Structural insights into acyl-ACP selective recognition by the Aeromonas hydrophila AHL synthase AhyI

Lei Jin,Yu Chen,Wenyi Du,Wenge Yang,Jingjiao Bao

doi:10.1186/s12866-021-02244-9

Abstract

BackgroundAeromonas hydrophila is a gram-negative bacterium and the major causative agent of the fish disease motile aeromonad septicemia (MAS). It uses N-acyl-homoserine lactone (AHL) quorum sensing signals to coordinate biofilm formation, motility, and virulence gene expression. The AHL signaling pathway is therefore considered to be a therapeutic target against pathogenic A. hydrophila infection. In A. hydrophila, AHL autoinducers biosynthesis are specifically catalyzed by an ACP-dependent AHL synthase AhyI using the precursors SAM and acyl-ACP. Our previously reported AhyI was heterologously expressed in E. coli, which showed the production characteristics of medium-long chain AHLs. This contradicted the prevailing understanding that AhyI was only a short-chain C4/C6-HSL synthase.ResultsIn this study, six linear acyl-ACP proteins with C-terminal his-tags were synthesized in Vibrio harveyi AasS using fatty acids and E. coli produced active holo-ACP proteins, and in vitro biosynthetic assays of six AHL molecules and kinetic studies of recombinant AhyI with a panel of four linear acyl-ACPs were performed. UPLC-MS/MS analyses indicated that AhyI can synthesize short-, medium- and long-chain AHLs from SAM and corresponding linear acyl-ACP substrates. Kinetic parameters measured using a DCPIP colorimetric assay, showed that there was a notable decrease in catalytic efficiency with acyl-chain lengths above C6, and hyperbolic or sigmoidal responses in rate curves were observed for varying acyl-donor substrates. Primary sequence alignment of the six representative AHL synthases offers insights into the structural basis for their specific acyl substrate preference. To further understand the acyl chain length preference of AhyI for linear acyl-ACP, we performed a structural comparison of three ACP-dependent LuxI homologs (TofI, BmaI1 and AhyI) and identified three key hydrophobic residues (I67, F125 and L157) which confer AhyI to selectively recognize native C4/C6-ACP substrates. These predictions were further supported by a computational Ala mutation assay.ConclusionsIn this study, we have redefined AhyI as a multiple short- to long-chain AHL synthase which uses C4/C6-ACP as native acyl substrates and longer acyl-ACPs (C8 ~ C14) as non-native ones. We also theorized that the key residues in AhyI would likely drive acyl-ACP selective recognition.

Highlights

Aeromonas hydrophila is a gram-negative bacterium and the major causative agent of the fish disease motile aeromonad septicemia (MAS)
Synthesis of acyl-Acyl carrier protein (ACP) substrates ACP was overexpressed in E. coli carrying the plasmid pET28a-acpP and isolated primarily in the apo-form
Six acyl-homoserine lactone (AHL) molecules were produced by the ACP-dependent AHL synthase AhyI via in vitro enzymatic reaction, demonstrating that it is a multiple shortto long-chain AHL synthase

Summary

Introduction

Aeromonas hydrophila is a gram-negative bacterium and the major causative agent of the fish disease motile aeromonad septicemia (MAS). It uses N-acyl-homoserine lactone (AHL) quorum sensing signals to coordinate biofilm formation, motility, and virulence gene expression. Aeromonas hydrophila is an opportunistic pathogen and ubiquitous inhabitant of various aquatic environments across the world It infects fish, reptiles, amphibians, and mammals (including humans) [1, 2], and causes motile aeromonad septicemia (MAS), the most important bacterial disease in fish, frequent outbreaks of which lead to huge economic losses [3, 4]. Targeting AHL signaling circuit asserts less selective pressure for developing drug resistances, and small-molecule antagonists of AHL signaling are an alternative to antibiotic usage to provide protection against the QS-dependent pathogenesis of A. hydrophila (Fig. S1)

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