A Novel Metagenomic Short-Chain Dehydrogenase/Reductase Attenuates Pseudomonas aeruginosa Biofilm Formation and Virulence on Caenorhabditis elegans

Abstract

In Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies.

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Interestingly, the authors proposed that in addition to 3OC12HSL, BpiB09 may also act on intermediates of the fatty acid cycle, which would indirectly inhibit AHL biosynthesis by lowering available 3-oxo-acyl-ACP in the cell, thereby hindering AHL signaling in two ways. Heterologous expression of bpiB09 in P. aeruginosa reduced AHL accumulation, consequently inhibiting swarming and swimming motilities, pyocyanin production, and biofilm formation in vitro (81). Furthermore, bpiB09-expressing P. aeruginosa was less pathogenic against C. elegans than WT bacteria (81), providing the first evidence that oxidoreductases can function to reduce virulence by inhibiting quorum sensing in vivo.…”

“…Heterologous expression of bpiB09 in P. aeruginosa reduced AHL accumulation, consequently inhibiting swarming and swimming motilities, pyocyanin production, and biofilm formation in vitro (81). Furthermore, bpiB09-expressing P. aeruginosa was less pathogenic against C. elegans than WT bacteria (81), providing the first evidence that oxidoreductases can function to reduce virulence by inhibiting quorum sensing in vivo.…”

“…(80), indicating that AHL oxidation by CYP102A1 will likely not be the most effective approach for quorum quenching. A third oxidoreductase, the NADH-dependent enzyme BpiB09, was identified by metagenomic analysis as able to inactivate 3OC12HSL (81). Interestingly, the authors proposed that in addition to 3OC12HSL, BpiB09 may also act on intermediates of the fatty acid cycle, which would indirectly inhibit AHL biosynthesis by lowering available 3-oxo-acyl-ACP in the cell, thereby hindering AHL signaling in two ways.…”

Novel Reporter for Identification of Interference with Acyl Homoserine Lactone and Autoinducer-2 Quorum Sensing

“…Because biofilms potentially have harmful effects on living organisms (in the case of pathogenic bacteria) and on yields in the industrial sector, an attractive novel strategy to control biofilm formation based on QS interference (quorum quenching [QQ]) has been proposed and established (12)(13)(14)(15)(16)(17)(18)(19). Synthesis of QS-interfering compounds has been demonstrated for bacteria and eukaryotes (20)(21)(22)(23)(24), leading to the hypothesis that eukaryotes have evolved such defense mechanisms as a strategy for protection and inhibition of association with bacterial pathogens (25).…”

RNA Sequencing Analysis of the Broad-Host-Range Strain Sinorhizobium fredii NGR234 Identifies a Large Set of Genes Linked to Quorum Sensing-Dependent Regulation in the Background of a traI and ngrI Deletion Mutant

“…One of the beststudied model organisms in this research field with respect to QS is the opportunistic pathogenic bacterium P. aeruginosa. Several studies have been published analyzing the P. aeruginosa QS-dependent transcriptome using microarray technologies and RNA sequencing (27,44,(47)(48)(49)(50). Since the overall setups of these studies were slightly different, the number of genes that were differentially regulated in response to QS processes varied.…”