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.
Highlights
The bacterial cell–cell communication (QS) is based on small signal molecules, so-called autoinducers, and represents a cell density-dependent process effecting gene regulation in Prokaryotes
Intra- and extra-cellular accumulation of autoinducers enables bacteria to detect an increasing cell density and allows changing their gene expression to coordinate behaviors that require high cell densities, e.g., pathogenicity and biofilm formation (Landini et al, 2010; Castillo-Juárez et al, 2015). Among those autoinducers are acyl-homoserine lactones (AHL) in Gram-negative bacteria, short peptide signals in Gram-positive bacteria, and furan molecules known as autoinducer-2 (AI-2) in both groups (Liu et al, 2012; Du et al, 2014; Brackman and Coenye, 2015)
Well-known naturally occurring examples for quorum quenching (QQ) proteins are (i) AHL-lactonases hydrolyzing the ester bond of the homoserine lactone (HL) ring to inactivate the signaling molecule (Dong et al, 2000; Chen et al, 2013), (ii) AHL-acylases inactivating AHL signals by cleaving its amide bond resulting in the corresponding fatty acids and HL which are not effective as signals (Leadbetter and Greenberg, 2000; Kalia et al, 2011), (iii) AHL-oxidoreductases reducing the 3-oxo group of AHLs to generate corresponding 3-hydroxy derivatives (Uroz et al, 2005; Bijtenhoorn et al, 2011b; Lord et al, 2014)
Summary
The bacterial cell–cell communication (QS) is based on small signal molecules, so-called autoinducers, and represents a cell density-dependent process effecting gene regulation in Prokaryotes. Intra- and extra-cellular accumulation of autoinducers enables bacteria to detect an increasing cell density and allows changing their gene expression to coordinate behaviors that require high cell densities (for review see Dickschat, 2010; Castillo, 2015), e.g., pathogenicity and biofilm formation (Landini et al, 2010; Castillo-Juárez et al, 2015) Among those autoinducers are acyl-homoserine lactones (AHL) in Gram-negative bacteria, short peptide signals in Gram-positive bacteria, and furan molecules known as autoinducer-2 (AI-2) in both groups (Liu et al, 2012; Du et al, 2014; Brackman and Coenye, 2015). Those quenching mechanisms are mainly based on interference with AI-2 synthesis by S-ribosylhomocysteine and transition state analogs (Shen et al, 2006; Singh et al, 2006; Widmer et al, 2007), or antagonistic small molecules as shown in V. harveyi and E. coli (Ganin et al, 2009; Lowery et al, 2009; Vikram et al, 2011; Roy et al, 2013; Yadav et al, 2014)
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