Expression Of Quorum Sensing Genes Research Articles

Quorum Sensing Inhibiting Activity of Streptomyces coelicoflavus Isolated from Soil.

Quorum sensing (QS) systems communicate bacterial population and stimulate microbial pathogenesis through signaling molecules. Inhibition of QS signals potentially suppresses microbial infections. Antimicrobial properties of Streptomyces have been extensively studied, however, less is known about quorum sensing inhibitory (QSI) activities of Streptomyces. This study explored the QSI potential of Streptomyces isolated from soil. Sixty-five bacterial isolates were purified from soil samples with morphological characteristics of Streptomyces. The three isolates: S6, S12, and S17, exhibited QSI effect by screening with the reporter, Chromobacterium violaceum. Isolate S17 was identified as Streptomyces coelicoflavus by sequencing of the hypervariable regions (V1–V6) of 16S rRNA and was assigned gene bank number KJ855087. The QSI effect of the cell-free supernatant of isolate S17 was not abolished by proteinase K indicating the non-enzymatic activity of QSI components of S17. Three major compounds were isolated and identified, using spectroscopic techniques (1D, 2D NMR, and Mass spectrometry), as behenic acid (docosanoic acid), borrelidin, and 1H-pyrrole-2-carboxylic acid. 1H-pyrrole-2-carboxylic acid inhibited QS and related virulence factors of Pseudomonas aeruginosa PAO1 including; elastase, protease, and pyocyanin without affecting Pseudomonas viability. At the molecular level, 1H-pyrrole-2-carboxylic acid suppressed the expression of QS genes (lasI, lasR, lasA, lasB, rhlI, rhlR, pqsA, and pqsR). Moreover, QSI activity of S17 was assessed under different growth conditions and ISP2 medium supplemented with glucose 0.4% w/v and adjusted at pH 7, showed the highest QSI action. In conclusion, 1H-pyrrole-2-carboxylic acid, one of the major metabolites of Streptomyces isolate S17, inhibited QS and virulence determinants of P. aeruginosa PAO1. The findings of the study open the scope to exploit the in vivo efficacy of this active molecule as anti-pathogenic and anti-virulence of P. aeruginosa.

Plant phenolic acids affect the virulence of Pectobacterium aroidearum and P. carotovorum ssp. brasiliense via quorum sensing regulation.

Several studies have reported effects of the plant phenolic acids cinnamic acid (CA) and salicylic acid (SA) on the virulence of soft rot enterobacteria. However, the mechanisms involved in these processes are not yet fully understood. Here, we investigated whether CA and SA interfere with the quorum sensing (QS) system of two Pectobacterium species, P. aroidearum and P. carotovorum ssp. brasiliense, which are known to produce N-acyl-homoserine lactone (AHL) QS signals. Our results clearly indicate that both phenolic compounds affect the QS machinery of the two species, consequently altering the expression of bacterial virulence factors. Although, in control treatments, the expression of QS-related genes increased over time, the exposure of bacteria to non-lethal concentrations of CA or SA inhibited the expression of QS genes, including expI, expR, PC1_1442 (luxR transcriptional regulator) and luxS (a component of the AI-2 system). Other virulence genes known to be regulated by the QS system, such as pecS, pel, peh and yheO, were also down-regulated relative to the control. In agreement with the low levels of expression of expI and expR, CA and SA also reduced the level of the AHL signal. The effects of CA and SA on AHL signalling were confirmed in compensation assays, in which exogenous application of N-(β-ketocaproyl)-l-homoserine lactone (eAHL) led to the recovery of the reduction in virulence caused by the two phenolic acids. Collectively, the results of gene expression studies, bioluminescence assays, virulence assays and compensation assays with eAHL clearly support a mechanism by which CA and SA interfere with Pectobacterium virulence via the QS machinery.

Expression of quorum-sensing related genes during Enterococcus faecalis biofilm formation

To investigate the relationship between the expression of the quorum-sensing related genes during Enterococcus faecalis(Ef) biofilm formation. Ef biofilms model was established in vitro and film formation process was observed by confocal laser scanning microscope at 6, 12, 24 and 48 hours respectively.Quantification of biofilms was achieved by staining with crystal violet.Real-time fluorescence quantitative PCR method was used to detect the expression of fsrB, gelE and sprE genes in the process of Ef biofilm formation. A lot of live and dead bacteria unevenly distributed in Ef biofilm. The quantity of biofilms increased with time within 24 hours and was 0 h:0.00 ± 0.00, 6 h:1.09 ± 0.13, 12 h:2.10 ± 0.79, 24 h:3.30 ± 0.13, which was significantly different among the 4 time period(P < 0.05). The quantity of biofilm at 48 h(3.51 ± 0.01) increased slightly compared with 24 h(3.30 ± 0.13) , but did not show significant difference.Quantitative real-time PCR showed that the expression of quorum-sensing related fsrB increased with time within 24 hours and was 0 h:9.98 ± 0.46, 6 h:23.45 ± 1.13, 12 h:47.30 ± 2.49, 24 h: 331.30 ± 2.18, which was significantly different among the 4 time period(P < 0.05). The expression of gelE was 0 h: 6.54 ± 0.73, 6 h: 14.26 ± 1.24, 12 h: 37.47 ± 2.35, 24 h:264.80 ± 5.10(P < 0.05). The expression of sprE was 0 h: 7.72 ± 0.74, 6 h: 21.15 ± 0.96, 12 h:49.87 ± 3.18, 24 h:441.89 ± 7.74, which was significantly different among the 4 time period(P < 0.05). The fsrB, gelE and sprE genes are closely related to the biofilm formation in Ef.

Quorum sensing-dependent virulence during Pseudomonas aeruginosa colonisation and pneumonia in mechanically ventilated patients

BackgroundPseudomonas aeruginosa frequently colonises intubated patients and causes life-threatening ventilator-associated pneumonia (VAP). The role of quorum sensing (QS), regulating virulence in this pathogen, during colonisation and development of VAP is...

A low concentration of azithromycin inhibits the mRNA expression of N-acyl homoserine lactone synthesis enzymes, upstream of lasI or rhlI, in Pseudomonas aeruginosa

Low-dose macrolides are effective therapy in patients with chronic lung infections, but the mechanisms of action are unclear. In this study, we performed DNA microarray analysis of Pseudomonas aeruginosa after treatment with a low concentration of azithromycin. We found that a sub-MIC of azithromycin didn't change mRNA expression of quorum-sensing related genes ( lasI, lasR, rhlI, rhlR, vft, rsaL), but lowered expression of most N-acyl homoserine lactone (AHL) synthesis enzymes upstream of lasI and rhlI. We propose that small down regulation of these enzymes cumulatively resulted in a larger decrease of AHL production and inhibition of quorum-sensing in P. aeruginosa.

Otopathogenic Pseudomonas aeruginosa Strains as Competent Biofilm Formers

To determine whether Pseudomonas aeruginosa, a common cholesteatoma pathogen, known to form biofilms in other chronic infections, is capable of contributing to biofilm formation in cholesteatoma. We tested 12 OPPA isolates for several aspects of biofilm formation, including adherence to human keratinocytes, expression of quorum-sensing genes, twitching motility, and production of extracellular matrix as determined by both crystal violet staining and carbazole reaction. Ten OPPA strains demonstrated increased adherence (1.5- to 12-fold) to human keratinocytes relative to PAO1, a laboratory strain. Expression of las and rhl quorum-sensing products were detected in 11 OPPA strains. By crystal violet staining, we found biofilm formation in all OPPA strains equal to or greater than that found in PAO1 (2- to 18-fold). In addition, OPPA strains demonstrated mucoid characteristics, including down-regulation of twitching motility and increased alginate production. Strains of OPPA isolated from cholesteatoma are strongly adherent to keratinocytes and capable of forming biofilm. In addition, OPPA strains have mucoid characteristics in vitro. When these bacteria assume a biofilm phenotype, they are highly resistant to antibiotics and host defenses. These data suggest that OPPA can contribute to biofilm formation in cholesteatoma, leading to the persistence of this infection.

8] Quorum sensing, gene expression, and Pseudomonas biofilms

Quorum sensing has been shown to be important for the development of a normal P. aeruginosa biofilm, and it follows that other microorganisms may employ a similar mechanism in the development of mature biofilms. To methods for detecting the presence of AI activity in biofilms are presented that employ an AI-responsive reporter strain harboring a lacZ fusion. Method 1 involves detection of AI activity in crude biofilms, whereas Method 2 employs an AI purification procedure. By using multiple indicator strains activated by AIs various acyl chain lengths, a wide range of AI molecules can be detected. Chromosomal knockout mutants are extremely useful for examining the contribution of a given gene to a specific phenotype. For quorum-sensing gene expression studies, mutants deficient in the production of AI offer more versatility than R-protein mutants. The main advantage of the AI mutants is that they can be complemented by either the AI synthase gene or the AI itself. Complementation with the AI circumvents having to grow the cells in the presence of antibiotics and allows experimental parameters such as AI concentration and time of addition to be manipulated easily. Finally, three reporter systems suitable for monitoring gene expression in P. aeruginosa biofilms are summarized in T Table II. The choice of reporter fusion depends mainly on whether in vivo analysis is required, whether temporal gene expression is to be examined, and the availability of equipment. In the case of P. aeruginosa, expression of quorum-sensing genes can be monitored either directly, by examining fusions of the R genes or AI synthase genes, or indirectly, by analyzing expression of genes controlled by these quorum-sensing systems.