Rhamnolipids are glycolipidic biosurfactants produced by various bacterial species. They were initially found as exoproducts of the opportunistic pathogen Pseudomonas aeruginosa and described as a mixture of four congeners: α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoyl-β-hydroxydecanoate (Rha-Rha-C₁₀-C₁₀), α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-β-hydroxydecanoate (Rha-Rha-C₁₀), as well as their mono-rhamnolipid congeners Rha-C₁₀-C₁₀ and Rha-C₁₀. The development of more sensitive analytical techniques has lead to the further discovery of a wide diversity of rhamnolipid congeners and homologues (about 60) that are produced at different concentrations by various Pseudomonas species and by bacteria belonging to other families, classes, or even phyla. For example, various Burkholderia species have been shown to produce rhamnolipids that have longer alkyl chains than those produced by P. aeruginosa. In P. aeruginosa, three genes, carried on two distinct operons, code for the enzymes responsible for the final steps of rhamnolipid synthesis: one operon carries the rhlAB genes and the other rhlC. Genes highly similar to rhlA, rhlB, and rhlC have also been found in various Burkholderia species but grouped within one putative operon, and they have been shown to be required for rhamnolipid production as well. The exact physiological function of these secondary metabolites is still unclear. Most identified activities are derived from the surface activity, wetting ability, detergency, and other amphipathic-related properties of these molecules. Indeed, rhamnolipids promote the uptake and biodegradation of poorly soluble substrates, act as immune modulators and virulence factors, have antimicrobial activities, and are involved in surface motility and in bacterial biofilm development.

Bacterial communities use “quorum sensing” (QS) to coordinate their population behavior through the action of extracellular signal molecules, such as the N -acyl- l -homoserine lactones (AHLs). The versatile and ubiquitous opportunistic pathogen Pseudomonas aeruginosa is a well-studied model for AHL-mediated QS. This species also produces an intercellular signal distinct from AHLs, 3,4-dihydroxy-2-heptylquinoline (PQS), which belongs to a family of poorly characterized 4-hydroxy-2-alkylquinolines (HAQs) previously identified for their antimicrobial activity. Here we use liquid chromatography (LC)/MS, genetics, and whole-genome expression to investigate the structure, biosynthesis, regulation, and activity of HAQs. We show that the pqsA-E operon encodes enzymes that catalyze the biosynthesis of five distinct classes of HAQs, and establish the sequence of synthesis of these compounds, which include potent cytochrome inhibitors and antibiotics active against human commensal and pathogenic bacteria. We find that anthranilic acid, the product of the PhnAB synthase, is the primary precursor of HAQs and that the HAQ congener 4-hydroxy-2-heptylquinoline (HHQ) is the direct precursor of the PQS signaling molecule. Significantly, whereas phnAB and pqsA-E are positively regulated by the virulence-associated transcription factor MvfR, which is also required for the expression of several QS-regulated genes, the conversion of HHQ to PQS is instead controlled by LasR. Finally, our results reveal that HHQ is itself both released from, and taken up by, bacterial cells where it is converted into PQS, suggesting that it functions as a messenger molecule in a cell-to-cell communication pathway. HAQ signaling represents a potential target for the pharmacological intervention of P. aeruginosa -mediated infections.

Background: Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important opportunistic human pathogen. Generally, the acquisition of genes in the form of pathogenicity islands distinguishes pathogenic isolates from nonpathogens. We therefore sequenced a highly virulent strain of P. aeruginosa, PA14, and compared it with a previously sequenced (and less pathogenic) strain, PAO1, to identify novel virulence genes. Results: The PA14 and PAO1 genomes are remarkably similar, although PA14 has a slightly larger genome (6.5 megabses [Mb]) than does PAO1 (6.3 Mb). We identified 58 PA14 gene clusters that are absent in PAO1 to determine which of these genes, if any, contribute to its enhanced virulence in a Caenorhabditis elegans pathogenicity model. First, we tested 18 additional diverse strains in the C. elegans model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of PA14 genes that are absent in PAO1 did not correlate with the virulence of these strains. Second, we utilized a full-genome nonredundant mutant library of PA14 to identify five genes (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans. Conclusion: Genes required for pathogenicity in one strain of P. aeruginosa are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is both multifactorial and combinatorial, the result of a pool of pathogenicity-related genes that interact in various combinations in different genetic backgrounds.

Opportunistic infections are often polymicrobial. Two of the most important bacterial opportunistic pathogens of humans, Pseudomonas aeruginosa and Staphylococcus aureus , frequently are coisolated from infections of catheters, endotracheal tubes, skin, eyes, and the respiratory tract, including the airways of people with cystic fibrosis (CF). Here, we show that suppression of S. aureus respiration by a P. aeruginosa exoproduct, 4-hydroxy-2-heptylquinoline- N -oxide (HQNO), protects S. aureus during coculture from killing by commonly used aminoglycoside antibiotics such as tobramycin. Furthermore, prolonged growth of S. aureus with either P. aeruginosa or with physiological concentrations of pure HQNO selects for typical S. aureus small-colony variants (SCVs), well known for stable aminoglycoside resistance and persistence in chronic infections, including those found in CF. We detected HQNO in the sputum of CF patients infected with P. aeruginosa , but not in uninfected patients, suggesting that this HQNO-mediated interspecies interaction occurs in CF airways. Thus, in all coinfections with P. aeruginosa , S. aureus may be underappreciated as a pathogen because of the formation of antibiotic-resistant and difficult to detect small-colony variants. Interspecies microbial interactions, analogous to those mediated by HQNO, commonly may alter not only the course of disease and the response to therapy, but also the population structure of bacterial communities that promote the health of host animals, plants, and ecosystems.

Pseudomonas aeruginosaproduces extracellular glycolipids composed ofl-rhamnose and 3-hydroxyalkanoic acid called rhamnolipids. Although these compounds are usually regarded as biosurfactants or haemolysins, their exact physiological function is not well understood. Rhamnolipids are synthesized by a rhamnosyltransferase, encoded by therhlABoperon, which catalyses the transfer of TDP-l-rhamnose to 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) moieties of various lengths. RhlB is the catalytic protein of the rhamnosyltransferase.rhlAis indispensable for rhamnolipid synthesis, but its function is unknown. Using a liquid chromatography/mass spectrometry method, the production of extracellular HAAs byP. aeruginosawas detected previously and it was demonstrated that they are the actual precursors of rhamnolipid biosynthesis. In this report, evidence is presented indicating thatrhlAis required for production of HAAs and that these HAAs display potent surface-active properties.P.aeruginosacan colonize surfaces by swarming motility, a form of organized translocation requiring the production of wetting agents. UsingrhlAandrhlBmutants it was observed that swarming requires the expression of therhlAgene but does not necessitate rhamnolipid production, as HAAs act as surfactants. Finally, it was shown that the use of ammonium instead of nitrate as source of nitrogen and an excess of available iron both decreaserhlAexpression and swarming motility.

Summary: The transcriptional regulator MvfR is required for full Pseudomonas aeruginosa virulence, the function of multiple quorum sensing (QS)‐regulated virulence factors and the synthesis of 4‐hydroxy‐2‐alkylquinolines (HAQs), including the Pseudomonas quinolone signal (PQS). Here we investigate the role of MvfR in the QS circuitry and P. aeruginosa pathogenesis. We demonstrate using a combination of biochemical and molecular approaches, including transcription profiling, that MvfR is involved in the regulation of multiple P. aeruginosa QS‐controlled genes without altering the expression of lasRI/rhlRI or the production of N‐acyl‐ l‐homoserine lactone (AHL) signals. Dissection of how mvfR is interwoven into the P. aeruginosa QS circuitry reveals that the MvfR system, through the essential contribution of PqsE, positively regulates a subset of genes dependant on both LasR and RhlR. Animal studies show that MvfR contributes to P. aeruginosa virulence by controlling the transcription of genes not under RhlR regulation, and that reduced virulence of a mvfR mutant is caused by the loss of pqsE expression and not only a deficiency in HAQs/PQS production. This study provides novel insights into the unique role of the MvfR system in AHL‐mediated QS and further supports its importance in P. aeruginosa pathogenesis.