Quorum Sensing and Virulence of Pseudomonas aeruginosa during Lung Infection of Cystic Fibrosis Patients

Pseudomonas aeruginosa is the predominant microorganism in chronic lung infection of cystic fibrosis patients. The chronic lung infection is preceded by intermittent colonization. When the chronic infection becomes established, it is well accepted that the isolated strains differ phenotypically from the intermittent strains. Dominating changes are the switch to mucoidity (alginate overproduction) and loss of epigenetic regulation of virulence such as the Quorum Sensing (QS). To elucidate the dynamics of P. aeruginosa QS systems during long term infection of the CF lung, we have investigated 238 isolates obtained from 152 CF patients at different stages of infection ranging from intermittent to late chronic. Isolates were characterized with regard to QS signal molecules, alginate, rhamnolipid and elastase production and mutant frequency. The genetic basis for change in QS regulation were investigated and identified by sequence analysis of lasR, rhlR, lasI and rhlI. The first QS system to be lost was the one encoded by las system 12 years (median value) after the onset of the lung infection with subsequent loss of the rhl encoded system after 17 years (median value) shown as deficiencies in production of the 3-oxo-C12-HSL and C4-HSL QS signal molecules respectively. The concomitant development of QS malfunction significantly correlated with the reduced production of rhamnolipids and elastase and with the occurrence of mutations in the regulatory genes lasR and rhlR. Accumulation of mutations in both lasR and rhlR correlated with development of hypermutability. Interestingly, a higher number of mucoid isolates were found to produce C4-HSL signal molecules and rhamnolipids compared to the non-mucoid isolates. As seen from the present data, we can conclude that P. aeruginosa and particularly the mucoid strains do not lose the QS regulation or the ability to produce rhamnolipids until the late stage of the chronic infection.     

Screening and anti-virulent study of N-acyl homoserine lactones DNA aptamers against Pseudomonas aeruginosa quorum sensing

In Pseudomonas aeruginosa, a quorum sensing (QS) system regulates the expression of many virulence factors. N-acyl homoserine lactone (HSL) is the signal molecule of QS system. In order to find a novel HSL binder to interfere with QS signaling and to attenuate P. aeruginosa virulence, an amino lactam surrogate (ALS) of HSL was used as a target to screen HSL aptamers with the technique of systematic evolution of ligands by exponential enrichment (SELEX). Eight HSL aptamers with high affinities for 3O-C12-HSL (20 nM ≤ K _d < 35 nM) or C4-HSL (25 nM < K _d < 50 nM) were finally obtained. In vitro QS-inhibiting study of P. aeruginosa showed that HSL aptamers could inhibit virulence in a dose-dependent manner. ALSap-8 which bound C4-HSL primarily acted on the rhl system and inhibited the secretion of pyocyanin. ALSap-5 which bound 3O-C12-HSL not only showed strong inhibitory activity on biofilm formation as well as secretions of LasA protease and LasB elastase, but also reduced pyocyanin secretion. Since the las system is capable of activating the rhl system mildly, we speculated that ALSap-5 can simultaneously interfere with the las and rhl systems. High-affinity aptamers against HSL in this study are novel QS and virulence-inhibitors, and may have potential as drug candidates for the treatment of P. aeruginosa infection.     

A Novel Signal Transduction Pathway that Modulates rhl Quorum Sensing and Bacterial Virulence in Pseudomonas aeruginosa

The rhl quorum-sensing (QS) system plays critical roles in the pathogenesis of P. aeruginosa. However, the regulatory effects that occur directly upstream of the rhl QS system are poorly understood. Here, we show that deletion of gene encoding for the two-component sensor BfmS leads to the activation of its cognate response regulator BfmR, which in turn directly binds to the promoter and decreases the expression of the rhlR gene that encodes the QS regulator RhlR, causing the inhibition of the rhl QS system. In the absence of bfmS, the Acka-Pta pathway can modulate the regulatory activity of BfmR. In addition, BfmS tunes the expression of 202 genes that comprise 3.6% of the P. aeruginosa genome. We further demonstrate that deletion of bfmS causes substantially reduced virulence in lettuce leaf, reduced cytotoxicity, enhanced invasion, and reduced bacterial survival during acute mouse lung infection. Intriguingly, specific missense mutations, which occur naturally in the bfmS gene in P. aeruginosa cystic fibrosis (CF) isolates such as DK2 strains and RP73 strain, can produce BfmS variants (BfmS_L181P, BfmS_L181P/E376Q, and BfmS_R393H) that no longer repress, but instead activate BfmR. As a result, BfmS variants, but not the wild-type BfmS, inhibit the rhl QS system. This study thus uncovers a previously unexplored signal transduction pathway, BfmS/BfmR/RhlR, for the regulation of rhl QS in P. aeruginosa. We propose that BfmRS TCS may have an important role in the regulation and evolution of P. aeruginosa virulence during chronic infection in CF lungs.     

Physiological Framework for the Regulation of Quorum Sensing-Dependent Public Goods in Pseudomonas aeruginosa

Many bacteria possess cell density-dependent quorum-sensing (QS) systems that often regulate cooperative secretions involved in host-microbe or microbe-microbe interactions. These secretions, or “public goods,” are frequently coregulated by stress and starvation responses. Here we provide a physiological rationale for such regulatory complexity in the opportunistic pathogen Pseudomonas aeruginosa. Using minimal-medium batch and chemostat cultures, we comprehensively characterized specific growth rate-limiting macronutrients as key triggers for the expression of extracellular enzymes and metabolites directly controlled by the las and rhl QS systems. Expression was unrelated to cell density, depended on the secreted product''s elemental composition, and was induced only when the limiting nutrient was not also a building block of the product; rhl-dependent products showed the strongest response, caused by the largely las-independent induction of the regulator RhlR and its cognate signal. In agreement with the prominent role of the rhl system, slow growth inverted the las-to-rhl signal ratio, previously considered a characteristic distinguishing between planktonic and biofilm lifestyles. Our results highlight a supply-driven, metabolically prudent regulation of public goods that minimizes production costs and thereby helps stabilize cooperative behavior. Such regulation would be beneficial for QS-dependent public goods that act broadly and nonspecifically, and whose need cannot always be accurately assessed by the producing cell. Clear differences in the capacities of the las and rhl systems to integrate starvation signals help explain the existence of multiple QS systems in one cell.     

Quorum-Sensing Genes in Pseudomonas aeruginosa Biofilms: Their Role and Expression Patterns

Acylated homoserine lactone molecules are used by a number of gram-negative bacteria to regulate cell density-dependent gene expression by a mechanism known as quorum sensing (QS). In Pseudomonas aeruginosa, QS or cell-to-cell signaling controls expression of a number of virulence factors, as well as biofilm differentiation. In this study, we investigated the role played by the las and rhl QS systems during the early stages of static biofilm formation when cells are adhering to a surface and forming microcolonies. These studies revealed a marked difference in biofilm formation between the PAO1 parent and the QS mutants when glucose, but not citrate, was used as the sole carbon source. To further elucidate the contribution of lasI and rhlI to biofilm maturation, we utilized fusions to unstable green fluorescent protein in concert with confocal microscopy to perform real-time temporal and spatial studies of these genes in a flowing environment. During the course of 8-day biofilm development, lasI expression was found to progressively decrease over time. Conversely, rhlI expression remained steady throughout biofilm development but occurred in a lower percentage of cells. Spatial analysis revealed that lasI and rhlI were maximally expressed in cells located at the substratum and that expression decreased with increasing biofilm height. Because QS was shown previously to be involved in biofilm differentiation, these findings have important implications for the design of biofilm prevention and eradication strategies.     

Influence of clove oil on certain quorum-sensing-regulated functions and biofilm of Pseudomonas aeruginosa and Aeromonas hydrophila

Quorum sensing (QS) plays an important role in virulence, biofilm formation and survival of many pathogenic bacteria including Pseudomonas aeruginosa. This signalling pathway is considered as novel and promising target for anti-infective agents. In the present investigation, effect of the Sub-MICs of clove oil on QS regulated virulence factors and biofilm formation was evaluated against P. aeruginosa PAO1 and Aeromonas hydrophila WAF-38 strain. Sub-inhibitory concentrations of the clove oil demonstrated statistically significant reduction of las- and rhl-regulated virulence factors such as LasB, total protease, chitinase and pyocyanin production, swimming motility and exopolysaccharide production. The biofilm forming capability of PAO1 and A. hydrophila WAF-38 was also reduced in a concentration-dependent manner at all tested sub-MIC values. Further, the PAO1-preinfected Caenorhabditis elegans displayed an enhanced survival when treated with 1.6% v/v of clove oil. The above findings highlight the promising anti-QS-dependent therapeutic function of clove oil against P. aeruginosa.     

Pulse Generation in the Quorum Machinery of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas aeruginosa is a Gram-negative bacterium that is responsible for a wide range of infections in humans. Colonies employ quorum sensing (QS) to coordinate gene expression, including for virulence factors, swarming motility and complex social traits. The QS signalling system of P. aeruginosa is known to involve multiple control components, notably the las, rhl and pqs systems. In this paper, we examine the las system and, in particular, the repressive interaction of rsaL, an embedded small regulative protein, employing recent biochemical information to aid model construction. Using analytic methods, we show how this feature can give rise to excitable pulse generation in this subsystem with important downstream consequences for rhamnolipid production. We adopt a symmetric competitive inhibition to capture the binding in the lasI–rsaL intergenic region and show our results are not dependent on the exact choice of this functional form. Furthermore, we examine the coupling of lasR to the rhl system, the impact of the predicted capacity for pulse generation and the biophysical consequences of this behaviour. We hypothesize that the interaction between the las and rhl systems may provide a quorum memory to enable cells to trigger rhamnolipid production only when they are at the edge of an established aggregation.     

Inhibitory effects of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) on acyl-homoserine lactone-mediated virulence factor production and biofilm formation in Pseudomonas aeruginosa PAO1

4-Hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF), a non-halogenated furanone found in a variety of fruits, has been shown to have antimicrobial activity. However, few studies have focused on its inhibitory effect on bacterial quorum sensing (QS) at levels below the non-inhibitory concentration. In this study, 0.1 μM HDMF decreased the production of QS signal molecules and inhibited QS-controlled biofilm formation by Pseudomonas aeruginosa PAO1 without causing growth inhibition. In the presence of 0.1 and 1.0 μM HDMF, biofilm production by PAO1 was reduced by 27.8 and 42.6%, respectively, compared to that by untreated control cells. HDMF (1.0 μM) also significantly affected virulence factor expression (regulated by the las, rhl, and pqs system), resulting in a significant reduction in the production of LasA protease (53.8%), rhamnolipid (40.9%), and pyocyanin (51.4%). This HDMF-dependent inhibition of virulence factor expression was overcome by increasing the levels of two QS signal molecules of P. aeruginosa, N-(3-oxo-dodecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, suggesting reversible competitive inhibition between HDMF and these molecules. The results of this study indicate that HDMF has great potential as an inhibitor of QS, and that it may be of value as a therapeutic agent and in biofilm control, without increasing selective pressure for resistance development.     

Study on pyoverdine and biofilm production with detection of LasR gene in MDR Pseudomonas aeruginosa

In cystic fibrosis individuals, chronic lung infections and hospital-acquired pneumonia are caused by Pseudomonas aeruginosa. P. aeruginosa generates siderophores such as pyoverdine (PVD) as iron uptake systems to cover its needs of iron ions for growth and infection. lasR quorum sensing (QS) gene has a crucial function in PVD production and biofilm generation in P. aeruginosa. Fifty isolates of P. aeruginosa were obtained from clinical specimens of sputum (collected from individuals suffering from pulmonary infections). Antibiotic sensitivity test was performed for 50P. aeruginosa isolates by using 10 different types of antibiotics. All isolates of P. aeruginosa showed resistance for all 10 using antibiotics in this study. Ten multidrug resistant isoloates of P. aeruginosa were selected for next tests. Virulence factors of ten multidrug resistant isolates of P. aeruginosa, such as biofilm generation, PVD production, and lasR gene were detected. From results, all 10P. aeruginosa isolates can produce biofilm, PVD, and contain lasR gene. The produced amplicon for the lasR gene was 725 bp. After mice injection by fresh and heated PVD produced by P. aeruginosa PS10 LC619328.2, the fresh PVD caused 100 % mortality within five days using 0.3 ml of its concentration (37.4 µM), while (15.3 µM) of heated PVD (toxoid) caused 50 % mortality.     

Cystic and Non-Cystic Fibrosis <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolates are not Differentiated by the Quorum-Sensing Signaling and Biofilm Production

The capability for biofilm and quorum-sensing (QS) signaling production among Pseudomonas aeruginosa isolates were evaluated. A total of 231 isolates were recovered from sputa of cystic fibrosis (CF, n = 104) and non-CF (non-CF, n = 127) patients. One hundred ninety-seven (85.3%; 95% CI 80.1–89.3%) were biofilm producers and 157 (68%; 95% CI 61.7–73.6%) were weak QS-producing. No difference was observed between CF and non-CF isolates regarding the ability to produce biofilm and QS-signaling. Interestingly, the degree of QS production appears to be related to the degree of biofilm production. Thus, blocking QS pathways may be crucial in the prevention and treatment of biofilm-related infections.     

Loss of Social Behaviours in Populations of Pseudomonas aeruginosa Infecting Lungs of Patients with Cystic Fibrosis

Pseudomonas aeruginosa, is an opportunistic, bacterial pathogen causing persistent and frequently fatal infections of the lung in patients with cystic fibrosis. Isolates from chronic infections differ from laboratory and environmental strains in a range of traits and this is widely interpreted as the result of adaptation to the lung environment. Typically, chronic strains carry mutations in global regulation factors that could effect reduced expression of social traits, raising the possibility that competitive dynamics between cooperative and selfish, cheating strains could also drive changes in P. aeruginosa infections. We compared the expression of cooperative traits - biofilm formation, secretion of exo-products and quorum sensing (QS) - in P. aeruginosa isolates that were estimated to have spent different lengths of time in the lung based on clinical information. All three exo-products involved in nutrient acquisition were produced in significantly smaller quantities with increased duration of infection, and patterns across four QS signal molecules were consistent with accumulation over time of mutations in lasR, which are known to disrupt the ability of cells to respond to QS signal. Pyocyanin production, and the proportion of cells in biofilm relative to motile, free-living cells in liquid culture, did not change. Overall, our results confirm that the loss of social behaviour is a consistent trend with time spent in the lung and suggest that social dynamics are potentially relevant to understanding the behaviour of P. aeruginosa in lung infections.     

铜绿假单胞菌群体感应信号分子PQS的功能多样性研究进展

铜绿假单胞菌(Pseudomonas aeruginosa)是一种革兰氏阴性条件致病菌,可对免疫功能低下或损伤的患者造成持续性感染。铜绿假单胞菌能成功感染离不开其自身产生的毒力因子,而这些毒力因子大多数都受群体感应系统(quorum sensing,QS)调控。铜绿假单胞菌有4个QS系统,分别为las系统、rhl系统、pqs系统和iqs系统。2-庚基-3-羟基-4-喹诺酮(Pseudomonas quinolone signal,PQS)作为铜绿假单胞菌pqs系统的信号分子,不仅能够调控许多毒力因子的表达,也能够影响一些微生物和宿主的多种生理过程。本文总结了PQS多种生物学功能,如介导QS系统、调控生物被膜形成、介导外膜囊泡产生及铁摄取、调节宿主免疫活性、介导细胞毒性作用,以及提供种群保护等。本文旨在突出铜绿假单胞菌PQS的功能多样性,并为PQS新功能研究和抗菌药物的研发提供指导。     

The Distinct Quorum Sensing Hierarchy of las and rhl in Pseudomonas sp. M18

Pseudomonas sp. M18 is a rhizosphere isolate capable of producing two kinds of antifungal agents: phenazine-1-carboxylic acid (PCA) and pyoluteorin. Recently, the two well-studied quorum sensing (QS) systems of Pseudomonas aeruginosa, LasR/LasI and RhlR/RhlI, have also been identified in this strain. However, in this study, through the use of lacZ translational fusion expression analysis and acyl-homoserine lactone thin-layer chromatography (TLC) bioassays, we clearly display a more complex and distinctive hierarchy of the las and rhl QS systems in strain M18. In this QS cascade, expression of rhlI was negatively controlled by the LasR/LasI QS system. In contrast with lasI, which negatively regulated the rhlR induction, lasR exerted a positive influence on rhlR expression during the log-phase. This interrelationship indicated that the response regulators (LasR and RhlR) of the QS system are expressed independently of their cognate synthases (LasI and RhlI). Furthermore, the las system also modulated the timing and magnitude of the rhlI and rhlR maximal expression. In addition, our data imply that the lasR gene exerts its negative control on PCA production through modulation of rhlI expression. Thus, interactions between the two QS systems are strain specific.     

Identification of a Pseudomonas sp. that Inhibits RHL System of Quorum Sensing

The production of many Pseudomonas aeruginosa virulence factors and secondary metabolites is regulated in concert with cell density by quorum sensing (QS). Therefore, strategies designed to inhibit QS are promising for the control of diseases. Here, we succeeded in isolating soil bacteria (56 out of 7,000 isolates) capable of inhibiting violacein production by Chromobacterium violaceum CV026. We focused on an isolate identified as a Pseudomonas sp. based on its 16S rRNA nucleotide sequence. A partially purified inhibitor factor(s) derived from culture supernatants consisted of at least three major components by HPLC analysis. A more highly purified preparation (16 μg/ml) specifically inhibited rhl-controlled pyocyanin and rhamnolipid production by wild type P. aeruginosa PAO1 (PAO1) and a QS double mutant PAO-MW1, without affecting growth. A significant inhibitory effect on elastase, protease and biofilm was also observed. These results provide compelling evidence that the inhibitor(s) interferes with the QS system. The identities of the inhibitors remain to be established.     

Equisetin as potential quorum sensing inhibitor of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Objective

To screen for the quorum-sensing (QS) inhibitors from marine-derived fungi and evaluate their anti-QS properties in Pseudomonas aeruginosa.

Results

QS inhibitory activity was found in secondary metabolites of a marine fungus Fusarium sp. Z10 using P. aeruginosa QSIS-lasI biosensor. The major active compound of this fungus was isolated by HPLC and identified as equisetin. Subinhibitory concentration of equisetin could inhibit the formation of biofilm, swarming motility, and the production of virulence factors in P. aeruginosa. The inhibition of las, PQS, and rhl system by equisetin were determined using Escherichia coli MG4/pKDT17, E.coli pEAL08-2, and E.coli pDSY, respectively. Real–time RT-PCR assays showed that equisetin could downregulate the mRNA expression of QS-related genes.

Conclusions

Equisetin proved its potential as an inhibitor against P. aeruginosa QS system and might also serve as precursor compound in development of novel therapeutics for infectious diseases by optimal design of structures.

     

铜绿假单胞菌生物被膜组成及其受群体感应系统和c-di-GMP调控的研究进展

作为人类条件性感染的前三大病原菌之一的铜绿假单胞菌,是一种革兰氏阴性细菌,对免疫功能低下和囊性纤维化患者可以造成严重和持续性感染。造成这种持续感染的原因主要是由于细菌接收外界信号后,在自身调控网络的协同作用下,会依附于固体表面,并产生胞外多糖、基质蛋白和胞外DNA等大分子物质形成高度结构化的膜状复合物将自身包裹形成生物被膜群体结构。生物被膜可以有效帮助细菌定殖、提高细菌对抗菌物质和宿主免疫反应的抵抗能力、促进群落细菌的细胞-细胞之间的信号交流等,是临床治疗中病原菌慢性感染和反复感染最重要的原因之一。本篇综述重点介绍了铜绿假单胞菌生物被膜的各组成成分及其在生物被膜形成中的重要功能,并进一步阐述了群体感应系统(las、rhl、pqs与iqs)和c-di-GMP对铜绿假单胞菌生物被膜形成的调控作用。通过本篇综述可以更清晰地了解细菌生物被膜形成和调控的过程,为开发新的治疗生物被膜感染策略提供帮助。     

Interspecific Small Molecule Interactions between Clinical Isolates of Pseudomonas aeruginosa and Staphylococcus aureus from Adult Cystic Fibrosis Patients

Pseudomonas aeruginosa and Staphylococcus aureus are the most prevalent pathogens in airway infections of cystic fibrosis (CF) patients. We studied how these pathogens coexist and interact with each other. Clinical isolates of both species were retrieved from adult CF patients. Culture supernatants from 63 P. aeruginosa isolates triggered a wide range of biofilm-stimulatory activities when added to the culture of a control S. aureus strain. The extent of biofilm formation by S. aureus was positively correlated to the levels of the 2-alkyl-4-(1H)-quinolones (AQs) Pseudomonas Quinolone Signal (PQS) and 2-heptyl-4-hydroxy quinoline N-oxide (HQNO) produced by the P. aeruginosa isolates. Supernatants from P. aeruginosa isogenic mutants deficient in PQS and HQNO production stimulated significantly less biofilm formation by S. aureus than that seen with the parental strain PA14. When studying co-isolated pairs of P. aeruginosa and S. aureus retrieved from patients showing both pathogens, P. aeruginosa supernatants stimulated less biofilm production by the S. aureus counterparts compared to that observed using the control S. aureus strain. Accordingly, some P. aeruginosa isolates produced low levels of exoproducts and also some of the clinical S. aureus isolates were not stimulated by their co-isolates or by PA14 despite adequate production of HQNO. This suggests that colonization of the CF lungs promotes some type of strain selection, or that co-existence requires specific adaptations by either or both pathogens. Results provide insights on bacterial interactions in CF.