The Structural Determinants Accounting for the Broad Substrate Specificity of the Quorum Quenching Lactonase GcL

Bergonzi, Céline; Schwab, Michael; Naik, Tikeswar; Elias, Mikael

doi:10.1002/cbic.201900024

Cited by 22 publications

(44 citation statements)

References 57 publications

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“…It is also possible that some of the observed changes are due to a different, unknown activity of these enzymes since both enzymes are known to be promiscuous. Indeed, both enzymes are known to proficiently hydrolyze various δand γ-lactones and to exhibit other low hydrolytic activities against phosphotriesters (both enzymes) and arylesters (SsoPox W263I) (Merone et al, 2005;Hiblot et al, 2013;Bergonzi et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

“…Although, it can also degrade C 4 HSL, SsoPox W263I is mainly active on 3-oxo-C 12 HSL while GcL can almost equally degrade both substrates. To take into account protein addition in the culture medium, SsoPox variant 5A8, which demonstrated no detectable activity on any AHL (Table 1) was used as a negative control, in the same amounts as active enzymes (Bergonzi et al, 2019).…”

Section: Lactonase Specificity On P Aeruginosa Ahlsmentioning

confidence: 99%

“…Among lactone-degrading enzymes, paraoxonases, metallo-β-lactamase like lactonases (MLL) and phosphotriesterase-like lactonases (PLL) are the main studied families (Elias and Tawfik, 2012). They share a common catalytic mechanism and their differences in AHL substrate preference mainly lie on how the acyl chain can be accommodated into the catalytic site (Elias and Tawfik, 2012;Bergonzi et al, 2019).…”

Section: Introductionmentioning

confidence: 99%

“…This lactonase was also shown to reduce mortality in a rat pneumonia model (Hraiech et al, 2014). The MLL GcL isolated from Parageobacillus caldoxylosilyticus which was recently characterized and exhibits broad AHL specificity was chosen as the second lactonase (Bergonzi et al, 2016(Bergonzi et al, , 2019Mahan et al, 2020). SsoPox W263I and GcL, which mainly differ in their ability to hydrolyze C 4 HSL, were used independently and in combination, to disrupt QS of P. aeruginosa PA14 and to investigate the role of lactonase specificity on phenotypes, gene expression and proteome regulation.…”

Section: Introductionmentioning

confidence: 99%

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Lactonase Specificity Is Key to Quorum Quenching in Pseudomonas aeruginosa

et al. 2020

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The human opportunistic pathogen Pseudomonas aeruginosa orchestrates the expression of many genes in a cell density-dependent manner by using quorum sensing (QS). Two acyl-homoserine lactones (AHLs) are involved in QS circuits and contribute to the regulation of virulence factors production, biofilm formation, and antimicrobial sensitivity. Disrupting QS, a strategy referred to as quorum quenching (QQ) can be achieved using exogenous AHL-degrading lactonases. However, the importance of enzyme specificity on quenching efficacy has been poorly investigated. Here, we used two lactonases both targeting the signal molecules N-(3-oxododecanoyl)-Lhomoserine lactone (3-oxo-C 12 HSL) and butyryl-homoserine lactone (C 4 HSL) albeit with different efficacies on C 4 HSL. Interestingly, both lactonases similarly decreased AHL concentrations and comparably impacted the expression of AHL-based QS genes. However, strong variations were observed in Pseudomonas Quinolone Signal (PQS) regulation depending on the lactonase used. Both lactonases were also found to decrease virulence factors production and biofilm formation in vitro, albeit with different efficiencies. Unexpectedly, only the lactonase with lower efficacy on C 4 HSL was able to inhibit P. aeruginosa pathogenicity in vivo in an amoeba infection model. Similarly, proteomic analysis revealed large variations in protein levels involved in antibiotic resistance, biofilm formation, virulence and diverse cellular mechanisms depending on the chosen lactonase. This global analysis provides evidences that QQ enzyme specificity has a significant impact on the modulation of QS-associated behavior in P. aeruginosa PA14.

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Section: Discussionmentioning

confidence: 99%

Section: Lactonase Specificity On P Aeruginosa Ahlsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Lactonase Specificity Is Key to Quorum Quenching in Pseudomonas aeruginosa

et al. 2020

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“…Here, we took advantage of the distinct substrate preference of the PLL, SsoPox (Hiblot et al, 2013), which prefers longer AHL molecules, and the MLL, GcL (Bergonzi et al, 2019), which exhibits very broad substrate specificity. We used these lactonases to study the effects of AHL signal disruption on P. aeruginosa clinical isolates from cystic fibrosis (CF) patients.…”

Section: Introductionmentioning

confidence: 99%

Effects of Signal Disruption Depends on the Substrate Preference of the Lactonase

et al. 2020

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Many bacteria produce and use extracellular signaling molecules such as acyl homoserine lactones (AHLs) to communicate and coordinate behavior in a cell-density dependent manner, via a communication system called quorum sensing (QS). This system regulates behaviors including but not limited to virulence and biofilm formation. We focused on Pseudomonas aeruginosa, a human opportunistic pathogen that is involved in acute and chronic lung infections and which disproportionately affects people with cystic fibrosis. P. aeruginosa infections are becoming increasingly challenging to treat with the spread of antibiotic resistance. Therefore, QS disruption approaches, known as quorum quenching, are appealing due to their potential to control the virulence of resistant strains. Interestingly, P. aeruginosa is known to simultaneously utilize two main QS circuits, one based on C4-AHL, the other with 3-oxo-C12-AHL. Here, we evaluated the effects of signal disruption on 39 cystic fibrosis clinical isolates of P. aeruginosa, including drug resistant strains. We used two enzymes capable of degrading AHLs, known as lactonases, with distinct substrate preference: one degrading 3-oxo-C12-AHL, the other degrading both C4-AHL and 3-oxo-C12-AHL. Two lactonases were used to determine the effects of signal disruption on the clinical isolates, and to evaluate the importance of the QS circuits by measuring effects on virulence factors (elastase, protease, and pyocyanin) and biofilm formation. Signal disruption results in at least one of these factors being inhibited for most isolates (92%). Virulence factor activity or production were inhibited by up to 100% and biofilm was inhibited by an average of 2.3 fold. Remarkably, the treatments led to distinct inhibition profiles of the isolates; the treatment with the lactonase degrading both signaling molecules resulted in a higher fraction of inhibited isolates (77% vs. 67%), and the simultaneous inhibition of more virulence factors per strain (2 vs. 1.5). This finding suggests that the lactonase AHL preference is key to its inhibitory spectrum and is an essential parameter to improve quorum quenching strategies.

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Engineering quorum quenching acylases with improved kinetic and biochemical properties

Sompiyachoke,

Elias

2024

Protein Science

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Many Gram‐negative bacteria use N‐acyl‐L‐homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum‐quenching enzymes, such as AHL acylases, chemically degrade these molecules which prevents signal reception by bacteria and inhibits undesirable biofilm‐related traits. These capabilities make acylases appealing candidates for controlling microbes, yet candidates with high activity levels and substrate specificity and that are capable of being formulated into materials are needed. In this work, we undertook engineering efforts against two AHL acylases, PvdQ and MacQ, to generate these improved properties using the Protein One‐Stop Shop Server. The engineering of acylases is complicated by low‐throughput enzymatic assays. Alleviating this challenge, we report a time‐course kinetic assay for AHL acylases that monitors the real‐time production of homoserine lactone. Using the assay, we identified variants of PvdQ that were significantly stabilized, with melting point increases of up to 13.2°C, which translated into high resistance against organic solvents and increased compatibility with material coatings. While the MacQ mutants were unexpectedly destabilized, they had considerably improved kinetic properties, with >10‐fold increases against N‐butyryl‐L‐homoserine lactone and N‐hexanoyl‐L‐homoserine lactone. Accordingly, these changes resulted in increased quenching abilities using a biosensor model and greater inhibition of virulence factor production of Pseudomonas aeruginosa PA14. While the crystal structure of one of the MacQ variants, M1, did not reveal obvious structural determinants explaining the observed changes in kinetics, it allowed for the capture of an acyl‐enzyme intermediate that confirms a previously hypothesized catalytic mechanism of AHL acylases.

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The Structural Determinants Accounting for the Broad Substrate Specificity of the Quorum Quenching Lactonase GcL

Cited by 22 publications

References 57 publications

Lactonase Specificity Is Key to Quorum Quenching in Pseudomonas aeruginosa

Lactonase Specificity Is Key to Quorum Quenching in Pseudomonas aeruginosa

Effects of Signal Disruption Depends on the Substrate Preference of the Lactonase

Engineering quorum quenching acylases with improved kinetic and biochemical properties

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