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:
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?
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.
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?
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:
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.
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?
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.