Engineering acyl-homoserine lactone-interfering enzymes toward bacterial control

Abstract

Enzymes able to degrade or modify acyl-homoserine lactones (AHLs) have drawn considerable interest for their ability to interfere with the bacterial communication process referred to as quorum sensing. Many proteobacteria use AHL to coordinate virulence and biofilm formation in a cell density-dependent manner; thus, AHL-interfering enzymes constitute new promising antimicrobial candidates. Among these, lactonases and acylases have been particularly studied. These enzymes have been isolated from various bacterial, archaeal, or eukaryotic organisms and have been evaluated for their ability to control several pathogens. Engineering studies on these enzymes were carried out and successfully modulated their capacity to interact with specific AHL, increase their catalytic activity and stability, or enhance their biotechnological potential. In this review, special attention is paid to the screening, engineering, and applications of AHL-modifying enzymes. Prospects and future opportunities are also discussed with a view to developing potent candidates for bacterial control.

Keywords: acyl-homoserine lactones quorum sensing; acylase; antibiotic resistance; biofilm; enzyme catalysis; lactonase; protein engineering; quorum quenching; virulence factor.

Figure 1.

Canonical quorum sensing in Gram-negative bacteria and quorum quenching. A, AHLs (blue triangles) are produced by cells and diffuse freely in and out cells. AHL concentration increases with cell concentration. Above a certain threshold, AHLs bind and activate the QS regulator, which in turn can bind QS promoter sequences and induce the expression of QS genes, such as the AHL synthase gene (I) and other target genes (T). QQ enzymes degrade extracellular AHLs, the QS regulator is not activated, and QS genes are not expressed. Strings, arrows, and boxes represent genetic arrangements. B, AHLs consist of a homoserine lactone ring with an acyl chain that can vary in length (green) or functionalization (red). AHLs can be differentially targeted by lactonase and acylase enzymes.

Figure 2.

Various Gram-negative bacteria that use AHL-based sensing to control pathogenicity. AHLs reported for each bacterium are highlighted in red (15).

Figure 3.

Structural overview of AHL-interfering enzymes. AiiA and SsoPox lactonases and the acylase PvdQ are presented using the same scale. A, crystal structure of the 28-kDa metalloenzyme lactonase (EC 3.1.1.81) AiiA mutant F107W from Bacillus thuringiensis with N-decanoyl-l-homoserine bound at the active site (PDB entry 4J5H). AiiA belongs to the metallo-β-lactamase superfamily and harbors two Zn(II) ions bound at the active site essential to catalytic activity. B, crystal structure of the 35-kDa metalloenzyme, PLL SsoPox W263I (EC 3.1.8.1) in complex with C₁₀-HTL (PDB entry 4KF1). SsoPox belongs to the amidohydrolase superfamily and exhibits a (α/β)8-barrel fold (the so-called TIM-barrel) and harbors a bicobalt active site. Loops 7 (red) and 8 (orange) play key roles in substrate recognition and protein flexibility. C, crystal structure of the acylase PvdQ (EC 3.5.1.97) with a covalently bound dodecanoic acid (PDB entry 2WYB). PvdQ is a member of the Ntn-hydrolase superfamily and is formed by an 18-kDa α-chain (purple) and a 60-kDa β-chain (pink).

Figure 4.

Screening approaches for identifying novel or improved AHL-interfering enzymes. A, in vivo assays based on natural QS systems. AHLs are perceived by biosensor cells that consist of a regulator that is activated upon AHL binding and in turn induces the expression of a reporter gene (luminescence, violacein, fluorescence, or β-gal). In the presence of active QQ enzymes, AHLs are degraded, and no signal is induced. B, in vitro assays. AHL degradation can be measured in vitro by colorimetric assays (cresol purple) or by fluorescent probes that recognize AHL-degradation products (fluorescamine) or react with them through copper competition (calcein).

Figure 5.

Catalytic performances and stability of native (57–68) and engineered AHL-interfering enzymes. Only enzymes with described k_cat/K_m values are represented in this figure. Enzymes are classified by their EC number. Catalytic efficiency (k_cat/K_m) on various lactones, corresponding to the highest values reported in the literature are presented using color gradients from blue to red diverging scale. Lactone names and structures are presented at the top. Melting temperature (T_m) values are presented with shades of green from light to dark. Colors and their respective values are detailed in the top left.