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Review 1: "Validation of DXS as An Attractive Drug Target in Mycobacteria"

The reviewers found the evidence that DXS knockdown leads to growth inhibition in Mycobacterium compelling, but raised several concerns regarding the subsequent sensitization experiments.

Published onJan 15, 2025
Review 1: "Validation of DXS as An Attractive Drug Target in Mycobacteria"
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Validation of DXS as an attractive drug target in mycobacteria
Validation of DXS as an attractive drug target in mycobacteria
Description

Abstract A rapid emergence in the incidences of Tuberculosis (TB) drug resistance undermines efforts to eradicate the disease and strengthens calls for development of new drugs with novel mechanisms of action. In drug discovery, finding an attractive drug target is as important as finding a good drug candidate. Hence more efforts are made to identify, validate and prioritize drug targets in TB drug discovery. Here, using CRISPRi technology, we showed that dxs1 transcriptional knockdown attenuated growth of both Mycobacterium smegmatis and Mycobacterium tuberculosis cultures, and the effect was more profound in the latter. Chemical supplementation of the growth medium with 10 μM of isoprenoid pyrophosphates, thiamine and thiamine pyrophosphate failed to rescue growth of M. smegmatis cultures, while partial rescue was observed with addition of menatetrenone, a menaquinone derivative with four isoprenyl groups. Similarly, culture growth could not be rescued by the addition of prenol and isoprenol, which suggested the lack of isoprenoid salvage pathway in mycobacteria. Importantly, and in the context of drug discovery, dxs1 depleted mutants displayed four-fold more sensitive towards a mixture of isoniazid, rifampicin and ethambutol, suggesting that inhibitors of DXS enzyme or other MEP pathway enzymes could potentiate antimycobacterial effect of the first-line TB drugs. Additionally, dxs1 depletion increased growth retardation of the mutant in acidic pH and under oxidative stress, conditions that are encountered in activated macrophage compartments. Taken together, our results validated DXS as an attractive drug target that should be prioritized for developments on new antitubercular agents.

RR\ID Evidence Scale rating by reviewer:

Not informative. The flaws in the data and methods in this study are sufficiently serious that they do not substantially justify the claims made. It is not possible to say whether the results and conclusions would match that of the hypothetical ideal study. The study should not be considered as evidence by decision-makers.

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Review: This study validates dxs1 as an interesting target for drug development against M. tuberculosis (Mtb). Given the extensive previous work that has been done on the MEP pathway as a Mtb drug target (summarized a.o. in PMID: 29390176), the work adds little new information to the field in its current form. The study can also be improved methodologically.

Major suggestions:

  1. Although M. smegmatis (Msmeg) can indeed be used as a model organism for Mtb, work aimed at advancing drug development for Mtb specifically benefits from using Mtb itself. For example, the MIC of rifampicin varies ~100-fold between Mtb and Msmeg. The study would thus be much stronger if the authors had continued working with their Mtb CRISPRi strains. Have the authors validated any of the dxs1 phenotypes, especially the chemical-genetic profile, in Mtb?

  2. Figure 2B shows the effect of dxs1 KD on the metabolic profile of the bacteria and quantified isopentenyl pyrophosphate by mass spectrometry. The experiments would benefit from a (CRISPRi-) complementation strain to show the extend of rescue at this downstream level given the findings on lack of supplementation with products of the MEP pathway and other downstream metabolites.

  3. Figure 5 shows the MIC shift of individual first-line drugs and their combination on a Msmeg dxs1 KD strain compared to a no ATc control. The figure needs a +ATc, empty vector control in panel A and B. This will allow quantification of the MIC shift for individual drugs (info lacking at the moment). Additionally, the findings are not surprising as they have been reported previously as cited by the authors in the paper. Of note, the authors did not mention the findings Li & Poulton et al. in this context. Li & Poulton et al. showed that dxs1 KD sensitizes to rifampicin and isoniazid over a spectrum of CRISPRi KD strengths in Mtb (PMID: 35637331). dxs1 KD also strongly sensitizes Mtb to clarithromycin, bedaquiline and vancomycin. Have the authors validated these additional sensitivities in their single strain cultures?

  4. The authors claim dxs1 KD sensitizes to low pH and oxidative stress. However, from the data presented in figures 6 and 7, KD of dxs1 does not seem to impose an additional fitness cost in lower pH and nitric oxide & hydrogen peroxide other than the already existing growth defect imposed by dxs1 KD. Also, is the relative growth inhibition dose-dependent on lower pH or increasing KNO2 or H202 concentrations?

Minor suggestions:

  1. The sequences of the reverse CRISPRi oligos used in this study seem to be incomplete (see Table S1). For correct ligation into the CRISPRi backbone, the reverse oligo has to start with 5'-AAACN...-3'. See also PMID: 34235662 for details.

  2. The authors mention that multi-drug resistance reduces treatment success rate from 86 % to 63%. Could they please include a reference for those data. How does this change in light of the introduction of BPaL?

  3. Although phenotypically the effect of dxs1 KD is larger in Mtb than in Msmeg, the authors have not quantified KD at the RNA or protein level in the Mtb strains. It is thus hard to directly compare both species from a single guide KD strains and claim the effect of dxs1 depletion is more profound in Mtb.

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