bioRxiv preprint doi: https://doi.org/10.1101/2020.08.26.267864; this version posted August 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Modulation of the cAMP levels with a conserved actinobacteria phosphodiesterase 2 enzyme reduces antimicrobial tolerance in mycobacteria 3 4 Michael Thomson1, Kanokkan Nunta1, Ashleigh Cheyne1, Yi liu1, Acely Garza-Garcia2 and 5 Gerald Larrouy-Maumus1† 6 7 1MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, 8 Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, UK 9 2The Francis Crick Institute, London NW1 1AT, United Kingdom. 10 11 †Corresponding author: 12 13 Dr Gerald Larrouy-Maumus 14 MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Faculty 15 of Natural Sciences, Imperial College London, London, SW7 2AZ, UK 16 Telephone: +44 (0) 2075 947463 17 E-mail: [email protected] 18 19 20 1 e ag P 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.26.267864; this version posted August 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 21 Abstract 22 Antimicrobial tolerance (AMT) is the gateway to the development of antimicrobial resistance 23 (AMR) and is therefore a major issue that needs to be addressed. 24 The second messenger cyclic-AMP (cAMP), which is conserved across all taxa, is involved 25 in propagating signals from environmental stimuli and converting these into a response. In 26 bacteria, such as M. tuberculosis, P. aeruginosa, V. cholerae and B. pertussis, cAMP has 27 been implicated in virulence, metabolic regulation and gene expression. However, cAMP 28 signalling in mycobacteria is particularly complex due to the redundancy of adenylate 29 cyclases, which are enzymes that catalyse the formation of cAMP from ATP, and the poor 30 activity of the only known phosphodiesterase (PDE) enzyme, which degrades cAMP into 5’- 31 AMP. 32 Based on these two features, the modulation of this system with the aim of investigating 33 cAMP signalling and its involvement in AMT in mycobacteria id difficult. 34 To address this pressing need, we identified a new cAMP-degrading phosphodiesterase 35 enzyme (Rv1339) and used it to significantly decrease the intrabacterial levels of cAMP in 36 mycobacteria. This analysis revealed that this enzyme increased the antimicrobial 37 susceptibility of M. smegmatis mc2155. Using a combination of metabolomics, RNA- 38 sequencing, antimicrobial susceptibility assays and bioenergetics analysis, we were able to 39 characterize the molecular mechanism underlying this increased susceptibility. 40 This work represents an important milestone showing that the targeting of cAMP signalling is 41 a promising new avenue for antimicrobial development and expands our understanding of 42 cAMP signalling in mycobacteria. 43 2 e ag P 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.26.267864; this version posted August 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 44 INTRODUCTION 45 Along with climate change and viral pandemics, antimicrobial resistance (AMR) is currently 46 one of the greatest threats to human health. Antimicrobial tolerance (AMT) precedes 47 antimicrobial resistance1-3, and although AMR stems from heritable genetic changes in 48 bacteria, such as the expression of efflux pumps or antibiotic degrading enzymes, AMT is 49 mediated by transient and reversible physiological adaptations in signalling systems that 50 orchestrate the remodelling of bacterial physiology to alter their susceptibility to 51 antimicrobials. 52 Cyclic AMP signalling, which is one of the best studied signalling pathways in biology, has 53 been implicated in AMR4,5. Several earlier studies have linked cAMP signalling to antibiotic 54 resistance in gram-negative bacteria6,7. For example, Alper et al. demonstrated that 55 Salmonella typhimurium with mutations in the cAMP control system, which comprises 56 adenylyl cyclase and the cAMP receptor protein, lead to a partial resistance to 20 commonly 57 used antibiotics6. Moreover, Kary et al. reported that Salmonella typhimurium strains lacking 58 Crp (a cAMP-activated global transcriptional regulator) or with altered levels of cAMP 59 exhibit reduced susceptibility to fluoroquinolones as a result of decreased permeability and 60 increased efflux of this class of antibiotics7. 61 Despite the above-mentioned research on gram-negative pathogens, the link between cAMP 62 signalling and AMR/AMT in mycobacteria, including the strict human pathogen 63 Mycobacterium tuberculosis, the vaccine strain Mycobacterium bovis BCG and the model 64 organism Mycobacterium smegmatis, has not been well studied. This lack of knowledge can 65 be explained by the complexity of the cAMP signalling system in mycobacteria and the lack 66 of effective tools for manipulating this system8-12. In contrast to the model organism E. coli, 67 which comprises one enzyme that generates cAMP, adenylate cyclase (Cya), and one enzyme 68 that hydrolyses cAMP, a phosphodiesterase (PDE) (CpdA)13. Conversely, the M. tuberculosis 69 H37Rv genome encodes 16 adenylate cyclases belonging to the class-III adenylate cyclase 70 enzymes, which typically harbour a cAMP-producing domain linked to a sensory domain12,14. 71 Interestingly, only one PDE (denoted Rv0805) found in mycobacteria belonging to the Mtb 72 complex15-19, M. leprae, M. marinum and M. ulcerans, has been characterized even though 73 cAMP PDE activity has also been reported in the model organism M. smegmatis20, which 74 suggesting the presence of an additional PDE that is likely ubiquitous in the mycobacteria 3 75 genus. Furthermore, Rv0805 exhibits more activity towards 2’3’-cAMP, which arises from e ag P 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.26.267864; this version posted August 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 76 RNA degradation, than towards the actual second messenger19. The high redundancy of 77 cAMP-producing enzymes and the poor activity of the known PDE make it difficult to 78 modulate this system with the aim of investigating the involvement of cAMP signalling with 79 AMR/AMT in mycobacteria. 80 To tackle this challenge, we first searched for a tool that would allow us to efficiently 81 modulate intracellular cAMP levels. The current exogenous strategies for altering cAMP 82 levels, including the use of adenylate cyclase activator (forskolin) and analogues such as 83 dibutyryl-cAMP, only result in modest changes in the intracellular cAMP levels21,22. Instead, 84 we focused on finding the “missing” cAMP PDE using a combination of conventional 85 biochemical approaches and mass spectrometry techniques (proteomics and metabolomics). 86 With this approach, we identified Rv1339, an uncharacterized cAMP phosphodiesterase that 87 is present in all mycobacteria. Phylogenetic analyses showed that Rv1339 is a member of a 88 poorly characterized group of PDE enzymes with a metallo-β-lactamase fold that is present in 89 several bacterial phyla. This group is different but evolutionarily related to the typical class-II 90 PDEs. 91 To demonstrate that Rv1339 can be used as a tool to alter the intracellular cAMP pool in 92 mycobacteria and evaluate its impact on physiology, we used the model organism M. 93 smegmatis mc2155. The results showed that the expression of Rv1339 leads to a 3-fold 94 decrease in the intracellular cAMP levels and thus indicate that Rv1339 likely plays a role in 95 cAMP signalling in mycobacteria. 96 By examining the bacterial transcriptome, metabolome, bioenergetics and permeability, we 97 characterized the mechanisms through which Rv1339 expression results in increased 98 permeability and decreased AMT to two common antibiotics with different modes of action, 99 ethambutol and streptomycin. These findings suggest that cAMP signalling might be a 100 promising new target for the development of compounds that inhibit the AMT mechanisms of 101 critical bacterial pathogens. 102 RESULTS 103 Rv1339 is an atypical class-II PDE 104 To identify novel sources of cAMP PDE activity in mycobacteria, we first utilized an 105 unbiased biochemical approach involving the sequential fractionation of M. bovis BCG 4 106 ΔRv0805 lysate coupled to a cAMP PDE activity assay20,23. The enrichment of cAMP PDE e ag P 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.26.267864; this version posted August 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 107 activity was achieved through two chromatography steps that separate proteins based on their 108 charge and molecular weight (Supplementary Fig. 1). After each purification step, the active 109 factions were identified through the cAMP PDE assay and pooled for further purification. 110 Subsequently, trypsin digestion and proteomic analysis were used to identify the proteins 111 present in the final set of active fractions. Out of eight proteins that were uniquely present in 112 the active fractions, only three were uncharacterized proteins: Rv0250, Rv2568 and Rv1339. 113 Himar-1 transposon mutagenesis studies have revealed that the uncharacterized proteins 114 Rv0250 and Rv2568 are nonessential24. Rv1339 is a conserved hypothetical protein that is 115 ubiquitous in mycobacteria and is predicted to have hydrolase activity. Based on these 116 observations, we decided to focus on the characterization of Rv1339. 117 Literature mining and phylogenetic analyses revealed that Rv1339, YfhI from Bacillus 118 subtilis25 and CpdA from Corynebacterium glutamicum26, are representatives of a previously 119 unrecognized class of PDEs defined by the signature metal-binding motif [T/S]HXHXDH, 120 where X tends to be a hydrophobic, small or very small residue.