Cyclic AMP is a critical mediator of intrinsic drug resistance and fatty acid metabolism in M. tuberculosis
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Evaluation Summary:
Bacteria living in stressful and fluctuating environments need to respond to changing conditions. Many species, including Mycobacterium tuberculosis, the causative agent of tuberculosis, use cAMP as a secondary messenger to sense and respond to specific stimuli. What distinguishes M. tuberculosis, is that its genome encodes for at least 15 adenylate cyclases, enzymes that synthesize cAMP from ATP. The authors characterized one specific adenylate cyclase, Rv3645, and demonstrate that it is the most significant contributor to cAMP levels and mediates fatty acid metabolism and antibiotic resistance. This manuscript will be of broad interest to readers in the field of tuberculosis drug discovery and bacterial metabolism.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
Cyclic AMP (cAMP) is a ubiquitous second messenger that transduces signals from cellular receptors to downstream effectors. Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, devotes a considerable amount of coding capacity to produce, sense, and degrade cAMP. Despite this fact, our understanding of how cAMP regulates Mtb physiology remains limited. Here, we took a genetic approach to investigate the function of the sole essential adenylate cyclase in Mtb H37Rv, Rv3645. We found that a lack of rv3645 resulted in increased sensitivity to numerous antibiotics by a mechanism independent of substantial increases in envelope permeability. We made the unexpected observation that rv3645 is conditionally essential for Mtb growth only in the presence of long-chain fatty acids, a host-relevant carbon source. A suppressor screen further identified mutations in the atypical cAMP phosphodiesterase rv1339 that suppress both fatty acid and drug sensitivity phenotypes in strains lacking rv3645 . Using mass spectrometry, we found that Rv3645 is the dominant source of cAMP under standard laboratory growth conditions, that cAMP production is the essential function of Rv3645 in the presence of long-chain fatty acids, and that reduced cAMP levels result in increased long-chain fatty acid uptake and metabolism and increased antibiotic susceptibility. Our work defines rv3645 and cAMP as central mediators of intrinsic multidrug resistance and fatty acid metabolism in Mtb and highlights the potential utility of small molecule modulators of cAMP signaling.
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Evaluation Summary:
Bacteria living in stressful and fluctuating environments need to respond to changing conditions. Many species, including Mycobacterium tuberculosis, the causative agent of tuberculosis, use cAMP as a secondary messenger to sense and respond to specific stimuli. What distinguishes M. tuberculosis, is that its genome encodes for at least 15 adenylate cyclases, enzymes that synthesize cAMP from ATP. The authors characterized one specific adenylate cyclase, Rv3645, and demonstrate that it is the most significant contributor to cAMP levels and mediates fatty acid metabolism and antibiotic resistance. This manuscript will be of broad interest to readers in the field of tuberculosis drug discovery and bacterial metabolism.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the …
Evaluation Summary:
Bacteria living in stressful and fluctuating environments need to respond to changing conditions. Many species, including Mycobacterium tuberculosis, the causative agent of tuberculosis, use cAMP as a secondary messenger to sense and respond to specific stimuli. What distinguishes M. tuberculosis, is that its genome encodes for at least 15 adenylate cyclases, enzymes that synthesize cAMP from ATP. The authors characterized one specific adenylate cyclase, Rv3645, and demonstrate that it is the most significant contributor to cAMP levels and mediates fatty acid metabolism and antibiotic resistance. This manuscript will be of broad interest to readers in the field of tuberculosis drug discovery and bacterial metabolism.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In addition to canonical bacterial signaling methods, two-component systems, and serine/threonine kinases, one of the most ubiquitous signal transduction modalities in M. tuberculosis is via adenylate cyclases. This study seeks to identify new adenylate cyclases of M. tuberculosis used to sense antibiotic treatment and resist its effects. To this end, authors employed cutting-edged techniques including genetic knock-out strategy, CRISPRi knock-down strategy, LC-MS-based target metabolite quantification, and various biochemical/microbiological methods. This study provides a conceptually novel strategy to kill M. tuberculosis with conventional tuberculosis chemotherapy.
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Reviewer #2 (Public Review):
The manuscript entitled" Cyclic AMP is a critical mediator of intrinsic drug resistance and fatty acid metabolism in M tuberculosis." is a well-written paper describing the previously unknown role of Rv3645. Using a CRISPRi screen the authors identify that the essential Rv3645 encoded adenylate cyclase causes susceptibility to several antibiotics when the gene is knocked down. Specifically, the authors found that Mtb is more susceptible to vancomycin, rifampicin, clarithromycin, bedaquiline, and meropenem but not other drugs such as isoniazid. The authors demonstrate that increased drug sensitivity in the Rv3645 knockdown strains is not due to large increases in envelope permeability by showing that fluorescently labelled vancomycin had only a modest increase in cellular uptake. Surprisingly, the authors …
Reviewer #2 (Public Review):
The manuscript entitled" Cyclic AMP is a critical mediator of intrinsic drug resistance and fatty acid metabolism in M tuberculosis." is a well-written paper describing the previously unknown role of Rv3645. Using a CRISPRi screen the authors identify that the essential Rv3645 encoded adenylate cyclase causes susceptibility to several antibiotics when the gene is knocked down. Specifically, the authors found that Mtb is more susceptible to vancomycin, rifampicin, clarithromycin, bedaquiline, and meropenem but not other drugs such as isoniazid. The authors demonstrate that increased drug sensitivity in the Rv3645 knockdown strains is not due to large increases in envelope permeability by showing that fluorescently labelled vancomycin had only a modest increase in cellular uptake. Surprisingly, the authors discover that Rv3645 essentiality is conditional to the presence of long-chain fatty acids. The authors were able to delete the Rv3645 strain if the Mtb strain is grown on a glucose-based media in the absence of oleic acid or palmitic acid. Moreover, the authors perform a CRISPR suppressor screen and identify that the loss of Rv1339 (Cyclic AMP phosphodiesterase) suppresses the fatty acid-sensitive phenotype. I thought the work is carefully performed and the conclusions are valid. It will be interesting to see what happens with these mutants in animal infection models. In the discussion, the authors describe the implications for other bacteria very well.
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Reviewer #3 (Public Review):
In a previous study, the authors screened a genome-wide CRISPRi library for sensitivity to a panel of antibiotics. One of the hits on this screen was found to be an essential adenylate cyclase, Rv3645. Rv3645 is a multidomain adenylate cyclase (AC), membrane-associated, and carries a HAMP domain (often associated with two-component signal transduction pathways). Surprisingly, Rv3645 was the only AC exhibiting this broad sensitivity to antibiotics. These observations were validated using a knock-down strategy and were also shown to be complemented by expressing a CRISPRi-resistant allele. To confirm that the sensitivity is not due to weakened cell walls or increased permeability of the cell to antibiotics, they measured the uptake of vancomycin using fluorescently conjugated vancomycin and by mass-spec. …
Reviewer #3 (Public Review):
In a previous study, the authors screened a genome-wide CRISPRi library for sensitivity to a panel of antibiotics. One of the hits on this screen was found to be an essential adenylate cyclase, Rv3645. Rv3645 is a multidomain adenylate cyclase (AC), membrane-associated, and carries a HAMP domain (often associated with two-component signal transduction pathways). Surprisingly, Rv3645 was the only AC exhibiting this broad sensitivity to antibiotics. These observations were validated using a knock-down strategy and were also shown to be complemented by expressing a CRISPRi-resistant allele. To confirm that the sensitivity is not due to weakened cell walls or increased permeability of the cell to antibiotics, they measured the uptake of vancomycin using fluorescently conjugated vancomycin and by mass-spec. Interestingly, the essentiality and drug sensitivity of rv3645KD was found to be dependent on long-chain fatty acids. When Mtb was cultured in absence of fatty acids, rv3645 was no longer essential which allowed them to construct an rv3645 deletion strain. To determine the role of AC in lipid metabolism, the authors carried out a suppressor screen to identify mutants that reversed the fatty-acid phenotype. Mutants were identified in fatty acid transporter genes and in a cAMP phosphodiesterase gene, rv1339. The role of cAMP levels in mediating fatty acid metabolism and antibiotic resistance was further confirmed through the measurement of cAMP levels using mass spectrometry and expression of an enzymatically inactive mutant of rv3645.
Overall, this is a very elegant study that uses cutting-edge bacterial genetics to address the role of cAMP in mycobacterial pathogenesis. All the experiments have been well designed with the necessary controls and rigor. The studies clearly establish the role of cAMP, especially mediated through rv3645 in fatty acid metabolism and resistance against different classes of antibiotics. While the upstream signal is still unknown this can be for future follow-up studies.
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