Highly Effective Inhibition of Biofilm Formation by the First Metagenome-Derived AI-2 Quenching Enzyme

Abstract

Bacterial cell–cell communication (quorum sensing, QS) represents a fundamental process crucial for biofilm formation, pathogenicity, and virulence allowing coordinated, concerted actions of bacteria depending on their cell density. With the widespread appearance of antibiotic-resistance of biofilms, there is an increasing need for novel strategies to control harmful biofilms. One attractive and most likely effective approach is to target bacterial communication systems for novel drug design in biotechnological and medical applications. In this study, metagenomic large-insert libraries were constructed and screened for QS interfering activities (quorum quenching, QQ) using recently established reporter strains. Overall, 142 out of 46,400 metagenomic clones were identified to interfere with acyl-homoserine lactones (AHLs), 13 with autoinducer-2 (AI-2). Five cosmid clones with highest simultaneous interfering activities were further analyzed and the respective open reading frames conferring QQ activities identified. Those showed homologies to bacterial oxidoreductases, proteases, amidases and aminotransferases. Evaluating the ability of the respective purified QQ-proteins to prevent biofilm formation of several model systems demonstrated highest inhibitory effects of QQ-2 using the crystal violet biofilm assay. This was confirmed by heterologous expression of the respective QQ proteins in Klebsiella oxytoca M5a1 and monitoring biofilm formation in a continuous flow cell system. Moreover, QQ-2 chemically immobilized to the glass surface of the flow cell effectively inhibited biofilm formation of K. oxytoca as well as clinical K. pneumoniae isolates derived from patients with urinary tract infections. Indications were obtained by molecular and biochemical characterizations that QQ-2 represents an oxidoreductase most likely reducing the signaling molecules AHL and AI-2 to QS-inactive hydroxy-derivatives. Overall, we propose that the identified novel QQ-2 protein efficiently inhibits AI-2 modulated biofilm formation by modifying the signal molecule; and thus appears particularly attractive for medical and biotechnological applications.

Quorum Sensing and the Use of Quorum Quenchers as Natural Biocides to Inhibit Sulfate-Reducing Bacteria

“…AI-2 is a product of SAM metabolism and may be indicative of the metabolic status of the cell and not present solely due to QS activity. Nevertheless, recent studies have shown that enzymes with AI-2 quenching capabilities have resulted in a significantly thinner biofilm that is more compact with a lower overall biovolume [58]. This finding suggests that, despite AI-2 having dual roles in both QS and metabolic pathways, quenching AI-2 may still be a good method to deter growth of unwanted bacterial populations.…”

“…Recently, by constructing large-insert cosmid libraries from metagenomes derived from various saline samples, enzymes displaying homologies to oxidoreductases, proteases, amidases, and aminotransferases were identified to exhibit a QQ effect against both AHL and AI-2 [58]. To exemplify, enzymes ranging from 177–478 aa that share close homology (42%–100% aa identity) with either aminotransferase, 3-hydroxy-2-methylbutyry-CoA dehydrogenase, ferredoxin reductase, 4-hydroxy-3-methybut-2-en-1-yl diphosphate synthase, 3-beta hydroxysteroid dehydrogenase, or N-acetylmuramoyl-L-alanine amidase were recovered from the Black Sea and salt marsh.…”

Evaluating the quorum quenching potential of bacteria associated toAurelia auritaandMnemiopsis leidyi

“…QQ assays on plates using strains AI1-QQ.1 and AI2-QQ.1 were performed with cell-free supernatants and cell extracts of fosmid clones and purified proteins as previously described in (25). In order to identify the respective ORFs of the fosmids conferring QQ activity, subcloning was used as previously described in (25,26). Putative QQ-ORFs were PCR-cloned into pMAL-c2X Nterminally fusing the QQ-ORFs to the maltose binding protein (MPB) using ORF-specific primers adding restriction recognition sites flanking the ORFs (see Tab.…”

“…Putative QQ-ORFs were PCR-cloned into pMAL-c2X Nterminally fusing the QQ-ORFs to the maltose binding protein (MPB) using ORF-specific primers adding restriction recognition sites flanking the ORFs (see Tab. S1); overexpressed and proteins purified as recently described in (26).…”

“…Many marine bacteria have evolved efficient strategies to combat neighboring bacteria, ultimately affecting the microbial community dynamics and patterns. Moreover, the identified high frequencies of QQ activities in marine bacteria disclose the enormous potential of marine bacteria as rich source for the identification of novel, biotechnologically relevant compounds, in particular QQ compounds for the inhibition of harmful biofilms(26, 60,65).…”