Inactivation of Pseudomonas aeruginosa 2-alkyl-4-hydroxyquinoline-type quorum sensing signals and antibiotics by Rhodococcus erythropolis and Mycobacterium abscessus
Final Report Abstract
The opportunistic pathogen Pseudomonas aeruginosa produces bioactive 2-alkyl-4(1H)-quinolones (AQ) and 2-alkyl-4-hydroxyquinoline-N-oxides. Acting as a major quorum sensing signal molecule, 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) is involved in the regulation of virulence factor production. 2- Heptyl-1-hydroxyquinolin-4(1H)-one (aka 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO)) has antibiotic effects, acting as inhibitor of respiratory electron transfer. In preliminary studies, we had observed that some actinobacteria are capable of HQNO conversion and PQS degradation. Arthrobacter and Rhodococcus spp. as well as Staphylococcus aureus were found to introduce a hydroxyl group at C-3 of HQNO. In contrast, Bacillus spp. transform HQNO to the glucosylated derivative 2-heptyl-1-(β-D-glucopyranosydyl)-4-oxoquinoline, which is significantly less inhibitory on respiration and menaquinol oxidase activity than HQNO. Three (putative) UDP-glycosyltransferases of B. subtilis 168 tested, namely YjiC, YdhE and YojK, catalyzed HQNO glucosylation. The substrate flexibility of these three enzymes suggested a physiological role in natural toxin resistance of B. subtilis. M. abscessus as well as Mycolicibacterium fortuitum and M. smegmatis modify HQNO by methylation of the N-hydroxy group, which results in substantial quenching of the inhibitory activity on quinol oxidase and significantly reduces the metabolite’s effect on formation of reactive oxygen species. We identified the MAB_2834c protein of M. abscessusT as HQNO methyltransferase. Both MAB_2834c and its homologue from Mycobacterium tuberculosis H37v (the Rv0560c protein) catalyze the methylation of different (antimicrobial) natural compounds as well as drugs, suggesting a role in response to chemical stress. In order to understand the molecular basis of substrate preference and catalysis in an HQNO-MTase, the crystal structure of Rv0560c has been solved in cooperation with the lab of Prof. Einsle. In co-culture experiments of P. aeruginosa PAO1 and different M. abscessus strains, those mycobacterial strains harboring the aqdRABC gene cluster (coding for a pathway-specific transcriptional regulator, a hydrolase, a monooxygenase, and a PQS dioxygenase) reduced PQS levels in the coculture, tentatively suggesting that the aqd cluster confers a competitive advantage. The key enzyme for PQS degradation is AqdC, a PQS dioxygenase catalyzing quinolone ring cleavage. When added to P. aeruginosa cultures, the AqdC protein quenched alkylquinolone and pyocyanin production but induced an increase in elastase levels. Combining AqdC with the lactonase QsdA which inactivates N- acylhomoserine lactone signals additionally retarded HQNO production and quenched rhamnolipid production and also elastase, however, the effects were moderate. In order to get insight into the molecular basis of PQS binding by AqdC and to pave the way for structure-based protein engineering, we cooperated with Dr. SHJ Smits to solve the crystal structure of AqdC. AqdC is a member of the α/β-hydrolase fold superfamily. The protein is traversed by a bipartite tunnel, with a funnel-like entry leading to an elliptical substrate cavity where PQS positioning is mediated by a combination of hydrophobic interactions and hydrogen bonds. Kinetic data confirmed the strict requirement of the active-site base H246 for catalysis, and suggested that evolution of the canonical nucleophile/His/Asp catalytic triad of the hydrolases to an Ala/His/Asp triad is favorable for catalyzing dioxygenolytic PQS ring cleavage. Functional annotation of dioxygenases among proteins of the α/β-hydrolase fold superfamily is a challenge. We defined search criteria for the primarily motif-based identification of 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases among the α/β-hydrolase fold proteins. The search criteria were validated by the successful identification of new PQS dioxygenases. In cooperation with Prof. Janssen and Dr. Wijma (Groningen), we applied the “FRESCO” workflow, which combines computational prediction methods and molecular dynamics-based screening, to predict and engineer more thermostable AqdC variants, and investigated their stability and catalytic activity. The approach resulted in two more stable variants of AqdC with extended half-lifes, and moreover yielded new insights on activity-stability relationships of α/β-hydrolase fold enzymes.
Publications
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(2017) Chemical modification and detoxification of the Pseudomonas aeruginosa toxin 2-heptyl-4-hydroxyquinoline N-oxide by environmental and pathogenic bacteria. ACS Chem Biol 12(9):2305-2312
Thierbach S, Birmes FS, Letzel MC, Hennecke U, Fetzner S
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(2017) Mycobacterium abscessus subsp. abscessus is capable of degrading Pseudomonas aeruginosa quinolone signals. Front Microbiol 8:339
Birmes FS, Wolf T, Kohl TA, Rüger K, Bange F, Kalinowski J, Fetzner S
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(2019) Interference with Pseudomonas aeruginosa quorum sensing and virulence by the mycobacterial PQS dioxygenase AqdC in combination with the N- acylhomoserine lactone lactonase QsdA. Infect Immun 87(10):e00278-19
Birmes FS, Säring R, Hauke MC, Ritzmann NH, Drees SL, Daniel J, Treffon J, Liebau E, Kahl BC, Fetzner S
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(2019) Structural basis for recognition and ring-cleavage of the Pseudomonas quinolone signal (PQS) by AqdC, a mycobacterial dioxygenase of the α/β-hydrolase fold family. J Struct Biol 207(3):287-294
Wullich SC, Kobus S, Wienhold M, Hennecke U, Smits SHJ, Fetzner S
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(2020) An α/β-hydrolase fold subfamily comprising Pseudomonas quinolone signal-cleaving dioxygenases. Appl Environ Microbiol 86(9):e00279-20
Wullich SC, Arranz San Martín A, Fetzner S
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(2020) Efficient modification of the Pseudomonas aeruginosa toxin 2-heptyl-1-hydroxyquinolin-4-one by three Bacillus glycosyltransferases with broad substrate ranges. J Biotechnol 308:74-81
Thierbach S, Sartor P, Yücel O, Fetzner S
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(2020). Modification of the Pseudomonas aeruginosa toxin 2-heptyl-1-hydroxyquinolin-4(1H)-one and other secondary metabolites by methyltransferases from mycobacteria. FEBS J. 2020, Oct 16
Sartor P, Bock J, Hennecke U, Thierbach S, Fetzner S
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Stabilizing AqdC, a Pseudomonas quinolone signal-cleaving dioxygenase from mycobacteria, by FRESCO-based protein engineering. ChemBioChem 2020 Oct 14
Wullich SC, Wijma HJ, Janssen DB, Fetzner S
