Acyl Homoserine Lactones Induce Early Response in the Airway

Acyl homoserine lactones (AHLs) are intercellular signaling molecules used in quorum sensing by Gram‐negative bacteria. We studied the early effects on the rat airway of in vivo intratracheal administration of AHLs (i.e., P. aeruginosa and B. cepacia) to test the hypothesis that AHLs also act on the airway cells, modifying secretory mechanisms which are important in mucosal defense. One hour after treatment, N‐butyryl‐homoserine lactone (C4‐HL) had caused dilated extracellular spaces, loss of cilia, reduction of secretory material, and the presence of prenecrotic elements in the epithelium, while N‐octanoyl‐homoserine lactone (C8‐HL) caused a mild lesion in the epithelium. After treatment with either C4‐ or C8‐HL, reduced immunoreactivity was found using CC10 antibody. At ultrastructural examination, dilatation of the mitochondria was evident in ciliate and secretory cells, while solitary chemosensory cells appeared better preserved, showing aspects of nucleocytoplasmic activation. Using microarray analysis, we found down‐regulation of early gene Fos and Egr1 in all AHL‐treated specimens. In vivo pharmacological magnetic resonance imaging after C4‐ or C8‐HL treatment showed a slight increase in tracheal secretion at a first evaluation 5 min after administration, with no increase in the following minutes. In conclusion, AHLs induce an early mucosal response, and the chondriomas of ciliate and secretory cells are the main cytological target of AHL action. Our results show that AHL action is not limited to activation of conspecific bacteria, but also modifies innate airway defense mechanisms. Anat Rec, 292:439–448, 2009. © 2009 Wiley‐Liss, Inc.     

Pseudomonas aeruginosa quorum-sensing signal molecule N-(3-oxododecanoyl)-L-homoserine lactone inhibits expression of P2Y receptors in cystic fibrosis tracheal gland cells.

ATP and UTP have been proposed for use as therapeutic treatment of the abnormal ion transport in the airway epithelium in cystic fibrosis (CF), the most characteristic feature of which is permanent infection by Pseudomonas aeruginosa. As for diverse gram-negative bacteria, this pathogenic bacterium accumulates diffusible N-acylhomoserine lactone (AHL) signal molecules, and when a threshold concentration is reached, virulence factor genes are activated. Human submucosal tracheal gland serous (HTGS) cells are believed to play a major role in the physiopathology of CF. Since ATP and UTP stimulate CF epithelial cells through P2Y receptors, we sought to determine whether CF HTGS cells are capable of responding to the AHLs N-butanoyl-L-homoserine lactone (BHL), N-hexanoyl-L-homoserine lactone (HHL), N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), and N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), with special reference to P2Y receptors. All AHLs inhibited ATP- and UTP-induced secretion by CF HTGS cells. The 50% inhibitory concentrations were as high as 10 and 5 microM for BHL and HHL, respectively, but were only 0.3 and 0.4 pM for OdDHL and OHHL, respectively. Furthermore, all AHLs down-regulated the expression of the P2Y2 and P2Y4 receptors. Ibuprofen and nordihydroguaiaretic acid were able to prevent AHL inhibition of the responses to nucleotides, but neither dexamethasone nor indomethacin was able to do this. These data indicate that AHLs may alter responsiveness to ATP and UTP by CF HTGS cells and suggest that, in addition to ATP and/or UTP analogues, ibuprofen may be of use for a combinational pharmacological therapy for CF.     

Construction and phenotypic characterization of M68, an RruI quorum sensing knockout mutant of the photosynthetic alphaproteobacterium Rhodospirillum rubrum

Many bacterial species communicate using a complex system known as quorum sensing (QS) in which gene expression is controlled in response to cell density. In this study an N-acylhomoserine lactone (AHL) synthase (Rru_A3396) knockout mutant (M68) of Rhodospirillum rubrum S1H (WT) was constructed and characterized phenotypically under light anaerobic conditions. Results showed that R. rubrum WT produces unsubstituted, 3-OH and 3-oxo-substituted AHLs with acyl chains ranging from 4 to 14 carbons, with 3-OH-C8 being the most abundant. Growth, pigment content and swimming motility were found to be under the control of this LuxI-type QS system. In addition, cultivation in a low shear environment put forward the aggregative phenotype of M68 and linked biofilm formation to QS in R. rubrum S1H. Interestingly, QS-mutant M68 continued to produce decreased levels of 3-OH-C8-HSL, probably due to the presence of an extra HdtS-type AHL synthase.     

Hormone fatty acid modifications: gram negative bacteria and vertebrates demonstrate common structure and function

Bacteria are known to regulate diverse physiological processes through a mechanism called quorum sensing (QS). Prokaryotes communicate by extracellular signalling compounds, i.e. autoinducers (acyl homoserine lactone, AHL of Gram negative bacteria) or pheromones (post-translationally modified peptides of Gram positive bacteria), which activate genetic pathways when they reach a sufficient concentration (QS). A large number of Gram-negative quorum-sensing systems studied so far utilize N-acyl homoserine lactones as signal molecules. In vertebrates small synthetic molecules called growth hormone secretagogues (GHSs) stimulate the release of growth hormone (GH) from the pituitary. GH release is stimulated by hypothalamic GH-releasing hormone (GHRH) and ghrelin (endogenous ligand of the GHS-receptor, GHS-R). Ghrelin is a 28-amino acid peptide, in which the serine-3 (Ser3) is n-octanoylated, and this modification is essential for ghrelin's activity. Ghrelin is the first known case of a peptide hormone modified by a fatty acid. The major active form of ghrelin is a 28-amino acid peptide with octanoylated Ser3; one of the more represented bacterial autoinducers is the N-Octanoyl-DL-homoserine lactone (C8-HL) molecule. The authors hypothesize that Gram-negative bacteria and vertebrates have a functional similarity in the search of food and an important structural homology of AHL and ghrelin for the highly conserved Serine-acylated motive in both molecules. Our suggestions could help one to understand the convergent origin and the biologic meaning of the Serine-acylated group in these organisms, a biologic meaning very important due to the high conservation in two kingdoms which are so different.     

Influence of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> quorum sensing signal molecule <Emphasis Type="Italic">N</Emphasis>-(3-oxododecanoyl) homoserine lactone on mast cells

Quorum sensing system is a cell-to-cell communication system that plays a pivotal role in virulence expression in bacteria. Recent advances have demonstrated that the Pseudomonas aeruginosa quorum sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC₁₂-HSL), exerts effects on mammalian cells and modulates host immune response. Mast cells (MCs) are strategically located in the tissues that are constantly exposed to external stimulus. Therefore, it is very much possible that 3OC₁₂-HSL may interact with MCs. Little is known, however, about specific effects of 3OC₁₂-HSL on MCs. To address this, we investigated the influence of 3OC₁₂-HSL on cell viability, apoptosis, intracellular calcium and cytokine release in MCs. We found that at high concentrations (100 μM), 3OC₁₂-HSL inhibited proliferation and induced apoptosis in P815. The 3OC₁₂-HSL treatment significantly increased intracellular calcium release in both P815 and HMC-1. We also observed that 3OC₁₂-HSL-induced histamine release and degranulation in HMC-1 cells. Furthermore, 3OC₁₂-HSL-induced IL-6 production at lower concentrations (6.25–12.5 μM) but steadily reduced IL-6 production at high concentration (50–100 μM). These data demonstrate that P. aeruginosa 3OC₁₂-HSL affects MCs function.     

Isolation and characterization of quorum‐sensing signalling molecules in Pseudomonas aeruginosa isolates recovered from nosocomial infections

Pseudomonas aeruginosa is one of the most common pathogens in nosocomial infections. Many studies have documented the role of quorum‐sensing (QS) systems in antibiotic tolerance of P. aeruginosa. N‐acyl homoserine lactones (AHLs) serve as QS signalling molecules and can be a target for modulating bacterial pathogenicity. In this study, nosocomial isolates of P. aeruginosa were characterized for the presence of different types of QS signalling molecules. AHLs were solvent extracted and quantified by determination of β‐galactosidase activity using the Escherichia coli MG4 reporter strain. Further characterization was performed by analytical thin layer chromatography coupled with detection using the Agrobacterium tumefaciens A136 biosensor strain. All P. aeruginosa isolates produced AHLs, but there were differences in the quantity and nature of AHLs. We identified AHLs belonging to C4‐homoserine lactone (HSL), C6‐HSL, C8‐HSL, C10‐HSL and C12‐HSL. AHL profiling of P. aeruginosa isolates showed differences in the amounts and types of AHLs, suggesting differences in the virulence factors and the potential for infection. Our results may be investigated further using animal model systems.     

铜绿假单胞菌密度感应信号分子OdDHL对小鼠肥大细胞作用观察

目的 通过化学合成铜绿假单胞菌密度感应(quorum sensing,QS)信号分子OdDHL,并观察该分子对小鼠肥大细胞生物学作用.方法 通过化学方法 合成OdDHL分子,经质谱、磁共振和高效液相色谱确定其结构和纯度,并采用OdDHL分子感应菌株检测其生物活性.以不同浓度OdDHL分子作用于小鼠肥大细胞系P815,观察不同时间点细胞活力改变、凋亡现象以及胞内钙离子变化.结果 成功合成具有生物活性的OdDHL分子,该分子能以时间、剂量依赖方式抑制细胞活力,并能诱导细胞凋亡,同时可上调胞内钙离子浓度.结论 铜绿假单胞菌OdDHL分子可诱导P815细胞凋亡并上调胞内钙离子浓度.     

Exogenous Yersinia pestis quorum sensing molecules N-octanoyl-homoserine lactone and N-(3-oxooctanoyl)-homoserine lactone regulate the LcrV virulence factor

LcrV is a key Yersinia pestis antigen, immune regulator, and component of the type III secretion system (T3SS). Researchers have shown that N-acyl-homoserine lactones (AHLs) can down-regulate the expression of the LcrV homolog, PcrV, in Pseudomonas aeruginosa. Using ELISA, western blot, DNA microarray analysis, and real time PCR we demonstrate that the addition of AHL molecules N-octanoyl-homoserine lactone (C8) or N-(3-oxooctanoyl)-homoserine lactone (oxo-C8) to Y. pestis cultures down-regulates LcrV protein expression. DNA microarray analysis shows 10 additional T3SS genes are consistently down-regulated by C8 or oxo-C8. This is the first report demonstrating that AHLs regulate Y. pestis virulence factor expression.     

Nodulation-gene-inducing flavonoids increase overall production of autoinducers and expression of N-acyl homoserine lactone synthesis genes in rhizobia

Legume-nodulating rhizobia use N-acyl homoserine lactones (AHLs) to regulate several physiological traits related to the symbiotic plant–microbe interaction. In this work, we show that Sinorhizobium fredii SMH12, Rhizobium etli ISP42 and Rhizobium sullae IS123, three rhizobial strains with different nodulation ranges, produced a similar pattern of AHL molecules, sharing, in all cases, production of N-octanoyl homoserine lactone and its 3-oxo and/or 3-hydroxy derivatives. Interestingly, production of AHLs was enhanced when these three rhizobia were grown in the presence of their respective nod-gene-inducing flavonoid, while a new molecule, C14-HSL, was produced by S. fredii SMH12 upon genistein induction. In addition, expression of AHL synthesis genes traI from S. fredii SMH12 and cinI and raiI from R. etli ISP42 increased when induced with flavonoids, as demonstrated by qRT-PCR analysis.     

Response of leaf-associated bacterial communities to primary acyl-homoserine lactone in the tobacco phyllosphere

The phyllosphere is inhabited by large populations of epiphytic bacteria that are able to modulate their phenotypes and behavior by quorum sensing (QS). However, the impact of acyl-homoserine lactones (AHLs) involved in QS on the ecology of bacteria in their natural habitat remains unclear. Therefore, we used a bioassay and liquid chromatography-mass spectrometry to detect AHLs in the tobacco phyllosphere. Our results identified several AHLs in the tobacco phyllosphere, the majority of which were short-chain AHLs. Furthermore, the addition of an exogenous N-(3-oxohexanoyl) homoserine lactone (3OC6HSL), which is seen in the naturally occurring tobacco phyllosphere, generated variability in the composition of the bacterial community as determined by denaturing gradient gel electrophoresis (DGGE) analysis and phospholipid fatty acid (PLFA) analysis. Notably, the ratio of Gram-positive (GP) bacteria increased in response to treatment with 1 μM AHL, but decreased incipiently when treated with 10 μM AHL. These observations provide insight into the composition of the leaf-colonizing epiphyte community responsible for AHLs, particularly GP bacteria as they do not use AHLs as signaling molecules for QS.     

Occurrence of diversified N‐acyl homoserine lactone mediated biofilm‐forming bacteria in rice rhizoplane

Quorum sensing (QS)‐mediated biofilm‐forming rhizobacteria are indispensable due to their competitiveness in the crop rhizosphere. In the present work, we have reported on the occurrence of diversified bacterial species capable of producing N‐acyl homoserine lactone (AHL) as the QS signal in the roots of a rice plant grown under field conditions. The AHL‐producing bacteria were directly isolated from the rice root by the biosensor reporter (Chromobacterium violaceum CV026) overlay method and characterized for biofilm production by the microtiter plate method. A total of 48 QS‐positive bacterial isolates were purified from different aged (7, 20, 24, 26, and 36 days) rice seedlings. The in vitro biofilm production and genetic diversity as revealed by BOX‐PCR fingerprinting showed high variability among the isolates. Most of the best biofilm‐forming isolates produced a N‐butyryl dl ‐homoserine lactone (a C4‐AHL type) signal in the medium. The 16S ribosomal RNA (rRNA) gene sequence of these putative elite isolates identified that they were close to Aeromonas hydrophila (QS7‐4; QS36‐2), A. enteropelongenes (QS20‐8), A. veronii (QS36‐3), Enterobacter sp. (QS20‐11), Klebsiella pneumoniae (QS24‐6), Kosakonia cowanii (QS24‐21), Providentia rettigeri (QS24‐2), Sphingomonas aquatilis (QS24‐17), and Pseudomonas sihuiensis (QS24‐20). These strains profusely colonized the rice root upon inoculation and formed biofilms on the surface of the root under gnotobiotic conditions. Developing inoculants from these strains would ensure competitive colonization on the rhizoplane of the crop through their biofilm and thereby improve plant growth and health.     

Pseudomonas aeruginosa quorum sensing molecule N-3-oxo-dodecanoyl-l-homoserine lactone activates human platelets through intracellular calcium-mediated ROS generation

Pseudomonas aeruginosa, an opportunistic pathogen release N-3-oxo-dodecanoyl-l-homoserine lactone (3-oxo-C₁₂HSL) and N-butyryl-l-homoserine lactone (C₄-HSL) quorum sensing (QS) molecules to regulate various virulence factors responsible for infection in the host. 3-oxo-C₁₂ HSL not only regulates the bacterial gene expression but also modulates the host cell system. Thus, it is pertinent to evaluate the effect of these QS molecules on blood platelets which is responsible for the maintenance of hemostasis and thrombus formation. Here, in the present study, we showed that 3-oxo-C₁₂ HSL activates platelets in a dose-dependent manner and induces intracellular calcium-mediated reactive oxygen species (ROS) release, whereas no such effect was observed with C₄-HSL. 3-oxo-C₁₂ HSL stimulated ROS release was mediated by NADPH oxidase. Results confirmed the involvement of phospholipase C (PLC) and IP₃ receptor behind intracellular calcium-mediated ROS generation. The impact of 3-oxo-C₁₂ HSL on platelet activation suggests that it could interfere and alter the normal function of platelet in individuals infected with P. aeruginosa.     

Bacterial communications in implant infections: a target for an intelligence war

The status of population density is communicated among bacteria by specific secreted molecules, called pheromones or autoinducers, and the control mechanism is called "quorum-sensing". Quorum-sensing systems regulate the expression of a panel of genes, allowing bacteria to adapt to modified environmental conditions at a high density of population. The two known different quorum systems are described as the LuxR-LuxI system in gram-negative bacteria, which uses an N-acyl-homoserine lactone (AHL) as signal, and the agr system in gram-positive bacteria, which uses a peptide-tiolactone as signal and the RNAIII as effector molecules. Both in gram-negative and in gram-positive bacteria, quorum-sensing systems regulate the expression of adhesion mechanisms (biofilm and adhesins) and virulence factors (toxins and exoenzymes) depending on population cell density. In gram-negative Pseudomonas aeruginosa, analogs of signaling molecules such as furanone analogs, are effective in attenuating bacterial virulence and controlling bacterial infections. In grampositive Staphylococcus aureus, the quorum-sensing RNAIII-inhibiting peptide (RIP), tested in vitro and in animal infection models, has been proved to inhibit virulence and prevent infections. Attenuation of bacterial virulence by quorum-sensing inhibitors, rather than by bactericidal or bacteriostatic drugs, is a highly attractive concept because these antibacterial agents are less likely to induce the development of bacterial resistance.     

N-acylhomoserine lactones undergo lactonolysis in a pH-, temperature-, and acyl chain length-dependent manner during growth of Yersinia pseudotuberculosis and Pseudomonas aeruginosa

In gram-negative bacterial pathogens, such as Pseudomonas aeruginosa and Yersinia pseudotuberculosis, cell-to-cell communication via the N-acylhomoserine lactone (AHL) signal molecules is involved in the cell population density-dependent control of genes associated with virulence. This phenomenon, termed quorum sensing, relies upon the accumulation of AHLs to a threshold concentration at which target structural genes are activated. By using biosensors capable of detecting a range of AHLs we observed that, in cultures of Y. pseudotuberculosis and P. aeruginosa, AHLs accumulate during the exponential phase but largely disappear during the stationary phase. When added to late-stationary-phase, cell-free culture supernatants of the respective pathogen, the major P. aeruginosa [N-butanoylhomoserine lactone (C4-HSL) and N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL)] and Y. pseudotuberculosis [N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL)] AHLs were inactivated. Short-acyl-chain compounds (e.g., C4-HSL) were turned over more extensively than long-chain molecules (e.g., 3-oxo-C12-HSL). Little AHL inactivation occurred with cell extracts, and no evidence for inactivation by specific enzymes was apparent. This AHL turnover was discovered to be due to pH-dependent lactonolysis. By acidifying the growth media to pH 2.0, lactonolysis could be reversed. By using carbon-13 nuclear magnetic resonance spectroscopy, we found that the ring opening of homoserine lactone (HSL), N-propionyl HSL (C3-HSL), and C4-HSL increased as pH increased but diminished as the N-acyl chain was lengthened. At low pH levels, the lactone rings closed but not via a simple reversal of the ring opening reaction mechanism. Ring opening of C4-HSL, C6-HSL, 3-oxo-C6-HSL, and N-octanoylhomoserine lactone (C8-HSL), as determined by the reduction of pH in aqueous solutions with time, was also less rapid for AHLs with more electron-donating longer side chains. Raising the temperature from 22 to 37 degrees C increased the rate of ring opening. Taken together, these data show that (i) to be functional under physiological conditions in mammalian tissue fluids, AHLs require an N-acyl side chain of at least four carbons in length and (ii) that the longer the acyl side chain the more stable the AHL signal molecule.     

Quorum sensing and the regulation of virulence gene expression in pathogenic bacteria

For many pathogens, the outcome of the interaction between host and bacterium is strongly affected by the bacterial population size. Coupling the production of virulence factors with cell population density ensures that the mammalian host lacks sufficient time to mount an effective defence against consolidated attack. Such a strategy depends on the ability of an individual bacterial cell to sense other members of the same species and in response, differentially express specific sets of genes. Such cell-cell communication is called "quorum sensing" and involves the direct or indirect activation of a response regulator by a small diffusible signal molecule. A number of chemically distinct quorum-sensing signal molecules have been described including the N-acyl-L-homoserine lactones (AHLs) in Gram-negative bacteria and post-translationally modified peptides in Gram-positive bacteria. For example, the human pathogens Pseudomonas aeruginosa and Staphylococcus aureus employ AHLs and peptides, respectively, to control the expression of multiple virulence genes in concert with cell population density. Apart from their role in signal transduction, certain quorum-sensing signal molecules, notably N-(3-oxododecanoyl)homoserine lactone, possess intrinsic pharmacological and immunomodulatory activities such that they may function as virulence determinants per se. While quorum-sensing signal molecules have been detected in tissues in experimental animal model and human infections, the mutation of genes involved in either quorum-sensing signal generation or signal transduction frequently results in the attenuation of virulence. Thus, interference with quorum sensing represents a promising strategy for the therapeutic or prophylactic control of infection.