Roles of Pseudomonas aeruginosa las and rhl quorum-sensing systems in control of twitching motility

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important human pathogen. The production of several virulence factors by P. aeruginosa is controlled through two quorum-sensing systems, las and rhl. We have obtained evidence that both the las and rhl quorum-sensing systems are also required for type 4 pilus-dependent twitching motility and infection by the pilus-specific phage D3112cts. Mutants which lack the ability to synthesize PAI-1, PAI-2, or both autoinducers were significantly or greatly impaired in twitching motility and in susceptibility to D3112cts. Twitching motility and phage susceptibility in the autoinducer-deficient mutants were partially restored by exposure to exogenous PAI-1 and PAI-2. Both twitching motility and infection by pilus-specific phage are believed to be dependent on the extension and retraction of polar type 4 pili. Western blot analysis of whole-cell lysates and enzyme-linked immunosorbent assays of intact cells were used to measure the amounts of pilin on the cell surfaces of las and rhl mutants relative to that of the wild type. It appears that PAI-2 plays a crucial role in twitching motility and phage infection by affecting the export and assembly of surface type 4 pili. The ability of P. aeruginosa cells to adhere to human bronchial epithelial cells was also found to be dependent on the rhl quorum-sensing system. Microscopic analysis of twitching motility indicated that mutants which were unable to synthesize PAI-1 were defective in the maintenance of cellular monolayers and migrating packs of cells. Thus, PAI-1 appears to have an essential role in maintaining cell-cell spacing and associations required for effective twitching motility.     

Furanone derivatives as quorum-sensing antagonists of Pseudomonas aeruginosa

The biofilm formation of Pseudomonas aeruginosa, an opportunistic human pathogen, is developed by cell-to-cell signaling, so-called quorum sensing (QS). To control the biofilm formation, we designed and synthesized new QS inhibitors of P. aeruginosa based on the structure of the previously known QS inhibitor, furanone. Newly synthesized compounds were a series of analogs of (5-oxo-2,5-dihydrofuran-3-yl)methyl alkanoate, and the structures of all six synthesized compounds was confirmed by NMR and GC/MS analyses. These new QS inhibitor candidates could remarkably inhibit both Pseudomonas QS signaling and biofilm formation, which were assayed by using the recombinant reporter system and flow cell confocal microscopy. The degree of QS inhibition by these new inhibitors varied from 20% to 90%. For the profound understanding about inhibition mechanism, we tried to estimate the binding energy between QS receptor, LasR, and our inhibitors from the in silico modeling system. The predicted binding pattern from the modeling system and our experimental data about QS inhibition were in good agreement. From these results, we suggest a new approach to develop the QS inhibitors and biofilm control agents based on structural modeling.     

Identification of a novel regulator of the quorum-sensing systems in Pseudomonas aeruginosa

Quorum sensing is a global gene-regulatory mechanism in bacteria that enables individual bacterial cells to communicate and coordinate their population behaviors. Quorum sensing is central to the pathogenesis of many bacterial pathogens including Pseudomonas aeruginosa and therefore has been exploited as a target for developing novel antipathogenic drugs. In P. aeruginosa , three intertwined quorum-sensing systems, las, rhl , and the 2-alkyl-4(1 H )-quinolone system, which includes the Pseudomonas quinolone signal (PQS), control virulence factor production, and pathogenesis processes. Previously, we obtained a mutant with diminished expression of the phzA1B1C1D1E1F1G1 operon that is involved in the production of virulence factor phenazine compounds. In this study, the mutant was further characterized, and evidence indicating that the disrupted gene PA1196 in the mutant is a potential regulator of the rhl and PQS systems is presented. PA1196 positively controls the expression of the rhl and PQS systems and affects bacterial motility and multiple virulence factor expression via the quorum-sensing systems. This adds an important new player in the complex quorum-sensing network in P. aeruginosa .     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extracellular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.     

Pseudomonas aeruginosa mutants resistant to urea inhibition of growth on acetanilide.

Pseudomonas aeruginosa AI 3 was able to grow in medium containing acetanilide (N-phenylacetamide) as a carbon source when NH4+ was the nitrogen source but not when urea was the nitrogen source. AIU mutants isolated from strain AI 3 grew on either medium. Urease levels in bacteria grown in the presence of urea were 10-fold lower when NH4+ or acetanilide was also in the medium, but there were no apparent differences in urease or its synthesis between strain AI 3 and mutant AIU 1N. The first metabolic step in the acetanilide utlization is catalyzed by an amidase. Amidases in several AIU strains showed altered physiochemical properties. Urea inhibited amidase in a time-dependent reaction, but the rates of the inhibitory reaction with amidases from the AIU mutants were slower than with AI 3 amidase. The purified amidase from AIU 1N showed a marked difference in its pH/activity profile from that obtained with purified AI 3 amidase. These observations indicate that the ability of strain AIU 1N and the other mutants to grow on acetanilide/urea medium is associated with a mutation in the amidase structural gene; this was confirmed for strain AIU 1N by transduction.     

In vitro biosynthesis of the Pseudomonas aeruginosa quorum-sensing signal molecule N-butanoyl-L-homoserine lactone

In Pseudomonas aeruginosa , synthesis of the quorum-sensing signal molecules N -butanoyl- L -homoserine lactone (BHL) and N -hexanoyl- L -homoserine lactone (HHL) requires the LuxI homologue RhlI(VsmI). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified RhlI can catalyse the biosynthesis of BHL and HHL using either S -adenosylmethionine (SAM) or homoserine lactone (HSL) but not homoserine as the source of the homoserine lactone moiety. As we were unable to detect homoserine lactone in cytoplasmic extracts of Escherichia coli , we conclude that SAM is the natural substrate for RhlI-directed N -acylhomoserine lactone (AHL) biosynthesis. The N -acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n -butanoyl-CoA or n -hexanoyl-CoA). The specificity of RhlI for charged CoA derivatives is demonstrated as RhlI was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n -octanoyl-CoA, n -decanoyl-CoA, DL-β-hydroxybutanoyl-CoA or crotonoyl-CoA. RhlI was also unable to use N -acetyl- S -3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the RhlI-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.     

The quorum-sensing negative regulator RsaL of Pseudomonas aeruginosa binds to the lasI promoter

A mutation in the rsaL gene of Pseudomonas aeruginosa produces dramatically higher amounts of N-acyl homoserine lactone with respect to the wild type, highlighting the key role of this negative regulator in controlling quorum sensing (QS) in this opportunistic pathogen. The DNA binding site of the RsaL protein on the rsaL-lasI bidirectional promoter partially overlaps the binding site of the LasR protein, consistent with the hypothesis that RsaL and LasR could be in binding competition on this promoter. This is the first direct demonstration that RsaL acts as a QS negative regulator by binding to the lasI promoter.