Peer review process
Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, public reviews, and a provisional response from the authors.
Read more about eLife’s peer review process.Editors
- Reviewing EditorAmit SinghIndian Institute of Science, Bangalore, India
- Senior EditorBavesh KanaUniversity of the Witwatersrand, Johannesburg, South Africa
Reviewer #1 (Public review):
Summary:
In this manuscript, the authors used a leucine/pantothenate auxotrophic strain of Mtb to screen a library of FDA-approved compounds for their antimycobacterial activity and found significant antibacterial activity of the inhibitor semapimod. In addition to alterations in pathways, including amino acid and lipid metabolism and transcriptional machinery, the authors demonstrate that semapimod treatment targets leucine uptake in Mtb. The work presents an interesting connection between nutrient uptake and cell wall composition in mycobacteria.
Strengths:
(1) The link between the leucine uptake pathway and PDIM is interesting but has not been characterized mechanistically. The authors discuss that PDIM presents a barrier to the uptake of nutrients and shows binding of the drug with PpsB. However it is unclear why only the leucine uptake pathway was affected. We still do not know what PpsB actually does for amino acid uptake - is it a transporter? Does semapimod binding affect its activity? Does the auxotrophic Mtb have lower PDIM levels compared to wild-type Mtb?
(2) The authors show an interesting result where they observed antibacterial activity of semapimod against H37Rv only in vivo and not in vitro. Why do the authors think this is the basis of this observation? It is possible semapimod has an immunomodulatory effect on the host since leucine is an essential amino acid in mice. The authors could check pro-inflammatory cytokine levels in infected mouse lungs with and without drug treatment.
(3) The authors show that the semapimod-resistant auxotroph lacks PDIM. The conclusions would be further strengthened by including validations using PDIM mutants, including del-ppsB Mtb and other genes of the PDIM locus, whether in vivo this mutant would be more susceptible (or resistant) to semapimod treatment.
(4) Prolonged subculturing can introduce mutations in PDIM, which can be overcome by supplementing with propionate (Mullholland et al, Nat Microbiol, 2024). Did the authors also supplement their cultures with propionate? It would be interesting to see what mutations would result in Semr strains with propionate supplementation along with prolonged semapimod treatment.
Weaknesses:
I have summarized the limitations above in my comments. Overall, it would be helpful to provide more mechanistic details to study the connection between leucine uptake and PDIM.
Reviewer #2 (Public review):
Summary
This important study uncovers a novel mechanism for L-leucine uptake by M. tuberculosis and shows that targeting this pathway with 'Semapimod' interferes with bacterial metabolism and virulence. These results identify the leucine uptake pathway as a potential target to design new anti-tubercular therapy.
Strengths
The authors took numerous approaches to prove that L-leucine uptake of M. tuberculosis is an important physiological phenomenon and may be effectively targeted by 'Semapimod'. This study utilizes a series of experiments using a broad set of tools to justify how the leucine uptake pathway of M. tuberculosis may be targeted to design new anti-tubercular therapy.
Weaknesses
The study does not explain how L-leucine is taken up by M. tuberculosis, leaving the mechanism unclear. Even though 'Semapimod' binds to the PpsB protein, the relevant connection between changes in PDIM and amino acid transport remains incomplete. Also, the fact that the drug does not function on WT bacteria makes it a weak candidate to consider its usefulness for a therapeutic option.
Reviewer #3 (Public review):
Agarwal et al identified the small molecule semapimod from a chemical screen of repurposed drugs with specific antimycobacterial activity against a leucine-dependent strain of M. tuberculosis. To better understand the mechanism of action of this repurposed anti-inflammatory drug, the authors used RNA-seq to reveal a leucine-deficient transcriptomic signature from semapimod challenge. The authors then measured a decreased intracellular concentration of leucine after semapimod challenge, suggesting that semapimod disrupts leucine uptake as the primary mechanism of action. Unexpectedly, however, resistant mutants raised against semapimod had a mutation in the polyketide synthase gene ppsB that resulted in loss of PDIM synthesis. The authors believe growth inhibition is a consequence of decreased accumulation of leucine as a result of an impaired cell wall and a disrupted, unknown leucine transporter. This study highlights the importance of branched-chain amino acids for M. tuberculosis survival, and the chemical genetic interactions between semapimod and ppsB indicate that ppsB is a conditionally essential gene in a medium depleted of leucine.
The conclusions regarding the leucine and PDIM phenotypes are moderately supported by experimental data. The authors do not provide experimental evidence to support a specific link between leucine uptake and impaired PDIM production. Additional work is needed to support these claims and strengthen this mechanism of action.
(1) Since leucine uptake and PDIM synthesis are important concepts of the manuscript, experiments would benefit from exploring other BCAAs to know if the phenotypes observed are specific to leucine, and adding additional strains to the 2D TLC experiments to provide confidence in the absence of the PDIM band.
(2) The intriguing observation that wild-type H37Rv is resistant to semapimod but the leucine-auxotroph is sensitive should be further explored. If the authors are correct and semapimod does inhibit leucine uptake through a specific transporter or disrupted cell wall (PDIM synthesis), testing semapimod activity against the leucine-auxotroph in various concentrations of BCAAs could highlight the importance of intracellular leucine. H37Rv is still able to synthesize endogenous leucine and is able to circumvent the effect of semapimod.