Characterization of a novel quorum quenching enzyme and determination of autoinducer signal receptor specificity

Abstract

Many bacterial species exhibit cell-density dependent traits that are most advantageous when expressed simultaneously throughout their population. Examples of these traits include the production of enzymes used to dismantle the defenses of a host organism, the formation of protective biofilm, and bioluminescence, among others. Detection of the required threshold cell density (a quorum) relies on chemical communication via the production of a variety of small molecule autoinducers such as N-acyl homoserine lactones (AHLs). This communication system is known as quorum sensing (QS), and its specificity depends on the ability of different bacteria to produce and sense distinct AHL molecules. In this work I focused on (i) the specificity of AHL receptors and (ii) the characterization of a novel enzyme to interfere with QS. Specifically, I investigated 4 receptors from different bacteria, and report their preference for range of AHLs. In particular, combined with a literature survey, my data suggest that receptors have broader AHL preference than what is typically reported. Additionally, I show that one of these receptors can bind and respond to lactone signals other than AHLs. Some bacterial enzymes can interfere with QS through the degradation of autoinducers, quorum quenching (QQ) the expression of the behaviors regulated by QS. The discovery of new enzymes capable of QQ is important to the development of fine-tuned control of bacterial communication and behaviors and can give hints to the biological importance of microbial signaling. Here we report the characterization of a novel quorum quenching enzyme, ZHD, from the fungi Clonostachys rosea, suggesting that interference in QS extends beyond bacteria. We found that ZHD is a broad spectrum AHL lactonase with kcat/KM values in the range of 104 to 105 M-1⋅s-1. ZHD hydrolyzes AHLs of various acyl chain lengths as evidenced by pH indicator assay measurements, biosensor measurements and mass spectrometry data. Analysis of ZHD bound to C8-AHL allowed for the identification of the substrate binding mode and proposal of a catalytic mechanism. ZHD is likely a novel representative of the α/β-hydrolase family of lactonases. Altogether, these results, i.e. the demonstration that AHL receptors that can respond to lactones other than AHLs and the identification of a quorum quenching enzyme from fungi, are evidence that the level of cross communication may not be limited to AHL-producing bacteria.