Elsevier

Analytical Biochemistry

Volume 566, 1 February 2019, Pages 10-12
Analytical Biochemistry

Optimisation of a high-throughput fluorescamine assay for detection of N-acyl-l-homoserine lactone acylase activity

https://doi.org/10.1016/j.ab.2018.10.029Get rights and content

Highlights

  • N-acyl-l-homoserine lactone (AHL) acylases degrade quorum sensing molecules.

  • A fluorescamine-based assay for the detection of homoserine lactone was optimised.

  • This assay can be used to screen potential acylases for activity towards AHLs.

Abstract

N-acyl-l-homoserine lactone (AHL) acylases are a well-known group of enzymes that disrupt quorum sensing in Gram-negative bacteria by degrading AHL signalling molecules. This degradation of signalling molecules (termed 'quorum quenching') has potential uses in the prevention or reduction of biofilm formation and/or bacterial infections. Therefore, there is a great deal of interest in the identification and characterisation of quorum quenching enzymes. Here, we present an optimised fluorescamine-based assay for the detection of AHL acylase activity and demonstrate it can be used in a high-throughput screening format.

Introduction

Bacteria use quorum sensing to coordinate behaviours such as biofilm formation and virulence factor secretion [1]. Quorum sensing relies on the production, release, accumulation and population-wide detection of signal molecules called autoinducers. The most common class of autoinducers in Gram-negative bacteria is the N-acyl-l-homoserine lactones (AHLs) [1]. AHLs consist of a conserved homoserine lactone moiety and a variable acyl side chain; the length and substitutions of acyl side-chains vary between species.

Enzymatic degradation of autoinducer molecules (termed 'quorum quenching') has many potential biotechnological applications including the prevention or treatment of bacterial infections, reduction of biofilm formation, and control of biofouling [2,3]. Therefore, there is a great deal of interest in the identification and/or directed evolution of quorum quenching enzymes [[4], [5], [6]].

Here, we present an optimised fluorescamine-based assay for the detection of quorum quenching AHL acylase activity in a high-throughput format. AHL acylases are one of the two major classes of quorum-quenching enzymes. They function by irreversibly cleaving the amide bond that joins the lactone moiety and acyl side-chain of the AHL substrate [7] (Fig. 1A). Fluorescamine is a non-fluorescent reagent that reacts rapidly with primary amines to form stable, highly fluorescent products [[8], [9], [10]]. The assay is based on the reaction of the primary amine of the homoserine lactone product with fluorescamine (Fig. 1B).

Fluorescamine has been commonly used in the detection of compounds containing primary amine moieties such as amino acids, amines, peptides and proteins [8,11,12]. Depending on the primary amine, assay conditions can affect both the signal intensity and the kinetics of the fluorescamine reaction [8,10]. For example, to detect amino acids and peptides, fluorescamine in acetonitrile, assayed in pH 8 buffer has been shown to be optimal [13]. In contrast, detection of β-lactams is optimal with acetone, buffered to pH 4 [14].

A previous study reported the use of fluorescamine to detect AHL-acylase activity [15]. However, it reported a single set of assay conditions (fluorescamine in acetone, assayed in acetate buffer, pH 4.5). Here, we have sought to optimise the assay systematically, in order to maximise its utility. In particular, we focused on developing an assay that was appropriate for use in high-throughput, microtitre plate-based screens for AHL-acylase activities.

First, we tested the reaction of homoserine lactone (HSL) and fluorescamine over a range of buffer conditions. In our initial tests, fluorescamine was dissolved in either acetone or acetonitrile. The latter consistently resulted in lower signal intensities, so acetone was used for all subsequent experiments. Acetone was used to dissolve fluorescamine to a concentration of 10 mM. In each reaction, one of 11 buffers that spanned the pH range 4.5–9.0 (each at a final concentration of 100 mM) was added to HSL (200 μM, final concentration). Fluorescamine was then added to a final concentration of 1 mM. After the addition of fluorescamine, the fluorescence was measured immediately (excitation at 390 nm; emission 480 nm), and again after 60 min. The relative fluorescence for each buffer condition and time point are shown in Fig. 2A.

Both pH and buffer composition affect the fluorescence signal intensity of the HSL-fluorescamine conjugate (Fig. 2A). The maximum fluorescence signal was obtained in potassium phosphate buffer, pH 6, while the lowest was in sodium borate buffer, pH 9. With respect to pH, differences in fluorescence signal intensity are likely due to variation in the protonation state of the homoserine lactone. Previous work has shown that protonation of primary amines affects their nucleophilicity and thus, their reactivity with fluorescamine for formation of the fluorophore [9,16]. Overall, the optimised reaction conditions are different to those previously reported for amino acids, peptides and/or beta-lactams; this emphasises the need to optimise the fluorescamine reaction for each primary amine of interest.

While obtaining maximum fluorescence intensity is desirable, it is also important to consider the stability of the HSL-fluorescamine conjugate when designing highthroughput assays. In these scenarios, large numbers of AHL acylase variants are screened in 96-well microtitre plates. In practice, it can take 30 min or more to set up such a high-throughput experiment. With this application in mind, we measured the change in fluorescence intensity of the HSL-fluorescamine conjugate over a period of 60 min. The final fluorescence signal, at t = 60 min, is compared to the initial (t = 0) signal in Fig. 2A. Irrespective of the buffer system, pH ≤ 6 caused a significant decrease in fluorescence intensity over time. In contrast, pH ≥ 7 yielded much more stable fluorescence intensities, particularly with phosphate or citrate-phosphate buffered solutions in the range of pH 7 to pH 8 (Fig. 2A).

Combining all of the data from these optimisation experiments, we concluded that the optimal conditions for detecting homoserine lactone are: (a) to dissolve the fluorescamine in acetone; (b) to use potassium phosphate buffer; and (c) to conduct assays at pH = 7.

Our ultimate goal is to implement a screen for the directed evolution of AHL acylases. Therefore, we next sought to develop a high-throughput assay format that can distinguish between active and inactive AHL acylases. While crude cell lysates are often used for high-throughput screening [17], we found that these lysates contain other reactive primary amines, which give rise to high levels of background fluorescence. Instead, it was necessary to purify each acylase variant before implementing the fluorescamine assay. However, it is important to note that the data from this high-throughput screen is only semi-quantitative, as the overall activities are dependent on both activity, solubility, and yield of the variants, which can vary from well-to-well.

In our proof-of-concept experiment, we compared the activities of Pseudomonas aeruginosa PAO1 HacB AHL acylase (HacB) and Pseudomonas sp. SY77 glutaryl-7-aminocephalosporanic acid acylase (GL7-ACA). These enzymes have been characterised previously: the former is known to hydrolyse octanoyl homoserine lactone (C8-AHL) [19] whereas the latter has no AHL acylase activity [20]. Each was expressed and purified using HisPur Cobalt Spin Plates (Thermo Fisher), using methods we have described previously [18]. Our protocol involves eluting from the HisPur Cobalt resin under mild conditions (75 mM imidazole). This is critical, because fluorescamine reacts with compounds that contain secondary amines, such as imidazole, to produce non-fluorescent products. In control experiments, we showed that – as predicted – fluorescence intensity does decrease with increasing imidazole concentration (data not shown), but that the effect was negligible under the conditions of our microplate-based assay (Suppl. Fig. 1).

To determine if the high-throughput assay format that can distinguish between active and inactive AHL acylases, the activities of our positive (HacB) and negative (GL7-ACA) control proteins were tested with C8-AHL. A master stock of the C8-AHL substrate (30 mM) was made by dissolving it in DMSO. This concentrated stock was diluted to 210 μM with potassium phosphate (100 mM, pH 7.0). The working stock of C8-AHL was dispensed into the first 47 wells of two flat-bottomed, black microtitre plates (190 μL per well, final C8-AHL concentration 200 μM). The next 47 wells received buffer and DMSO, but no C8-AHL. The final two wells were left empty at this stage. A 10-μL aliquot of purified enzyme was added to the first 94 wells of each plate (one plate with HacB and the other with GL7-ACA). Thus, 47 “plus substrate” samples were compared with 47 “minus substrate” controls, to assess variation in the amount of protein eluted from each well of the HisPur Cobalt Spin Plate. The plates were sealed to minimise evaporation and incubated at 30 °C, with shaking at 100 rpm, for 24 h. After this incubation, 200 μL of homoserine lactone (200 μM), made up in potassium phosphate (100 mM, pH 7.0) was added to the 95th well of each plate. Finally, 25 μL of a fluorescamine stock (10 mM; dissolved in 100% acetone) was added to all 95 wells of each plate, to give a final fluorescamine concentration of 1 mM. The relative fluorescence in each well was measured using the CLARIOstar microplate reader, with the HSL standard in the 95th well being used to set the gain on the instrument.

The AHL acylase activity of HacB against C8-AHL was detectable under our optimised conditions, and this signal was distinguishable from the controls lacking substrate (Fig. 2B). This showed that background fluorescence from the purified enzymes was negligible. As expected, the GL7ACA acylase showed no significant activity with C8-AHL (Fig. 2B). We calculated the Z′ statistic of the assay data; a Z′ value provides a measure of assay quality, taking into account both the signal-to-noise ratio and assay variability. For a high throughput screening assay a Z′ value of >0.5 is generally considered acceptable [21]. The Z’ value for the assay data presented is 0.6 ± 0.1, indicating that the assay identifies hits with an acceptable level of confidence. However, as with any high-throughput screen, any hits should be validated and the activity quantified using additional methods.

Previous high-throughput assays to detect new activities from variants of the Pseudomonas sp. SY77 glutaryl-7-aminocephalosporanic acid acylase required aliquoting samples into plates containing the required buffer, adding fluorescamine and incubating for an hour before measurement [20]. We optimised our high-throughput assay such that fluorescamine can be added directly to samples in black 96-well plates and then measured immediately. Another recently described method for screening quorum-quenching enzymes uses calcein [22]. It reacts with homoserine to produce a fluorescent product. However, calcein is used to detect homoserine in the micromolar range, and imidazole strongly quenches fluorescence of calcein. In contrast, our protocol for protein purification and high-throughput assays is designed to use imidazole at a concentration that does not significantly affect the fluorescence signal, and detects HSL in the nanomolar range (Suppl. Fig. 1).

In conclusion, we have optimised the reaction of HSL with fluorescamine. We have also shown this fluorescamine assay can be used as a high-throughput screen to detect enzymes with AHL acylase activity.

Section snippets

Funding

This research was supported by the Maurice Wilkins Centre for Molecular Biodiscovery, New Zealand; and a Smart Ideas grant from the Ministry of Business, Innovation and Employment, New Zealand. S.M. was supported by a University of Otago Doctoral Scholarship, New Zealand.

Declarations of competing interests

None.

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    These authors contributed equally to this work.

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