Identifying a novel mechanism of L-leucine uptake in Mycobacterium tuberculosis using a chemical genomic approach

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.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

n 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:

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 observe interference of L-leucine, but not of pantothenate, uptake in mc2 6206 strain upon semapimod treatment. At present, we do not have any clue whether PDIM presents a barrier exclusively to the uptake of L-leucine. Further studies may shed a light on underlying mechanism(s) by which L-leucine uptake is modulated by this small molecule.

We still do not know what PpsB actually does for amino acid uptake - is it a transporter?

By BLI-Octet we do not find any interaction between L-leucine and PpsB. Therefore, we doubt that PpsB is a transporter of L-leucine.

Does semapimod binding affect its activity?

Our study suggests that semapimod treatment alters PDIM architecture which becomes restrictive to L-leucine. However, at present the exact mechanism is not clear. Further studies are required to thoroughly examine the effect of semapimod on Mtb PpsB activity and alterations in PDIM by mass spectrometry.

Does the auxotrophic Mtb have lower PDIM levels compared to wild-type Mtb?

As per the published report by Mulholland et al, and by vancomycin susceptibility phenotype in our study, both the strains appear to have comparable PDIM levels.

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.

Semapimod inhibits production of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6, which would indeed help pathogen establish chronic infection. However, a significant reduction in bacterial loads in lungs and spleen upon semapimod treatment despite inhibition of proinflammatory cytokines clearly indicates bacterial dependence on host-derived exogenous leucine during intracellular growth.

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.

PDIM is a virulence factor, and plays an important role in the intracellular survival of the TB pathogen. Mtb strains lacking PDIM are expected to show attenuated growth during infection, even without semapimod treatment. In such a case, it might be difficult to draw any conclusions about the effect of semapimod against PDIM(-) strains in vivo.

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.

Considering the fact that extensive subculturing may result in loss of PDIM, we avoided prolonged subculturing of bacteria. As presented in Fig. 6b, the WT bacteria retain PDIM. While performing the initial screening of drugs, we did not anticipate such phenotype, and hence bacteria were cultured in regular 7H9-OADS medium without propionate supplementation.

A comprehensive future study would help examining the effect of propionate on generation of semapimod resistant mutants in Mtb mc2 6206.

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.

While Leucine uptake involves specific transporters in other bacteria, such transport system is not known in Mtb. By screening small molecule inhibitors, we came across a molecule, semapimod, which selectively kills the leucine auxotroph (mc2 6206), but not the WT Mtb. To understand the underlying mechanism of differential susceptibility of the WT and auxotrophic strains to this molecule, we evaluated the effect of restoration of leuCD and panCD expression on susceptibility of the auxotrophic strain to semapimod. Interestingly, our results demonstrated that upon endogenous expression of leuCD genes, mc2 6206 strain becomes resistant to killing by semapimod. In contrast, no effect of panCD expression was observed on semapimod susceptibility of mc2 6206. These findings were further substantiated by gene expression analysis of semapimod treated mc2 6206, which exhibits differential regulation of a set of genes that are altered upon leucine depletion in Mtb as well as in other bacteria. Overall results thus provide first evidence of perturbation of L-leucine uptake by semapimod treatment of the leucine auxotroph.

To further gain mechanistic insights into the effect of semapimod on leucine uptake in Mtb, we generated the semapimod resistant strain which exhibits point mutation in 4 genes including ppsB. Interestingly, overexpression of wild-type ppsB, but not of other genes, restored susceptibility of the resistant bacteria to semapimod. Our observations that semapimod interacts with PpsB, and semapimod resistant strain accumulates mutation in PpsB resulting in loss of PDIM together support the involvement of cell-wall PDIM in regulation of L-leucine transport in Mtb.

As mentioned above, we anticipate that semapimod treatment brings about certain modifications in PDIM which becomes more restrictive to L-leucine. A comprehensive future study will be helpful to examine the effect of semapimod on Mtb physiology.

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.

We agree that semapimod is not an appropriate drug candidate against TB owing to its inhibitory effect on production of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 that help pathogen establish chronic infection. However, a significant reduction in bacterial loads in lungs and spleen upon semapimod treatment despite inhibition of proinflammatory cytokines clearly indicates bacterial dependence on host-derived exogenous leucine during intracellular growth. Therefore targeting L-leucine uptake can be a novel therapeutic strategy against TB.

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.

As mentioned above, overall results from this study provide first evidence of perturbation of L-leucine uptake by semapimod treatment of the leucine auxotroph. Our observations that semapimod interacts with PpsB, and semapimod resistant strain accumulates mutation in PpsB resulting in loss of PDIM together support the involvement of cell-wall PDIM in regulation of L-leucine transport in Mtb.

As hitherto mentioned, it appears that semapimod treatment brings about certain modifications in PDIM which becomes restrictive to L-leucine. Future studies are required to gain detailed mechanistic insights into the effect of semapimod on Mtb physiology.

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.

We thank the peer reviewer for this suggestion. We would be happy to analyse the effect of semapimod on the level of other amino acids including BCAA by mass spectrometry.

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.

We thank the peer reviewer for this suggestion. We would explore the possibility of analysing the effect of increasing concentrations of BCAAs on mc2 6206 susceptibility to semapimod.