can specifically decrease mistranslation due to the indirect tRNA aminoacylation pathway. This, in turn could limit emergence of rifampicin resistance in vitro and increased mycobacterial susceptibility to rifampicin both in vitro and in a murine model of infection.

The study opens a potential approach to saving rifampicin, which is one of the best TB drugs we have today. Of course, much more follow-up and clinical work in humans is necessary to follow up on this proof of concept work.

As regards the lab methods, I will defer to expert reviewers working in this area.

Reviewer #2:

Overall this is a very nice article trying to decipher the action of kasugamycin and its synergist effects with rifampicin. Considering the high death toll of Tuberculosis and the danger of MDR and XMDR Tb strains this is an important field of wide interest.

In order to provide more context for the general reader I would suggest to expand the discussion on other approaches to combat MDR TBR from a mere list of papers to a concise explanation of alternative strategies, as little as one sentence may be sufficient. I couldn't help noticing that recent excellent work from the Baulard group (ie. Blondiaux et al., 2017) could be mentioned.

The conclusions by the authors appear to be justified, however, I feel that the statistical treatment of a sometimes limited set of experiments needs to be explained in more detail. At several cases it is not clear how many independent measurements were taken to calculate the reported mean values and the error bars. I would suggest that the authors state that explicitly including the experiments in the supplementary material. I can only assume that the figures with individual data points refer to individual mice, so the authors should clarify that.

Given that the paper is not very long, I would also suggest that the authors consider which of the figures on the supplementary material should be in the main text.

In order to provide more molecular insight I'd like to suggest that the authors discuss the actual binding site of kasugamycin in the ribosome. This may provide further evidence for the hypothesis put forward. This would not require additional experiments but a very careful analysis of the structures in the data bases and possible some sequence alignments to see how conserved the binding site is.

Reviewer #3:

Kasugamycin potentiates rifampicin in Mycobacterium tuberculosis by specifically decreasing mycobacterial mistranslation

Overall Comments:

This interesting, content-rich article, draws logical conclusions as to how kasugamycin reduces mistranslation and enhances the effects of rifampicin on Mycobacterium tuberculosis (Mtb). The detailed methods investigate these effects genotypically, phenotypically and using a murine model comparing the means in the effects of rifampicin +/- kasugamycin +/- streptomycin.

Presentation of data

This is clear, but due to the density of information and number of methods used, it might be useful to have clearly stated objectives and how these were achieved. For example, in the third objective: "Kasugamycin increases rifampicin susceptibility in vivo", it could be noted that this was achieved through pharmacokinetic characterization of Kasugamycin and streptomycin, assessing CFUs from lung and spleen tissue, and determining toxicity. Similarly, if the figures and tables are displayed according to the objectives, it would be easier to cross-reference.

Figures

Compressing the display information into 2 figures makes it difficult to read. Since up to 4 figures / tables are allowed, perhaps rearrange this information to enhance the text. Again, consider linking the displayed items to the objectives.

Methods

In the Materials and methods section, describing where different doses of Kasugamycin were used and how these were determined, would help clarify the figures and results.

Conclusions

The information related to Kasugamycin increasing the susceptibility of M tuberculosis to Rifampicin in vitro was convincing in both methods, results and interpretation. I had some concerns regarding the mouse model work and information relating to Figure 2 and related tables/figures.

If co-administration of Kasugamycin resulted in a 30-fold boosting of rifampicin killing of Mtb in mouse lungs (but was toxic), and Kasugamycin pre-treatment decreases rifampicin resistance, can the authors explain why sequential treatment with Kasugamycin and rifampicin in the murine model only occurred in spleen but not lungs? It is noted that this was not explained by differences in tissue PK, but are there any other possibilities?

I was also a bit surprised to see, in Figure 2C and the Supplementary file 2 that provides the statistical detail, that killing with both strep and ksg was significant but "strep vs ksg" was not different. In subsection Kasugamycin increases rifampicin susceptibility in vivo”, where the experiment is described, the aminoglycoside concentrations for the experiment appeared to have been chosen to show that the ksg-enhanced rif activity was not due to inhibition of protein synthesis alone (by aminoglycosides). If I understand the intention correctly, the concentration of streptomycin was chosen to demonstrate that such inhibition can be shown in the mouse model (at 7xMIC thus inhibitory). The concentration of Kasugamycin, conversely, was such that killing was not likely to occur (0.8xMIC, non-inhibitory). Yet killing is shown by both equally in Figure 2C. How can this be explained? Is the MIC for Kasugamycin correct? At 0.8xMIC no such direct killing should occur, without RIF?

I found it equally intriguing, looking at Figure 2C, that the Rif+Ksg effect was seen in spleen but not lung, which, as mentioned above, was not explained by drug levels. Yet, looking at the figure, it seems that Rif+Ksg were equally effective in both organs reducing counts by about 1.5 logs (eyeballing the figure). Rif alone and Rif+ Strep were much less effective in spleen than lung, helping the effect of the Rif+Ksg in spleen to statistical significance. There seem to be mixed messages in here that could be carved out a bit better.

Reviewer #4:

In the present study, Swarnava Chaudhuri and colleagues explore the therapeutic potential of kasugamycin in tuberculosis treatment. They use in vitro and in vivo cultures of the tuberculosis agent, Mycobacterium tuberculosis, to show that kasugamycin administration prevents M. tuberculosis growth and sensitizes M. tuberculosis to another drug, RNA polymerase inhibitor rifampicin. Finally, the authors use in vitro translation system and laboratory strains M. tuberculosis in an attempt to test their hypothesis that kasugamycin alters M. tuberculosis growth and rifampicin sensitivity by suppressing GatCAB-mediated mistranslation.

The current manuscript is unacceptable for publication due to the poor quality of its experimental design, non-justified conclusions and misleading content.

Firstly, kasugamycin has been extensively studied over the past few decades. It was shown that this drug is not simply a molecule that increases translation accuracy – as Chaudhuri and colleagues make the reader think, referring to (van Buul et al., 1984). Instead, kasugamycin was shown to:

1) Inhibit protein synthesis by suppressing translation initiation of canonical mRNAs (Okuyama et al., 1971, Poldermans et al., 1979, Moll et al., 2002, Schluenzen et al., 2006, Kaberdina et al., 2009, Surkov et al., 2010).

2) Allow translation of some leaderless mRNAs (Moll et al., 2002, Kaberdina et al., 2009, Lange et al., 2017).

3) Rapidly alter protein content of bacterial cells and trigger a complex stress-like response, which nature we still do not fully understand (Kaberdina et al., 2009, Muller et al., 2016, Lange et al., 2017).

Chaudhuri and colleagues totally disregard four decades of kasugamycin research, apart from (van Buul et al., 1984) and a structural study by (Schurwirth et al., 2006). Did the authors ignore the kasugamycin studies that disagree with kasugamycin as a specific inhibitor of mistranslation? Or is it due to ignorance about the major object of their study?

Secondly, the major conclusion of the manuscript – that kasugamycin inhibits M. tuberculosis by reducing Asn-to-Asp or Gln-to-Glu mistranslation – is not justified by their experimental data. I found no proof that kasugamycin specifically alters Asn-to-Asp or Gln-to-Glu mistranslation, because the read-through assay used in this study is not capable to discriminate Asn-to-Asp or Gln-to-Glu mistranslation from overall changes in the accuracy of protein synthesis. To prove that a small molecule alters one specific type of mistranslation (e.g. Asn-to-Asp and Gln-to-Glu but not other types of mistranslation) people typically use quantitative mass-spectrometry (for instance, Cvetesic et al., 2016).

Thirdly, I found several overstatements. For instance, the idea that "reducing mistranslation may be a novel mechanism for targeting bacterial adaptation" (the last sentence of the abstract) is by no means novel. It was pronounced in numerous papers by Paul Schimmel, Mike Ibba, Susan Martinis and others, including Babak Javid's group (e.g. Su et al., 2016).

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for resubmitting your work entitled "Kasugamycin potentiates rifampicin in Mycobacterium tuberculosis by specifically decreasing mycobacterial mistranslation" for further consideration at eLife. Your article has been favorably evaluated by two new reviewers together with Madhukar Pai (Reviewing Editor), and Gisela Storz (Senior Editor).

Based on the new evaluations, your article will be considered for publication in eLife. However, the issues raised by the new reviewers need to be addressed, as outlined below. Please address these comments as well as the comments of the original reviewers in a revised version of your manuscript. (We realize this is a little non-standard for eLife but your manuscript has taken an unusual path.)

Reviewer #5:

The revised manuscript by Chaudhuri et al., describes a synergistic role of kasugamycin with rifampicin to inhibit mycobacteria by reducing mistranslation. I feel this work could potentially be an important contribution to understanding the role of translational fidelity in bacteria-host interactions as well as developing a combinatory treatment for mycobacteria infections.

The major criticism of the previous submission comes from reviewer #4, and I share some of the same concerns. The authors convinced me that kasugamycin decreases the level of mistranslation in mycoplasma, which is supported by the in vivo and in vitro experiments using split and fused reporters. The challenge is to demonstrate that the potentiating effect of kasugamycin is due to reducing mistranslation. The results of the mutant strains with mutations in gatA and rpoB genes (Figure 1D, Figure 2C, and 2E) are particularly interesting and provide support for their conclusion. However, tolerance to antibiotics is very complicated and not fully understood. The use of an additional antibiotic or introducing a mutation in the RNA polymerase may change cellular responses (e.g., toxin/antitoxin levels or efflux) that lead to altered sensitivity to rifampicin. As the authors also rightfully admitted, other effects of kasugamycin cannot be ruled out. Kasugamycin inhibits translation initiation. I feel it would strengthen their conclusion by using other non-aminoglycoside antibiotics that inhibit initiation (e.g., thermorubin) and other steps of translation (Wilson, 2014) in the potentiating assays (Figure 2A). This would reveal if inhibiting translation in general has synergistic effects with rifampicin to limit the growth of mycobacteria. In this manuscript, the authors used streptomycin as a control, which enhances mistranslation itself and therefore is not a proper control for general translation inhibition.

To summarize, I feel this work is worth reconsidering to be published in eLife if the authors can provide further data (as suggested above and below) to strengthen the conclusion statement in the title. These experiments are not expected to take a long time.

Reviewer #6:

Despite being generally viewed as negative outcome, inaccurate translation of mRNA (commonly referred to as mistranslation) may in certain cases be beneficial for some organisms. One such case is Mycobacterium tuberculosis, in which mistranslation is related to emergence of rifampicin resistance. It has been proposed that mistranslation expands the phenotypic heterogeneity of the proteome, hence increasing the chances of antibiotic-resistant proteins. Based on this, Chaudhuri et al., hypothesize that decreasing mistranslation would lessen the occurrence of resistance. In this paper, the effect of kasugamycin on rifampicin resistance emergence is explored in pathogenic Mycobacterium strains. Using a fluorescence-based reporting system, the authors show that the addition of the antibiotic kasugamycin reduces indirect -tRNA mistranslation and prevents rifampicin-resistant strains emerging in vitro and in vivo. Finally, they are able to replicate these findings in a mouse model, in which they show that rifampicin killing potential is increased by addition of kasugamycin.

*Strengths in vitro…: The fluorescence-based assay used is this paper is an elegant and robust way of measuring the effects of mistranslation.

in vivo: The most interesting point of the paper, in my opinion, is how they try to translate their in vitro findings to a live model. Despite some mixed results (such as clearing the Mycobacterium in the spleen but not in the lungs) and the toxicity of the co-administration of kasugamycin and rifampicin, they show that these two drugs are able to partially clear the infection. This is a very exciting and promising finding and it would be interesting to see if they can improve and replicate this in humans in the future.

Assessment: The study of the "beneficial" effects of mistranslation is gaining a lot of attention. This study adds another piece of evidence about the link between translation accuracy and fitness. In this paper, the authors focus on the mistranslation product of the indirect pathway used for charging tRNA-Asn and tRNA-Gln. This is a very particular and well characterized "controlled" event of tRNA mischarging. The results presented in the first part of the paper (fluorescence-based assays and survival experiments) are adequate for the point the authors try to make, yet it is true that they are somewhat overused.

It is worth mentioning the effort the authors have placed to use a live mouse model to further test their findings. Physiology and pharmacokinetics are outside of my field of expertise, so I do not have anything meaningful to add on the second part of the study, but it seems promising. The authors seem to have problems delivering the antibiotics to high enough levels that do not cause toxicity as well, which casts some doubts about its possible application in humans.

*Weaknesses

Lack of alternative methods: the fluorescence-based method, despite its usefulness, is only an indirect way of detecting mistranslation events. Although I think that asking for additional, more direct experiments would be out of the scope of this work, it would be something to consider in the future.

https://doi.org/10.7554/eLife.36782.023

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

As you can see below, we received divergent feedback from the peer reviewers (reviewer #4, in particular, has raised several major concerns), and our final decision was reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

Reviewer #1:

I am not a basic TB scientist, and my comments are focused more on the relevance of this study and its potential implications for TB treatment.

Current TB treatment is long, and therapy for drug-resistant TB is particularly long, toxic, and expensive. Success rates are only about 50% and identification of new TB drug targets is a key priority for the TB field.

In this novel study, the authors have identified kasugamycin as a small molecule that can specifically decrease mistranslation due to the indirect tRNA aminoacylation pathway. This, in turn could limit emergence of rifampicin resistance in vitro and increased mycobacterial susceptibility to rifampicin both in vitro and in a murine model of infection.

The study opens a potential approach to saving rifampicin, which is one of the best TB drugs we have today. Of course, much more follow-up and clinical work in humans is necessary to follow up on this proof of concept work.

As regards the lab methods, I will defer to expert reviewers working in this area.

We thank Dr. Pai for his positive comments regarding our work and its potential implications for salvaging rifampicin within the standard TB regimen.

Reviewer #2:

Overall this is a very nice article trying to decipher the action of kasugamycin and its synergist effects with rifampicin. Considering the high death toll of Tuberculosis and the danger of MDR and XMDR Tb strains this is an important field of wide interest.

We thank the reviewer for their positive assessment of our work, that our conclusions appear to be justified and for stating that the data are of potentially wide interest.

In order to provide more context for the general reader I would suggest to expand the discussion on other approaches to combat MDR TBR from a mere list of papers to a concise explanation of alternative strategies, as little as one sentence may be sufficient. I couldn't help noticing that recent excellent work from the Baulard group (ie. Blondiaux et al., 2017) could be mentioned.

Thank you for this comment. We have now included a brief discussion of alternative strategies to rescue existing anti-TB drugs, including the recent important paper from Baulard and colleagues (see Discussion section).

The conclusions by the authors appear to be justified, however, I feel that the statistical treatment of a sometimes limited set of experiments needs to be explained in more detail. At several cases it is not clear how many independent measurements were taken to calculate the reported mean values and the error bars. I would suggest that the authors state that explicitly including the experiments in the supplementary material. I can only assume that the figures with individual data points refer to individual mice, so the authors should clarify that.

Thank you. We have now added additional details to the Materials and methods section and Figure legends to improve clarity of how the data were generated, including explicitly stated (as correctly surmised by the reviewer) that individual mouse organ bacterial burdens are presented in Figure 2.

Given that the paper is not very long, I would also suggest that the authors consider which of the figures on the supplementary material should be in the main text.

Thank you for this suggestion on how to improve the readability of our manuscript. The revised manuscript is now composed of three main figures, with some of the more important supplemental data moved to the main figures to assist the reader.

In order to provide more molecular insight I'd like to suggest that the authors discuss the actual binding site of kasugamycin in the ribosome. This may provide further evidence for the hypothesis put forward. This would not require additional experiments but a very careful analysis of the structures in the data bases and possible some sequence alignments to see how conserved the binding site is.

Thank you for this suggestion. There are two published structures of kasugamycin bound to the ribosome: Schuwirth et al., 2006, which was cited in the originally submitted manuscript, showing the structure of the E. coli ribosome and kasugamycin, and Schluenzen et al., 2006, which shows two potential kasugamycin binding sites in the T. thermophilus ribosome.

These sites are highly conserved in all bacteria, as is the resistance mechanism to

kasugamycin: kasugamycin action requires N6 methylation of two adjacent adenosine nucleotides (A1518 and A1519, E. coli numbering) in 16S rRNA, which is mediated by the KsgA methyltransferase. These prior data suggest that kasugamycin is likely to bind to the same primary site in all bacterial ribosomes. We have added a brief discussion of these points in the revised manuscript (see Discussion section).

Reviewer #3:

Kasugamycin potentiates rifampicin in Mycobacterium tuberculosis by specifically decreasing mycobacterial mistranslation

We thank Dr. Diacon for his comments on our manuscript, in particular his assessment that our study is both logical in its deductions, and multi-layered in approach.

Specifically:

Overall Comments:

This interesting, content-rich article, draws logical conclusions as to how kasugamycin reduces mistranslation and enhances the effects of rifampicin on Mycobacterium tuberculosis (Mtb). The detailed methods investigate these effects genotypically, phenotypically and using a murine model comparing the means in the effects of rifampicin +/- kasugamycin +/- streptomycin.

Presentation of data

This is clear, but due to the density of information and number of methods used, it might be useful to have clearly stated objectives and how these were achieved. For example, in the third objective: "Kasugamycin increases rifampicin susceptibility in vivo", it could be noted that this was achieved through pharmacokinetic characterization of Kasugamycin and streptomycin, assessing CFUs from lung and spleen tissue, and determining toxicity. Similarly, if the figures and tables are displayed according to the objectives, it would be easier to cross-reference.

Thank you for these comments to improve the readability of our manuscript. We have now revised the manuscript, explicitly stating our objectives at the beginning of each subsection of the Results section.

Figures

Compressing the display information into 2 figures makes it difficult to read. Since up to 4 figures / tables are allowed, perhaps rearrange this information to enhance the text. Again, consider linking the displayed items to the objectives.

Thank you. As also suggested by reviewer #2, the revised manuscript comprises three main figures.

Methods

In the Materials and methods section, describing where different doses of Kasugamycin were used and how these were determined, would help clarify the figures and results.

Thank you for this suggestion. The Materials and methods section and/ or Figure legends now include this information including the rationale for dose selection.

Conclusions

The information related to Kasugamycin increasing the susceptibility of M tuberculosis to Rifampicin in vitro was convincing in both methods, results and interpretation. I had some concerns regarding the mouse model work and information relating to Figure 2 and related tables/figures.

If co-administration of Kasugamycin resulted in a 30-fold boosting of rifampicin killing of Mtb in mouse lungs (but was toxic), and Kasugamycin pre-treatment decreases rifampicin resistance, can the authors explain why sequential treatment with Kasugamycin and rifampicin in the murine model only occurred in spleen but not lungs? It is noted that this was not explained by differences in tissue PK, but are there any other possibilities?

a) Lung vs. Spleen Efficacy in alternate dosing model: We can only speculate regarding the reason for lack of efficacy in kasugamycin boosting of rifampicin in lungs at 1 month in the alternate dosing regimen. The results are consistent over several replications. Although measurement of bulk tissue PK (i.e. in homogenates) did not reveal any differences between lungs and spleens in kasugamycin dosing, such bulk measurements would not be able to detect any intra-tissue heterogeneity: as such, it is possible that kasugamycin and rifampicin in the alternate dosing schedule were targeting similar bacterial populations within spleens, but different bacterial populations within the lungs, potentially explaining the results. (see Discussion section).

I was also a bit surprised to see, in Figure 2C and the Supplementary file 2 that provides the statistical detail, that killing with both strep and ksg was significant but "strep vs ksg" was not different. In subsection Kasugamycin increases rifampicin susceptibility in vivo”, where the experiment is described, the aminoglycoside concentrations for the experiment appeared to have been chosen to show that the ksg-enhanced rif activity was not due to inhibition of protein synthesis alone (by aminoglycosides). If I understand the intention correctly, the concentration of streptomycin was chosen to demonstrate that such inhibition can be shown in the mouse model (at 7xMIC thus inhibitory). The concentration of Kasugamycin, conversely, was such that killing was not likely to occur (0.8xMIC, non-inhibitory). Yet killing is shown by both equally in Figure 2C. How can this be explained? Is the MIC for Kasugamycin correct? At 0.8xMIC no such direct killing should occur, without RIF?

b) Ksg vs Strep alone significance: For almost all anti-TB drugs, peak plasma concentrations at least 1-2 logs greater than in vitro MIC are required for in vivo efficacy (Mitchison, 2012, Pasipanodya et al., 2013). The streptomycin Cmax/MIC was ~7 and had no statistically significant efficacy. Although not measured by us, the standard rifampicin dose given typically achieves a Cmax/MIC of ~100. This makes the kasugamycin (alone) result even more remarkable. As correctly observed, the plasma Cmax never reaches MIC, and the tissue-specific PK measurements suggest tissue concentrations of Ksg are approximately 30-40x lower than the in vitro MIC – although with more stable kinetics than plasma concentrations. We have now expanded our discussion of these findings, which although not the central focus of the current work, are nonetheless intriguing. Although purely speculation at this point, and the subject of future study, we believe that the kasugamycin alone is not acting as a typical antibiotic, but may be interfering with “adaptive mistranslation/ translation” of the M. tuberculosis (see also comments to reviewer # 4 below). This may explain why it is able to restrict M. tuberculosis growth without achieving MIC. Alternative explanations could be that kasugamycin gets massively concentrated in tissues (not the case from our bulk tissue PK studies), or in macrophages – although we haven’t measured macrophage concentrations of kasugamycin, for all aminoglycosides where it has been measured, aminoglycoside intra-cellular penetration is extremely poor, making this unlikely. Nonetheless, we have now included all these possibilities in our discussion.

We have now clarified our streptomycin dosing choice: we wanted to ensure that any effects of rifampicin+kasugamycin were not due to non-specific aminoglycoside translation inhibition or post-antibiotic effects. The maximum Cmax/MIC achievable with kasugamycin was 0.8. We measured the MIC of M. tuberculosis-H37Rv to kasugamycin several times and by different methods. These varied from 400-1000 µg/ml according to method, but we stated the most conservative (i.e. lowest MIC) measurement rather than a mean in the manuscript so as to avoid under-estimating the Cmax/ MIC of 0.8. The PK equivalent dosing of streptomycin would be 0.4mg/kg. However, we wished to not under-estimate streptomycin effects, therefore used a dose of 3mg/kg (Cmax/MIC of ~ 7): this would be within an order of magnitude (but higher) than the equivalent kasugamycin dose, but would not be so high as to confound rifampicin potentiation by simple additive effects of what is known to be a bactericidal anti-TB drug. Since streptomycin is fairly well tolerated, much higher doses (1560 mg/kg) have historically been used to achieve efficacy (equivalent to Cmax/MIC of 30120, i.e. 35-150x equivalent kasugamycin dose).

These points aside: the absolute lung and spleen bacterial burdens were 6.1 and 4.8 log10 CFU for streptomycin alone and 6.0 and 4.5 log10 CFU for kasugamycin alone (compared with no drug: 6.4 and 4.9 log10 CFU) respectively. The absolute reduction in lung and spleen bacterial burdens were 0.34 and 0.14 log10 CFU for streptomycin alone and 0.45 and 0.42 log10 CFU for kasugamycin alone (compared with no drug), suggesting greater efficacy of kasugamycin, but the difference between the two groups was not statistically significant. Nonetheless, kasugamycin alone (at 9x lower effective dose than streptomycin) but not streptomycin alone did achieve significance threshold compared with the no drug group.

I found it equally intriguing, looking at Figure 2C, that the Rif+Ksg effect was seen in spleen but not lung, which, as mentioned above, was not explained by drug levels. Yet, looking at the figure, it seems that Rif+Ksg were equally effective in both organs reducing counts by about 1.5 logs (eyeballing the figure). Rif alone and Rif+ Strep were much less effective in spleen than lung, helping the effect of the Rif+Ksg in spleen to statistical significance. There seem to be mixed messages in here that could be carved out a bit better.

The reviewer is correct that the relative magnitude of the RIF+Ksg effect was approximately 1.5 log CFU reduction in both lungs and spleens. However, the lack of significance in the lungs is due to the fact the RIF alone had almost identical efficacy in the lungs, but was significantly less effective in the spleens. It’s possible that rifampicin’s superior efficacy in the lungs is that it’s intra-organ distribution within lungs better matches where M. tuberculosis reside, or that there is a greater mismatch between rifampicin/ kasugamycin distribution within lungs compared with spleens, and these are now discussed in the revised manuscript.

Reviewer #4:

In the present study, Swarnava Chaudhuri and colleagues explore the therapeutic potential of kasugamycin in tuberculosis treatment. They use in vitro and in vivo cultures of the tuberculosis agent, Mycobacterium tuberculosis, to show that kasugamycin administration prevents M. tuberculosis growth and sensitizes M. tuberculosis to another drug, RNA polymerase inhibitor rifampicin. Finally, the authors use in vitro translation system and laboratory strains M. tuberculosis in an attempt to test their hypothesis that kasugamycin alters M. tuberculosis growth and rifampicin sensitivity by suppressing GatCAB-mediated mistranslation.

The current manuscript is unacceptable for publication due to the poor quality of its experimental design, non-justified conclusions and misleading content.

We thank the reviewer for taking the time to read our manuscript and their observations, which we have addressed below with the aim of improving the readability and interpretation of our data.

Firstly, kasugamycin has been extensively studied over the past few decades. It was shown that this drug is not simply a molecule that increases translation accuracy – as Chaudhuri and colleagues make the reader think, referring to (van Buul et al., 1984). Instead, kasugamycin was shown to:

1) Inhibit protein synthesis by suppressing translation initiation of canonical mRNAs (Okuyama et al., 1971, Poldermans et al., 1979, Moll et al., 2002, Schluenzen et al., 2006, Kaberdina et al., 2009, Surkov et al., 2010).

2) Allow translation of some leaderless mRNAs (Moll et al., 2002, Kaberdina et al., 2009, Lange et al., 2017).

3) Rapidly alter protein content of bacterial cells and trigger a complex stress-like response, which nature we still do not fully understand (Kaberdina et al., 2009, Muller et al., 2016, Lange et al., 2017).

Chaudhuri and colleagues totally disregard four decades of kasugamycin research, apart from (van Buul et al., 1984) and a structural study by (Schurwirth et al., 2006). Did the authors ignore the kasugamycin studies that disagree with kasugamycin as a specific inhibitor of mistranslation? Or is it due to ignorance about the major object of their study?

We thank the reviewer for these important observations. Aminoglycosides are familiar as a chemical group to both TB biologists (the first anti-TB medication was streptomycin) and to translation biologists (as translation inhibitors), the two groups most likely to have interest in our findings. We made reference to both these general properties of aminoglycosides, including kasugamycin and streptomycin in our manuscript. We chose to emphasise kasugamycin’s previously described role in reducing ribosomal misreading errors, since it was relevant to our rationale and this property may have been less familiar to most readers. As rightly pointed out, we apologise that we did not more fully cite some of the seminal prior work on kasugamycin’s other described activities in our manuscript. We have now expanded our paper and discussion of kasugamycin considerably, including discussion of some of these important papers (see Results section and Discussion section).

However, although these prior data (all performed in E. coli except for one crystal structure) do point to additional functions of kasugamycin, we believe that the rifampicin potentiation effects in particular, which is the central focus of our current study, are specifically and at least for the most part mediated by kasugamycin’s actions in reducing mistranslation due to the indirect pathway.

This is because:

a) In Figure 1—figure supplement 5 of the original manuscript (now Figure 2E), we utilised a strain we had previously described (in Su et al., 2016), M. smegmatis-RpoB-N434T (N434T). This strain has a single point mutation in the rpoB gene, altering an asparagine residue critical for rifampicin binding, to threonine. Aspartate, but not asparagine or threonine at residue 434 disrupts rifampicin binding to RNAP, and in our prior published work we showed by use of this strain and other genetic tools that mistranslation of this ASN residue in wild-type mycobacteria to ASP resulted in significant rifampicin tolerance. The N434T variant of RpoB can still bind rifampicin, but the threonine residue can no longer be mistranslated by the indirect tRNA aminoacylation pathway to aspartate, by definition, since mistranslation via that pathway is specific and limited to aspartate for asparagine and glutamate for glutamine misincorporations. This strain is more susceptible to rifampicin killing but is relatively resistant to kasugamycin potentiation of rifampicin.

The reported alternative actions of kasugamycin (e.g. on specific inhibition of translation of canonical but not leaderless mRNAs, non-specific stress responses etc) would not be altered by this single amino acid substitution. These data strongly argue that the rifampicin potentiation effects of kasugamycin, for the most part, are mediated by inhibition of mistranslation of this residue, which physiologically occurs in mycobacteria, due to the indirect tRNA aminoacylation pathway.

b) Furthermore, all of the effects in M. smegmatis and M. tuberculosis on potentiation of rifampicin in vitro occur at concentrations of kasugamycin between 30-150 µg/ml, when kasugamycin has no anti-bacterial activity in vitro. In data that I share below, but which was not included in the originally submitted manuscript, we tested two further activities of kasugamycin in M. smegmatis and failed to find evidence of either, even up to concentrations of 1500-2000 µg/ml kasugamycin.

Moll and colleagues and others (Moll et al., 2002, Kaberdina et al., 2009 and others) described that kasugamycin specifically inhibited translation of canonical mRNAs, but not leaderless transcripts. Of note, a large proportion of mycobacterial transcripts are leaderless. To investigate whether kasugamycin could inhibit canonical but not leaderless translation in mycobacteria, we constructed an M. smegmatis strain expressing GFP and mCherry from the same basic promoter (Psmyc), but GFP from a canonical version of the promoter and mCherry from a leaderless version of the promoter. 5’RACE verified the transcription start sites of the two promoters as described. At 1500 µg/ml kasugamycin, fully 10-50 times the concentration required to potentiate rifampicin or to decrease mistranslation, kasugamycin only slightly slowed bacterial growth, and failed to inhibit translation of either fluorescent protein (See Author response image 1 and Figure 1—figure supplement 2 in the revised manuscript).

Author response image 1
Kasugamycin does not significantly inhibit translation of a canonical or leaderless transcript in M. smegmatis at doses much higher than required to reduce mistranslation.

A strain of M. smegmatis was transformed with an episomal plasmid expressing gfp from the promoter Psmyc (canonical promoter with 5’ UTR) and a leaderless version of Psmyc driving mCherry. Chloramphenicol inhibited translation of both transcripts, whereas kasugamycin at 1500 µg/ml failed to significantly attenuate translation from either transcript.

https://doi.org/10.7554/eLife.36782.021

In Kaberdina et al., 2009 Moll and colleagues showed that kasugamycin treatment in vitro resulted in intriguing 61S alternate ribosomes. We investigated whether we could identify such specialised ribosomes in M. smegmatis, and despite numerous attempts and different kasugamycin concentrations and time points, failed to do so (see Author response image 2 as a representative example), suggesting that mycobacteria may not form these specialised ribosomal structures.

Author response image 2
Kasugamycin does not cause formation of 61S mycobacterial ribosomes.

Ribosomal profiles of M. smegmatis ribosomes generated by sucrose gradient density centrifugation +/- kasugamycin treatment prior to isolation.

https://doi.org/10.7554/eLife.36782.022

Whilst we are confident that the rifampicin potentiation effects of kasugamycin are due to its effects on mistranslation for the reasons cited in (a) above, we cannot 100% exclude other mechanisms mediating the kasugamycin only effect, which was only witnessed in vivo and is not the main focus of this paper. Although we have no evidence for these alternative mechanisms at play in mycobacteria, absence of evidence does not fully imply evidence of absence, therefore we have extended our discussion of the kasugamycin only phenotype to include the possibility that these other mechanisms may potentially play a role.

Secondly, the major conclusion of the manuscript – that kasugamycin inhibits M. tuberculosis by reducing Asn-to-Asp or Gln-to-Glu mistranslation – is not justified by their experimental data. I found no proof that kasugamycin specifically alters Asn-to-Asp or Gln-to-Glu mistranslation, because the read-through assay used in this study is not capable to discriminate Asn-to-Asp or Gln-to-Glu mistranslation from overall changes in the accuracy of protein synthesis. To prove that a small molecule alters one specific type of mistranslation (e.g. Asn-to-Asp and Gln-to-Glu but not other types of mistranslation) people typically use quantitative mass-spectrometry (for instance, Cvetesic et al., 2016).

To address this issue, which is, as the reviewer rightly surmises, central to the arguments of the manuscript, we would ask that the reviewer re-examines the data that we resummarise below, which were all included in the original submission.

a) First, we made no claims that kasugamycin only decreases mistranslation due to the indirect tRNA aminoacylation pathway and apologise if this was not communicated clearly enough. In fact, we cited the 1984 van Buul et al., paper to specifically address that kasugamycin had been previously implicated in decreasing errors in ribosomal misreading. However, no previous studies (to our knowledge) have ever examined the effects of kasugamycin in decreasing errors in the indirect tRNA aminoacylation pathway, which as the Reviewer mentions, is via potentially an entirely different mechanism.

b) To specifically address whether kasugamycin decreases mistranslation via the indirect tRNA aminoacylation pathway (a central claim of the paper), we used reporters that measure misincorporation (not readthrough) of asparagine to aspartate (or glutamate for glutamine in one of the Ren-FF reporters). A dual Renilla-Firefly luciferase reporter system has been characterised in detail in prior publications (Javid et al., 2014 and Su et al., 2016). The Nluc/GFP reporter is used for the first time here. Both sets of reporters work via the same principle. Both Nluc and Renilla luciferases have aspartate residues (D140 in Nluc, and D120 in Renilla) critical for enzyme activity. Mutation of these residues to asparagine results in 2-3 orders of magnitude loss of function. Substitution of the coded asparagine (AAC codon) for aspartate during translation by translational error would result in gain in function of the mistranslated subpopulation of luciferase enzyme, which can be sensitively detected. The reporter by itself cannot formally distinguish whether substitution of asparagine for aspartate is via misincorporation of misacylated tRNA (Asp-tRNAAsn) or ribosomal misreading of AAC codon for GAC (Asp) – although we have previously demonstrated (in Leng et al., 2015), that codon position 1 ribosomal misreading errors in mycobacteria occur at far lower frequencies than the aspartate for asparagine substitution error rates shown here and in Su et al. 2016. It should be noted that any other detection method of amino acid substitution (e.g. the suggested mass spectrometry route) would not be able to distinguish the source of mistranslation either. However, we have two strong lines of evidence that together unequivocally suggest that kasugamycin is able to decrease mistranslation of the indirect pathway i.e. misincorporation of misacylated tRNA (AsptRNAAsn) at AAC codons:

i) In Su et al., 2016 we characterised strains of M. smegmatis with partial loss of function in GatCAB (the key enzyme in the indirect pathway) due to mutations in gatA. In that work we showed using gatA mutations on an otherwise isogenic background, as well as genetic complementation, that mutations in gatA were sufficient for significantly increasing mistranslation of aspartate for asparagine/ glutamate for glutamine, and that in the mutant strains, the defect in translational fidelity is specifically in the indirect tRNA aminoacylation pathway (see Figure 1 and Figure 1—figure supplement 2 in that work).

In this work, we use kasugamycin to measure the change in mistranslation rate in strain HWS.4 (gatA-V405D), which, as described above, has an increased mistranslation rate solely due to a defect in the indirect tRNA aminoacylation pathway. We show that kasugamycin can significantly reduce the mistranslation rate in this strain by a greater degree than the total mistranslation rate of the corresponding parent strain (in absolute terms, by > 4%/ codon, which is higher than the total mistranslation rate in the wild-type mycobacterial strain under the same conditions). It should be noted that in other data from Su et al., 2016, we showed that even in wild-type mycobacteria, the major source of this specific type of error is due to the indirect tRNA aminoacylation pathway, which provided the rationale for using kasugamycin to target mistranslation in wild-type M. tuberculosis. See Figure 1D of the revised manuscript. Our interpretation of these data, are that the reduction in mistranslation as measured by the reporter, must at least partly (and we would argue for the most part) be due to reduction in mistranslation due to the indirect tRNA aminoacylation pathway.

ii) The hybrid cell-free translation system provides the strongest evidence that kasugamycin can decrease error that is generated from misacylated Asp-tRNAAsn (Figure 1D in the original manuscript now 1E). This E. coli translation systems lacks the indirect pathway. The corrected Nluc activity (as measured by Nluc/ GFP – and a sensitive measure of misincorporation, by any mechanism, of aspartate for asparagine) is low but measurable (since the abrogation of Nluc activity in the D140N substitution results in 100-fold loss of activity, the high potency of the Nluc enzyme means that residual activity can still be measured sensitively). A non-discriminatory aspartyl synthetase specifically misacylates tRNAAsn to Asp-tRNAAsn, with no opportunity for correction due to lack of GatCAB in the system, and addition of this enzyme to the cell-free translation system increases the reporter measured Nluc activity (i.e. mistranslation error rate) three-fold. The only source of increased error in this system is misacylated Asp-tRNAAsn. Kasugamycin, at doses that do not inhibit translation of GFP in the Nluc-GFP fusion protein, specifically decrease the measured gain in Nluc activity in a dose-dependent manner to baseline. These data strongly and unequivocally argue that kasugamycin is capable of increasing fidelity against mistranslation generated by the indirect tRNA aminoacylation pathway.

Although mass-spectrometry is sensitive at detecting many misincorporations, the detection of aspartate for asparagine or glutamate for glutamine pose specific technical challenges. First, the mass shift of these two mistranslation events is 1 Dalton, which makes it difficult to distinguish mistranslation events of a small (typically 1%) subpopulation of otherwise identical peptides from the naturally occurring isotope envelopes in the spectra. Secondly, and more importantly, sample preparation for mass spectrometry routinely encounters high (5%+/ asparagine or glutamine residue) post-lysis deamidation (see e.g. PMID 1678690) which is identical to the mistranslation that occurs in the indirect pathway. As such, measurement of physiological mistranslation due to the indirect pathway is technically challenging by mass spectrometry and at any rate, as mentioned above, detection of aspartate for asparagine substitutions by mass spectrometry would not be able to infer the molecular mechanistic source of the substitution. We agree that mass spectrometry would be able to detect other potentially easier to detect substitutions, but we have not argued that kasugamycin solely increases discrimination of errors from the indirect pathway. We have argued that kasugamycin’s activity in reducing error in this pathway is responsible for the potentiation of rifampicin killing for the reasons outlined in point (1a) above.

We stand by our claim that our data supports the interpretation that kasugamycin can increase discrimination of misacylated tRNAs generated by the indirect pathway. We do not claim that kasugamycin does so exclusively and does not increase fidelity of translation due to ribosomal misreading, and we have added a note in the Discussion to address this point specifically.

Thirdly, I found several overstatements. For instance, the idea that "reducing mistranslation may be a novel mechanism for targeting bacterial adaptation" (the last sentence of the abstract) is by no means novel. It was pronounced in numerous papers by Paul Schimmel, Mike Ibba, Susan Martinis and others, including Babak Javid's group (e.g. Su et al., 2016)

Thank you for your comment. Of course, and as clearly stated throughout the manuscript, the rationale for identifying a small molecule that targets bacterial mistranslation was provided by both our own prior work (specifically in the mycobacterial system) as well as the work of Ibba, Schimmel, Martinis and others in other bacterial systems (all cited and discussed in our prior review, which we cite in the manuscript: Ribas de Pouplana et al., 2014). However, to our knowledge, all prior work had been genetic validation of the principles of “adaptive mistranslation”. We therefore believe the discovery that bacterial adaptive mistranslation is amenable to pharmacological targeting is both novel, and an important proof of principle, and therefore worth emphasising, in the hope that our work spurs further research in this area. We have, however, changed the wording to “pharmacologically reducing mistranslation may be a novel mechanism for targeting bacterial adaptation” in order to be explicit in our intention.

[Editors' note: the author responses to the re-review follow.]

Based on the new evaluations, your article will be considered for publication in eLife. However, the issues raised by the new reviewers need to be addressed, as outlined below. Please address these comments as well as the comments of the original reviewers in a revised version of your manuscript. (We realize this is a little non-standard for eLife but your manuscript has taken an unusual path.)

I would like to thank you as Senior Editor, and Dr. Madhukar Pai as Reviewing Editor of our recently submitted manuscript to eLife entitled “Kasugamycin potentiates rifampicin and limits emergence of resistance in Mycobacterium tuberculosis by specifically decreasing mycobacterial mistranslation”. We appreciate the opportunity to revise our manuscript in light of comments from additional reviewers following the initial resubmission.

Reviewer #5:

The revised manuscript by Chaudhuri et al. describes a synergistic role of kasugamycin with rifampicin to inhibit mycobacteria by reducing mistranslation. I feel this work could potentially be an important contribution to understanding the role of translational fidelity in bacteria-host interactions as well as developing a combinatory treatment for mycobacteria infections.

We thank the reviewer for their assessment of our work as making a potentially important contribution in understanding the role of translational fidelity in host-pathogen interactions. With regards to specific comments raised:

The major criticism of the previous submission comes from reviewer #4, and I share some of the same concerns. The authors convinced me that kasugamycin decreases the level of mistranslation in mycoplasma, which is supported by the in vivo and in vitro experiments using split and fused reporters. The challenge is to demonstrate that the potentiating effect of kasugamycin is due to reducing mistranslation. The results of the mutant strains with mutations in gatA and rpoB genes (Figures 1D, 2C, and 2E) are particularly interesting and provide support for their conclusion. However, tolerance to antibiotics is very complicated and not fully understood. The use of an additional antibiotic or introducing a mutation in the RNA polymerase may change cellular responses (e.g., toxin/antitoxin levels or efflux) that lead to altered sensitivity to rifampicin. As the authors also rightfully admitted, other effects of kasugamycin cannot be ruled out. Kasugamycin inhibits translation initiation. I feel it would strengthen their conclusion by using other non-aminoglycoside antibiotics that inhibit initiation (e.g., thermorubin) and other steps of translation (Wilson, 2014) in the potentiating assays (Figure 2A). This would reveal if inhibiting translation in general has synergistic effects with rifampicin to limit the growth of mycobacteria. In this manuscript, the authors used streptomycin as a control, which enhances mistranslation itself and therefore is not a proper control for general translation inhibition.

Thank you for these comments. There are four well-characterised 30S translation initiation inhibitors in the literature (as in e.g. the Wilson review, cited above), being: edeine, thermorubin, pactamycin and kasugamycin. Of these, only kasugamycin is currently commercially available. We were unable to identify commercial sources of any of the other three inhibitors, in China, the US or the UK, and none of the 6 or so chemistry CROs that we approached in China were willing or able to synthesise any of the three reagents for us. Nonetheless, we eventually identified Prof. Ian Brierley at the University of Cambridge, who has published on edeine in cell-free translation systems. He had purchased a batch of edeine >> 5 years ago when it was still commercially available, and he generously provided us with a small quantity of the reagent for evaluation. We have now tested Edeine, as well as chloramphenicol, an inhibitor of peptide bond formation in both the mistranslation assay (Figure 1—figure supplement 3) and rifampicin potentiating assay (Figure 2—figure supplement 2). Intriguingly, at subMIC concentrations, Edeine also decreases mistranslation rates and potentiates rifampicin, but chloramphenicol does not. These data suggest that inhibitors of 30S initiation, but not other translation inhibitors may increase ribosomal discrimination of misacylated Asp-tRNAAsn and Glu-tRNAGln during translation. We have now added a note in the Discussion section regarding these observations.

https://doi.org/10.7554/eLife.36782.024

Article and author information

Author details

  1. Swarnava Chaudhuri

    Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine, Beijing, China
    Contribution
    Formal analysis, Investigation, Methodology, Writing—original draft
    Contributed equally with
    Liping Li
    Competing interests
    No competing interests declared
  2. Liping Li

    Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, United States
    Contribution
    Investigation, Methodology
    Contributed equally with
    Swarnava Chaudhuri
    Competing interests
    No competing interests declared
  3. Matthew Zimmerman

    Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  4. Yuemeng Chen

    Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine, Beijing, China
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  5. Yu-Xiang Chen

    Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine, Beijing, China
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  6. Melody N Toosky

    1. Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine, Beijing, China
    2. Department of Immunology and Infectious Diseases, Harvard TH Chan School of Public Health, Boston, United States
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  7. Michelle Gardner

    Department of Immunology and Infectious Diseases, Harvard TH Chan School of Public Health, Boston, United States
    Contribution
    Investigation
    Competing interests
    No competing interests declared
  8. Miaomiao Pan

    Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine, Beijing, China
    Contribution
    Investigation, Methodology
    Competing interests
    No competing interests declared
  9. Yang-Yang Li

    Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine, Beijing, China
    Contribution
    Resources
    Competing interests
    No competing interests declared
  10. Qingwen Kawaji

    Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine, Beijing, China
    Contribution
    Methodology
    Competing interests
    No competing interests declared
  11. Jun-Hao Zhu

    Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine, Beijing, China
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  12. Hong-Wei Su

    Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine, Beijing, China
    Contribution
    Resources
    Competing interests
    No competing interests declared
  13. Amanda J Martinot

    Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, United States
    Contribution
    Formal analysis
    Competing interests
    No competing interests declared
  14. Eric J Rubin

    Department of Immunology and Infectious Diseases, Harvard TH Chan School of Public Health, Boston, United States
    Contribution
    Formal analysis, Supervision, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5120-962X
  15. Veronique Anne Dartois

    Public Health Research Institute, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, United States
    Contribution
    Formal analysis, Supervision, Methodology, Project administration, Writing—review and editing
    For correspondence
    veronique.dartois@rutgers.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9470-5009
  16. Babak Javid

    1. Centre for Global Health and Infectious Diseases, Collaborative Innovation Centre for the Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Medicine, Beijing, China
    2. Department of Immunology and Infectious Diseases, Harvard TH Chan School of Public Health, Boston, United States
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing, Conceived the study
    For correspondence
    bjavid@gmail.com
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6354-6305

Funding

Bill and Melinda Gates Foundation (OPP1109789)

  • Babak Javid

Wellcome (207487/B/17/Z)

  • Babak Javid

National Natural Science Foundation of China (31570129)

  • Babak Javid

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was in part funded by grants from the Bill and Melinda Gates Foundation (OPP1109789) and from the National Natural Science Foundation of China (31570129) to BJ. BJ is an Investigator of the Wellcome Trust (207487/B/17/Z). The Edeine was generously provided as a kind gift by Prof. Ian Brierley, University of Cambridge. We thank Martin Gengenbacher for helpful discussion regarding analysis of the data and Jiazi Wang, Jansy Sarathy and Jiye Yin for technical assistance.

Ethics

Animal experimentation: All mouse infection and treatment experiments were approved by the Institutional Animal Care and Use committee of Rutgers University and mouse toxicity studies were approved by the Institutional Animal Care and Use Committee of Tsinghua University under protocol number 17-BJ2.

Senior Editor

  1. Gisela Storz, National Institute of Child Health and Human Development, United States

Reviewing Editor

  1. Madhukar Pai, McGill University, Canada

Reviewers

  1. Madhukar Pai, McGill University, Canada
  2. Andreas Diacon, Stellenbosch University, South Africa

Publication history

  1. Received: March 19, 2018
  2. Accepted: August 27, 2018
  3. Accepted Manuscript published: August 28, 2018 (version 1)
  4. Version of Record published: September 27, 2018 (version 2)

Copyright

© 2018, Chaudhuri et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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