Cyclic di-GMP regulates Mycobacterium tuberculosis resistance to ethionamide

Zhang, Hainan; Xu, Zhaowei; Jiang, He‐wei; Wu, Fanlin; He, Xiang; Liu, Yin; Guo, Shujuan; Li, Yang; Bi, Lijun; Deng, Jiao-Yu; Zhang, Xian-En; Tao, Sheng-Ce

doi:10.1038/s41598-017-06289-7

Cited by 30 publications

(25 citation statements)

References 55 publications

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“…Recently, we constructed a functional Mtb proteome microarray (with Ͼ95% coverage of the Mtb proteome) (38). The power of this Mtb proteome microarray has already been demonstrated in investigations of PPI (39) and small molecule/drug-protein binding (40).…”

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Systematic Identification of Mycobacterium tuberculosis Effectors Reveals that BfrB Suppresses Innate Immunity

Jiang

Chen

et al. 2017

Molecular & Cellular Proteomics

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(Mtb) has evolved multiple strategies to counter the human immune system. The effectors of Mtb play important roles in the interactions with the host. However, because of the lack of highly efficient strategies, there are only a handful of known Mtb effectors, thus hampering our understanding of Mtb pathogenesis. In this study, we probed Mtb proteome microarray with biotinylated whole-cell lysates of human macrophages, identifying 26 Mtb membrane proteins and secreted proteins that bind to macrophage proteins. Combining GST pull-down with mass spectroscopy then enabled the specific identification of all binders. We refer to this proteome microarray-based strategy as SOPHIE (Systematic unlOcking of Pathogen and Host Interacting Effectors). Detailed investigation of a novel effector identified here, the iron storage protein BfrB (Rv3841), revealed that BfrB inhibits NF-κB-dependent transcription through binding and reducing the nuclear abundance of the ribosomal protein S3 (RPS3), which is a functional subunit of NF- κB. The importance of this interaction was evidenced by the promotion of survival in macrophages of the mycobacteria, by overexpression of BfrB. Thus, beyond demonstrating the power of SOPHIE in the discovery of novel effectors of human pathogens, we expect that the set of Mtb effectors identified in this work will greatly facilitate the understanding of the pathogenesis of Mtb, possibly leading to additional potential molecular targets in the battle against tuberculosis.

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Systematic Identification of Mycobacterium tuberculosis Effectors Reveals that BfrB Suppresses Innate Immunity

Jiang

Chen

et al. 2017

Molecular & Cellular Proteomics

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“…This motif is located in the N-terminal tail of this protein that we found is responsible for its dimerization (Supplementary Figure 3) . Since many of the c-di-GMP binding proteins are dimers or tetramers 4, 31, 45 , a plausible explanation for this effect of c-di-GMP described here is that c-di-GMP interferes with the dimerization of CobB.…”

Section: Discussionmentioning

confidence: 77%

Protein Deacetylase CobB Interplays with c-di-GMP

Zhang

et al. 2018

Preprint

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45As a ubiquitous bacterial secondary messenger, c-di-GMP plays key 46 regulatory roles in processes such as bacterial motility and transcription 47 regulation. CobB is the Sir2 family protein deacetylase that controls 48 energy metabolism, chemotaxis and DNA supercoiling in many bacteria. 49 Using an E.coli proteome microarray, we found that c-di-GMP strongly 50 binds to CobB. Protein deacetylation assays showed that c-di-GMP 51 inhibits CobB activity and thereby modulates the biogenesis of 52 acetyl-CoA. Through mutagenesis studies, residues R8, R17 and E21 of 53 CobB were shown to be required for c-di-GMP binding. Next, we found 54 that CobB is an effective deacetylase of YdeH, a major diguanylate 55 cyclase (DGC) of E.coli that is endogenously acetylated. Mass 56 spectrometry analysis identified YdeH K4 as the major site of acetylation, 57 and it could be deacetylated by CobB. Interestingly, deacetylation of 58 YdeH enhances its stability and cyclase activity in c-di-GMP production. 59 Thus, our work establishes a novel negative feedback loop linking 60 c-di-GMP biogenesis and CobB-mediated protein deacetylation. 61 62 Key words 63 c-di-GMP; CobB; diguanylate cyclase; protein acetylation; negative 64 feedback loop 65 66 4 / 56 82 c-di-GMP is synthesized by diguanylate cyclases (DGCs) 10 and 83 degraded by specific phosphodiesterases (PDEs) 11-13 . In E.coli, the 84 dominant DGCs are YdeH (also known as DgcZ) 14 and DosC 15 , and the 85 major PDEs are YhjH 16 and DosP 15 . Each of these proteins has been 86shown to be modulated by "first" messengers such as light, oxygen and 87 temperature 2 . Yet, owing to the involvement of c-di-GMP in the 88 5 / 56 aforementioned fundamental biological processes that are all 89 well-regulated by many basic metabolic mechanisms 17, 18 , there is also 90 the possibility that c-di-GMP biosynthesis is also regulated via 91 metabolism-related intracellular signals. 92 The Sir2 family protein CobB is a NAD + -dependent deacetylase that is 93 highly conserved in prokaryotes 19, 20 . In E.coli, CobB is the sole Sir2 94 homolog, although there are two forms of CobB, namely, CobB and 95 CobB S , the former of which has an additional 37 aa N-terminal tail 21 . 96 CobB exhibits protein deacetylation activity and it regulates a variety of 97 physiological functions. For example, CobB deacetylates lysine-609 of 98 acetyl-coenzyme A synthetase (Acs) to activate its activity 22 , resulting in 99 an increased cellular concentration of acetyl-coenzyme A (acetyl-CoA), 100 which is a central component of energy metabolism. In addition, CobB 101 regulates E.coli chemotaxis by deacetylating the chemotaxis response 102 regulator protein (CheY) 23 , as well as the activity of N-hydroxyarylamine 103 O-acetyltransferase (NhoA) 24 and topoisomerase I (TopA) 25 . Yet, despite 104 the critical role that CobB plays in many biological processes, its inherent 105 regulation remains poorly understood 26-28 . 106 To globally identify c-di-GMP effectors and explore new functions of 107 c-di-GMP...

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“…Our characterization, supplemented with existing observations that PdtaR is constitutively expressed during macrophage infection (7), highly expressed during infection in SCID mice (37) and is a key secreted antigen and potential vaccine candidate (38) strengthens the case for the exploration of PdtaS-PdtaR inhibitors as a drug target. Development of c-di-GMP mimetics as PdtaS inhibitors also promise to affect a wider range of Mycobacterial functions such as lipid transport and metabolism (19), multi-drug resistance, biofilm formation, cell wall polar lipid and glycopeptidolipid content (20, 21, 39) and dormancy and survival under nutrient deprivation (22, 33), reducing the chances of drug resistance through target mutation. Overall, we have unravelled the details of PdtaS-PdtaR signalling, added to our knowledge of ligands for Mycobacterial SKs and uncovered a promising target for future anti-tubercular therapy.…”

Section: Discussionmentioning

confidence: 99%

The histidine kinase PdtaS is a cyclic di-GMP binding metabolic sensor that controls mycobacterial adaptation to nutrient deprivation

Hariharan

Thakur

Singh

et al. 2019

Preprint

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Cell signalling relies on second messengers to transduce signals from the sensory 16 apparatus to downstream components of the signalling pathway. In bacteria, one of the most 17 important and ubiquitous second messengers is the small molecule cyclic diguanosine 18 monophosphate (c-di-GMP). While the biosynthesis, degradation and regulatory pathways 19 controlled by c-di-GMP are well characterized, the mechanisms through which c-di-GMP 20 controls these processes is not completely understood. Here we present the first report of a c-21 di-GMP regulated sensor histidine kinase previously named PdtaS (Rv3220c), which binds to 22 c-di-GMP at sub-micromolar concentrations, subsequently perturbing signalling of the PdtaS-23 PdtaR (Rv1626) two component system. Aided by biochemical analysis, molecular docking 24 and structural modelling, we have characterized the binding site of c-di-GMP in the GAF 25 domain of PdtaS. We show that a pdtaS knockout in M. smegmatis is severely compromised in 26 growth on amino acid deficient media and exhibits global transcriptional dysregulation. 27Perturbation of the c-di-GMP-PdtaS-PdtaR axis results in a cascade of cellular changes 28 recorded by a multi-parametric systems approach of transcriptomics, unbiased metabolomics 29 and lipid analyses. 30One-sentence summary: The universal bacterial second messenger cyclic di-GMP controls 31 the mycobacterial nutrient stress response 32Among the less understood TCSs of Mtb is the highly conserved PdtaS-PdtaR TCS. Studies 48 have shown PdtaR to be essential for survival on medium containing cholesterol as the sole 49 carbon source (6) and intracellular growth during macrophage infection (7). Its cognate SK 50PdtaS has been implicated in controlling ribosomal protein composition and sensitivity to 51 ribosome targeting antibiotics, membrane transport inhibitors and respiratory chain antagonists 52in Mycobacterium smegmatis (8). However, neither the ligands that triggers the activation of 53 this TCS nor the adaptive responses that result from PdtaS-PdtaR signalling are known. 54In this study, we use a knockout of pdtaS in Mycobacterium smegmatis to identify the role of 55 this conserved TCS in mycobacterial physiology. Using comparative metabolomics and in 56 silico docking, we have identified c-di-GMP as a ligand for PdtaS. Furthermore, we show that 57PdtaS phosphorylation and integrity of the c-di-GMP binding site are essential for growth, lipid 58 homeostasis and transcriptional adaptation during nutrient deprivation. Finally, using protein-59 protein interaction network modelling of the transcriptomic data, we uncover a novel 60 interactome that connects diverse metabolic pathways in mycobacteria. 61 Results 62 PdtaS deficiency results in poor growth during nutrient deprivation 63In order to study the role of PdtaS and PdtaS-PdtaR signalling in the physiology of 64 mycobacteria, we deleted pdtaS (MSMEG_1918) in M. smegmatis using specialized 65 transduction as described previously (9) and verified the deletion using PCR and Southern blot 66...

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Cyclic di-GMP regulates Mycobacterium tuberculosis resistance to ethionamide

Cited by 30 publications

References 55 publications

Systematic Identification of Mycobacterium tuberculosis Effectors Reveals that BfrB Suppresses Innate Immunity

Systematic Identification of Mycobacterium tuberculosis Effectors Reveals that BfrB Suppresses Innate Immunity

Protein Deacetylase CobB Interplays with c-di-GMP

The histidine kinase PdtaS is a cyclic di-GMP binding metabolic sensor that controls mycobacterial adaptation to nutrient deprivation

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