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 â microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug speci®c-ally targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa bio®lms 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.

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Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.

Acylated homoserine lactones (AHLs) play a widespread role in intercellular communication among bacteria. The Australian macroalga Delisea pulchra produces secondary metabolites which have structural similarities to AHL molecules. We report here that these metabolites inhibited AHL-controlled processes in prokaryotes. Our results suggest that the interaction between higher organisms and their surface-associated bacteria may be mediated by interference with bacterial regulatory systems.Acylated homoserine lactones (AHLs) serve as signals in bacterial communication. AHLs and their derivatives regulate bioluminescence, Ti plasmid transfer, production of virulence factors and antibiotics (for reviews, see references 15 and 24), and swarming motility (14). These bacterial processes are fundamental to the interaction of bacteria with each other, their environment, and, particularly, higher organisms. It might therefore be expected that plants or animals would have evolved strategies to interfere with bacterial AHL-mediated processes. The seaweed Delisea pulchra (Rhodophyta) produces a number of halogenated furanones (9), which are structurally similar to the bacterial AHLs ( Fig. 1) and have strong biological activity (7), including antifouling and antimicrobial properties (8,23). We hypothesized that these metabolites could interfere with bacterial processes which involve AHLdriven quorum-sensing systems. This hypothesis was tested in terms of responses known to be regulated by AHLs; swarming motility in Serratia liquefaciens (14) and bioluminescence produced by the marine bacterial strains Vibrio fischeri and Vibrio harveyi (15).Bacterial swarming is a multicellular, density-dependent behavior that is induced in response to recognition of surfaces with a certain viscosity. Cells differentiate into a multinucleated, elongated, and hyperflagellated form, orient themselves lengthwise in close contact with each other, and then move rapidly in a coordinated fashion over the surface of the growth substratum. Swarming has been described for a variety of bacteria, including members of the genera Serratia, Proteus, Vibrio, Bacillus, Escherichia, and Salmonella (1,2,17). For Proteus mirabilis and Vibrio parahaemolyticus, the ability to differentiate into the swarmer cell state plays an important role in bacterial virulence, surface adsorption, and colonization (3-5).S. liquefaciens is a suitable model organism, because members of the genus can colonize a wide variety of surfaces in water, soil, plants, insects, fish, and humans (16). The formation of a swarming colony of S. liquefaciens was recently shown to involve two genetic switches, the first of which encodes a quorum-sensing control mechanism employing at least two extracellular signal molecules, N-butanoyl-L-homoserine lactone (BHL) and N-hexanoyl-L-homoserine lactone ( Fig. 1 and reference 14). The second involves the flhDC master operon, which regulates expression of the flagellar regulon and governs control over swim and swarm cell differentiation (13).Development ...

With the widespread appearance of antibiotic-resistant bacteria, there is an increasing demand for novel strategies to control infectious diseases. Furthermore, it has become apparent that the bacterial life style also contributes significantly to this problem. Bacteria living in the biofilm mode of growth tolerate conventional antimicrobial treatments. The discovery that many bacteria use quorum-sensing (QS) systems to coordinate virulence and biofilm development has pointed out a new, promising target for antimicrobial drugs. We constructed a collection of screening systems, QS inhibitor (QSI) selectors, which enabled us to identify a number of novel QSIs among natural and synthetic compound libraries. The two most active were garlic extract and 4-nitro-pyridine-N-oxide (4-NPO). GeneChip-based transcriptome analysis revealed that garlic extract and 4-NPO had specificity for QS-controlled virulence genes in Pseudomonas aeruginosa. These two QSIs also significantly reduced P. aeruginosa biofilm tolerance to tobramycin treatment as well as virulence in a Caenorhabditis elegans pathogenesis model. Several bacteria show organized behavior when they establish themselves in the eukaryotic host (22). The invading bacteria express a battery of tissue-damaging virulence factors in accordance with their numbers in a process termed quorum sensing (QS) (16). This is accomplished by sensing the concentration of small, diffusible signal molecules produced by the bacteria themselves. In gram-negative bacteria, the signals are N-acyl homoserine lactones (AHLs), which are produced by the LuxI family of AHL synthases. The signal molecules differ with respect to the length of their side chains (C4 to C16) and with various degrees of substitution and saturation (34). Shortchain AHLs are freely diffusible over the cell membranes, whereas long-chain AHLs are the substrate of efflux pumps, such as mexAB-oprM (36). The AHLs are sensed by proteins belonging to the LuxR family of response regulators. LuxR homologues contain two domains, an AHL binding domain and a DNA binding domain. When AHL is bound, it alters the configuration of the LuxR homologue protein, enabling it to interact with DNA and act as a transcriptional activator (16). It should be noted that some LuxR homologues acts as repressors, blocking transcription in the absence of AHL and, when sufficient AHL is present, derepressing the target gene(s) (6). The two key components of the QS system, the luxI and luxR homologues, are often linked genes, whereas the QS target genes are localized elsewhere on the genome. In case of Vibrio fischeri, the AHL synthase gene itself is a target gene of the QS mechanism, creating an autoinduction loop, which at the triggering (or threshold) AHL concentration gives rise to a burst in AHL production and QS-controlled gene expression.It has recently become evident that QS target genes are not generally activated at a certain threshold concentration but merely become activated as a continuum at different AHL-cell concentrations (23,40). Pseud...