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Review 2: "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 2: "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:

  • Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review: This manuscript is well written and describes a justified and well-thought-out set of experiments to explore Mycobacterium dxs as a target. The authors report depletion of dxs using CRISPRi inhibits growth of M. smegmatis, and to a greater extent, M. tuberculosis.  This is certainly the most likely explanation for their data (and would be consistent with previous reports) but the specificity of the growth inhibitory effect is not shown without doubt. This is because although the authors show with RT-PCR that the dxs transcript is depleted in M. smegmatis by 18 h, by this time growth is already inhibited and it is not shown using a negative control gene that the reduction in transcript is dxs-specific.  They also do not show that in their hands the ATC is without any inhibitory effect; this could have been done, for example, with a construct with a non-targeting gRNA.  Nonetheless, the partial rescue by menatetrenone does help build the case that the gRNA is on-target, at least for M. smegmatis.

The authors show that under the dxs knockdown condition M. smegmatis is sensitised to a mixture of rifamipicin, isoniazid and ethambutol.  The evidence for sensitisation to dxs inhibitors, low pH and oxidative stress is, however, less compelling.  This is because, by contrast with what is done in the case of the rifampicin, isoniazid and ethambutol, the experiments are not performed and/or the data not analysed in a way that considers the reduced growth caused by the knock-down.  Hence, whether sensitisation occurs is difficult to see, and I would argue that there is no difference for nitrosobenzene, with reducing pH, or oxidants.  There were also no statistical analyses performed to support differences reported in the text. For the tests of clomazone and nitrosobenzene it would have been better to show dose response curves with growth reported as a percentage of growth in the absence of inhibitor for each condition, or at least to do this for the single inhibitor concentrations shown.  A similar correction for differences in growth should also be done for the pH and oxidant data.

The manuscript additionally lacks details on the number of times each experiment was performed and what the means represent – i.e. it should be stated that means are averages from X independent experiments or if representative data are shown.  This is critical for understanding the reproducibility of their findings. As mentioned above, statistical tests to show whether there are differences between means, and to support statements made, have also not been performed.

One further limitation of this study is that M. smegmatis is quite a different bacterium to M. tuberculosis, and only one experiment is performed with M. tuberculosis, the organism an antituberculosis drug would need to be active against.  Ideally more experiments would be confirmed with M. tuberculosis, but the challenge associated with working with M. tuberculosis is recognised and we appreciate this may fall out of the scope of the present study.

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