Leo Eberl

Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors

Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip® microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response.

Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound

Novel molecular tools have been constructed which allow for in situ detection of N-acyl homoserine lactone (AHL)-mediated quorum sensing in Pseudomonas aeruginosa biofilms. The reporter responds to AHL activation of LasR by expression of an unstable version of the green-fluorescent protein (Gfp). Gfp-based reporter technology has been applied for non-destructive, single-cell-level detection of quorum sensing in laboratory-based P. aeruginosa biofilms. It is reported that a synthetic halogenated furanone compound, which is a derivative of the secondary metabolites produced by the Australian macroalga Delisea pulchra, is capable of interfering with AHL-mediated quorum sensing in P. aeruginosa. It is demonstrated that the furanone compound specifically represses expression of a PlasB-gfp reporter fusion without affecting growth or protein synthesis. In addition, it reduces the production of important virulence factors, indicating a general effect on target genes of the las quorum sensing circuit. The furanone was applied to P. aeruginosa biofilms established in biofilm flow chambers. The Gfp-based analysis reveals that the compound penetrates microcolonies and blocks cell signalling and quorum sensing in most biofilm cells. The compound did not affect initial attachment to the abiotic substratum. It does, however, affect the architecture of the biofilm and enhances the process of bacterial detachment, leading to a loss of bacterial biomass from the substratum.

The cep quorum-sensing system of Burkholderia cepacia H111 controls biofilm formation and swarming motility

Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, the isolation of random mini-Tn5 insertion mutants of B. cepacia H111 defective in biofilm formation on an abiotic surface is reported. It is demonstrated that one of these mutants no longer produces N-acylhomoserine lactones (AHLs) due to an inactivation of the cepR gene. cepR and the cepI AHL synthase gene together constitute the cep quorum-sensing system of B. cepacia. By using a gene replacement method, two defined mutants, H111-I and H111-R, were constructed in which cepI and cepR, respectively, had been inactivated. These mutants were used to demonstrate that biofilm formation by B. cepacia H111 requires a functional cep quorum-sensing system. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains suggested that the quorum-sensing system is not involved in the regulation of initial cell attachment, but rather controls the maturation of the biofilm. Furthermore, it is shown that B. cepacia is capable of swarming motility, a form of surface translocation utilized by various bacteria to rapidly colonize appropriate substrata. Evidence is provided that swarming motility of B. cepacia is quorum-sensing-regulated, possibly through the control of biosurfactant production. Complementation of the cepR mutant H111-R with different biosurfactants restored swarming motility while biofilm formation was not significantly increased. This result suggests that swarming motility per se is not essential for biofilm formation on abiotic surfaces.

Involvement of N‐acyl‐l‐homoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens

Several bacterial species possess the ability to differentiate into highly motile swarmer cells capable of rapid surface colonization. In Serratia liquefaciens, we demonstrate that initiation of swarmer‐cell differentiation involves diffusible signal molecules that are released into the growth medium. Using high‐performance liquid chromatography (HPLC), high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, we identified N‐butanoyl‐l‐homoserine lactone (BHL) and N‐hexanoyl‐l‐homoserine lactone (HHL) in cell‐free Serratia culture supernatants. BHL and HHL are present in a ratio of approximately 10:1 and their structures were unequivocally confirmed by chemical synthesis. The swrlswarmer initiation) gene, the predicted translation product of which exhibits substantial homology to the Luxl family of putative Nacyl homoserine lactone (AHL) synthases is responsible for directing synthesis of both BHL and HHL. In an swrl mutant, swarming motility is abolished but can be restored by the addition of an exogenous AHL. These results add swarming motility to the rapidly expanding list of phenotypes known to be controlled through quorum sensing.

How Delisea pulchra furanones affect quorum sensing and swarming motility in Serratia liquefaciens MG1

Halogenated furanones produced by the benthic marine macroalga Delisea pulchra inhibit swarming motility of Serratia liquefaciens MG1. This study demonstrates that exogenously added furanones control transcription of the quorum sensing regulated gene swrA in competition with the cognate signal molecule N:-butanoyl-L-homoserine lactone. This in turn results in reduced production of the surface-active compound serrawettin W2, which is crucial for surface translocation of the differentiated swarm cells. It is demonstrated that furanones interfere with interspecies communication during swarming of mixed cultures and that the mode of interference in quorum-sensing control and interspecies communication is not through inhibition of autoinducer synthesis.

Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa.

The pathogenesis of Pseudomonas aeruginosa is associated with expression of virulence factors, many of which are controlled by two N:-acylhomoserine lactone (AHL)-based quorum-sensing systems. Escherichia coli strains equipped with a luxR-based monitor system expressing green fluorescent protein (GFP) in the presence of exogenous AHL molecules were used to detect the production of AHLs from P. aeruginosa in vivo. Mice were challenged intratracheally with alginate beads containing P. aeruginosa and E. coli and killed on different days after the challenge. By means of confocal scanning laser microscopy, GFP-expressing E. coli bacteria could be detected in the lung tissues, indicating production and excretion of AHL molecules in vivo by the infecting P. aeruginosa. AHL signals were detected mainly in lung tissues exhibiting severe pathological changes. These findings support the view that expression of AHL molecules by P. aeruginosa during infection coincides with its pathogenesis.

N-Acyl Homoserinelactone-mediated Gene Regulation in Gram-negative Bacteria

The view of bacteria as unicellular organisms has strong roots in the tradition of culturing bacteria in liquid media. However, in nature microbial activity is mainly associated with surfaces where bacteria form highly structured and cooperative consortia which are commonly referred to as biofilms. The ability of bacteria to organize structurally and to distribute metabolic activities between the different members of the consortium demands a high degree of coordinated cell-cell interaction. Recent work has established that many bacteria employ sophisticated intercellular communication systems that rely on small signal molecules to control the expression of multiple target genes. In Gram-negative bacteria, the most intensively investigated signal molecules are N-acyl-L-homoserine lactones (AHLs), which are utilized by the bacteria to monitor their own population densities in a process known as quorum sensing. These density-dependent regulatory systems rely on two proteins, an AHL synthase, usually a member of the LuxI family of proteins, and an AHL receptor protein belonging to the LuxR family of transcriptional regulators. At low population densities cells produce a basal level of AHL via the activity of an AHL synthase. As the cell density increases, AHL accumulates in the growth medium. On reaching a critical threshold concentration, the AHL molecule binds to its cognate receptor which in turn leads to the induction/repression of AHL-regulated genes. To date, AHL-dependent quorum sensing circuits have been identified in a wide range of gram-negative bacteria where they regulate various functions including bioluminescence, plasmid conjugal transfer, biofilm formation, motility, antibiotic biosynthesis, and the production of virulence factors in plant and animal pathogens. Moreover, AHL signal molecules appear to play important roles in the ecology of complex consortia as they allow bacterial populations to interact with each other as well as with their eukaryotic hosts.

Genetic analysis of functions involved in the late stages of biofilm development in Burkholderia cepacia H111

Burkholderia cepacia and Pseudomonas aeruginosa often co‐exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, we report the isolation of 13 random mini‐Tn 5 insertion mutants of B. cepacia H111 that are defective in biofilm formation on a polystyrene surface. We show that the screening procedure used in this study is biased towards mutants defective in the late stages of biofilm development. A detailed quantitative analysis of the biofilm structures formed by wild‐type and mutant strains revealed that the isolated mutants are impaired in their abilities to develop a typical three‐dimensional biofilm structure. Molecular investigations showed that the genes required for biofilm maturation fall into several classes: (i) genes encoding for surface proteins; (ii) genes involved in the biogenesis and maintenance of an integral outer membrane; and (iii) genes encoding regulatory factors. It is shown that three of the regulatory mutants produce greatly reduced amounts of N ‐octanoylhomoserine lactone (C8‐HSL). This compound serves as the major signal molecule of the cep quorum‐sensing system. As this density‐dependent regulatory system is involved in the regulation of biofilm maturation, we investigated the interplay between the three regulatory genes and the quorum‐sensing cascade. The results of these investigations show that the identified genes encode for regulatory elements that are positioned upstream of the cep system, indicating that the quorum‐sensing system of B. cepacia is a major checkpoint for biofilm formation.

Killing of Caenorhabditis elegans by Burkholderia cepacia is controlled by the cep quorum-sensing system

Burkholderia cepacia H111, which was isolated from a cystic fibrosis patient, effectively kills the nematode Caenorhabditis elegans. Depending on the medium used for growth of the bacterium two different killing modes were observed. On high‐osmolarity medium the nematodes became paralysed and died within 24u2003h. Using filter assays we provide evidence that this killing mode involves the production of an extracellular toxin. On nematode growth medium killing occurs over the course of 2–3u2003days and involves the accumulation of bacteria in the intestinal lumen of C. elegans. We demonstrate that the cep quorum‐sensing system of H111 is required for efficient killing of C. elegans under both killing conditions. Using the C. elegans phm‐2 mutant that has a non‐functional grinder evidence is provided that the cep system is required to enter the intestinal lumen but is dispensable for the colonization of the gut. Furthermore, we demonstrate that the type II secretion machinery is not essential for nematode killing.

An inhibitor of bacterial quorum sensing reduces mortalities caused by Vibriosis in rainbow trout (Oncorhynchus mykiss, Walbaum).

The fish pathogen Vibrio anguillarum produces quorum sensing signal molecules, N-acyl homoserine lactones (AHLs), which in several Gram-negative human and plant pathogenic bacteria regulate virulence factors. Expression of these factors can be blocked using specific quorum-sensing inhibitors (QSIs). The purpose of this study was to investigate the effect of a QSI, furanone C-30, on mortality of rainbow trout during challenge with V. anguillarum. Addition of 0.01 or 0.1 microM furanone C-30 to rainbow trout infected by cohabitation caused a significant reduction in accumulated mortality from 80-100% in challenge controls to 4-40% in treated groups. Furanone C-30 had no effect in an immersion challenge system, probably due to a very high water exchange and a rapid dilution of furanone C-30. Growth and survival of V. anguillarum were not affected by the concentrations of furanone C-30 used in the challenge experiments, thus avoiding selection for resistance. To elucidate the mechanism of disease control by furanone C-30, we determined its effect on the bacterial proteome, motility, and respiration. No effects were seen of furanone C-30 in any of these experiments. Although no cytotoxic effect on HeLa cells were observed, exposure to 1 microM (or higher) concentrations of furanone C-30 had detrimental effects on the rainbow trout. Our results indicate that QSIs can be used in non-antibiotic based control of fish diseases. However, they also underline the need for development of novel, less toxic QSI compounds and the need for understanding the exact mechanism(s) of action.