The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone stimulates phagocytic activity in human macrophages through the p38 MAPK pathway

Quorum-sensing is an important mechanism for the regulation of bacteria-to-bacteria communication. Recent advances have demonstrated that the Pseudomonas aeruginosa signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone (3O-C(12)-HSL) is also a potent modulator of eukaryotic cells and may thus play an important role in the host response during P. aeruginosa infections. Little is known, however, about specific effects of 3O-C(12)-HSL molecules on human macrophages. To address this issue, we investigated the influence of 3O-C(12)-HSL on the phagocytic activity, production of reactive oxygen species, and activation of p38 and p42/44 MAPK signaling pathways in human macrophages. We show an effect of 3O-C(12)-HSL on the phagocytic capacity in human macrophages, which depends on concentration and time of exposure. When cells were exposed to 100 microM 3O-C(12)-HSL for 30 min or 1 h, the phagocytic activity increased 1.8 and 1.6 times, respectively. The 3O-C(12)-HSL treatments had no significant effect on the level of reactive oxygen species production. Furthermore, the p38 MAPK, but not the p42/44 MAPK, signaling pathway was activated in response to 3O-C(12)-HSL. In addition, specific blocking of p38 MAPK activation with 10 microM SB 203580 prevented the 3O-C(12)-HSL-induced increase in the phagocytic activity. These findings demonstrate that the bacterial quorum-sensing can play a significant role also in regulation of macrophage activity during infections caused by P. aeruginosa.     

铜绿假单胞菌3-氧十二烷酰高丝氨酸内酯信号分子对单核细胞Toll样受体2和4的影响

目的研究铜绿假单胞菌群体感应系统信号分子3-氧十二烷酰高丝氨酸内酯（3-O-C12-HSL）对人外周血单核细胞凋亡以及1]LR2／4mRNA表达的影响。方法自健康献血者血液中分离单核细胞进行体外培养,并以0、1、10、50和100μmol／L3-O-C12-HSL作用,采用MTT法,观察不同浓度的3-O-C12-HSL分别作用对人单核细胞生长增殖的影响。用RT-PCR测定单核细胞TLR2／4mRNA表达。结果3-O-C12-HSL刺激单核细胞12h后TLR2／4mRNA表达降低,且呈剂量依赖关系,TLR2／4mRNA在100μmol／L时最为显著（0．3494±0．0979,0．3351±0．1121）。3-O-C12-HSL对单核细胞增殖有抑制作用,呈剂量依赖关系,差异有显著性（P≤0．05）。结论3-O-C12-HSL可以激发单核细胞细胞凋亡,下调Toll样受体的表达水平,从而削弱单核细胞对铜绿假单胞菌的应答能力,这可能是铜绿假单胞菌逃逸免疫清除的重要原因之一。     

The Pseudomonas aeruginosa quorum-sensing signal N-(3-oxododecanoyl) homoserine lactone can accelerate cutaneous wound healing through myofibroblast differentiation in rats

Quorum sensing is a cell density-dependent gene regulation system in bacteria. N-(3-oxododecanoyl) homoserine lactone (3-oxo-C12-HSL) is used in the las quorum-sensing system in Pseudomonas aeruginosa, which is an opportunistic pathogen that causes many human diseases. Although many studies have investigated the sole effects of quorum sensing on several types of mammalian cells, including lung cells, little is known about the effects of quorum sensing on the cells associated with wound healing. To better understand the mechanism of bacterial wound infection, we investigated the effects of 3-oxo-C12-HSL on cells using a rat full-thickness wound-healing model. We found that the wound contraction was significantly increased at 24 h after the administration of 3-oxo-C12-HSL to the surface of granulation tissue. Differentiation of fibroblasts to myofibroblasts was induced in the in vivo wound-healing model and was confirmed in vitro using the rat fibroblastic cell line Rat-1. Cyclooxygenase (Cox)-2 expression was also induced in Rat-1 cells by 3-oxo-C12-HSL. This finding suggested that Cox-2 upregulation may be related to the inflammatory findings in the histological examinations, in which infiltrating polymorphonuclear neutrophils were observed at the wound site. Taken together, these results imply that mammals have a potential defense system against invading pathogens by responding to the presence of 3-oxo-C12-HSL and inducing the differentiation of fibroblasts to myofibroblasts as well as inflammation for accelerating wound healing.     

Influence of Pseudomonas autoinducer N-3-oxododecanoyl homoserine lactone on human corneal epithelial cells

The quorum-sensing (QS) signaling-dependent extracellular virulence factors of Pseudomonas aeruginosa can cause infections such as P. aeruginosa keratitis. P. aeruginosa communicates by secreting and sensing small chemical molecules called autoinducers in QS system. The key QS signal molecule, N-3-oxododecanoyl-homoserine lactone (3OC12HSL), can affect the behavior of host cells and initiate immune response. In this report we investigated the influence of 3OC12HSL on human corneal epithelial cells (HCECs) and the mechanisms of 3OC12HSL on activated toll-like receptor 2 (TLR2)-dependent interleukin-8 (IL-8) secretion in HCECs. Cells were cultured under different concentrations of 3OC12HSL. Cell viability was assessed using Crystal violet staining and the cell counting kit-8 assay. We demonstrated the administration of 3OC12HSL decreased HCEC viability and survival in a concentration- and time-dependent manner. At high concentrations, 3OC12HSL rapidly promoted a time-dependent increase in the expressions of TLR2 and TLR4. It was found that the nuclear translocation and expression of nuclear factor-κB (NF-κB) were also increased in response to 3OC12HSL treatment. The significantly elevated expressions of TLR2, TLR4, and NF-κB, encouraged us to further test their mechanisms that cause inflammatory response. Among the inflammatory factors examined (IL-6, IL-8, IL-10, and TNF-α), we found that IL-8 was significantly increased after treatment with 3OC12HSL and its expression was inhibited when TLR2 was specifically blocked or silenced. These results indicated that the QS signaling molecule 3OC12HSL could be recognized by the host innate immune system in HCECs. This recognition then triggered an immune inflammatory response involving the activation of TLR2 and an increase in expression of IL-8. This crosstalk between 3OC12HSL and host immunity in HCECs contributes to the development and progression of P. aeruginosa keratitis.     

Pseudomonas aeruginosa quorum sensing molecule N-(3 oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells

Acyl-homoserine lactone (HSL) quorum sensing molecules play an important role in regulation of virulence gene expression in Pseudomonas aeruginosa. Here, we show that 3O-C(12)-HSL can disrupt barrier integrity in human epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER), increased paracellular flux, reduction in the expression and distribution of ZO-1 and occludin, and reorganization of F-actin. P. aeruginosa 3O-C(12)-HSL activate p38 and p42/44 kinases, and inhibition of these kinases partly prevented 3O-C(12)-HSL-induced changes in TER, paracellular flux and expression of occludin and ZO-1. These findings demonstrate that P. aeruginosa 3O-C(12)-HSL can modulate tight junction integrity of Caco-2 cells.     

N-(3-oxododecanoyl)-homoserine lactone regulates osteoblast apoptosis and differentiation by mediating intracellular calcium

Pseudomonas aeruginosa (P. aeruginosa) is associated with periapical periodontitis. The lesions are characterized by a disorder in osteoblast metabolism. Quorum sensing molecular N-(3-oxododecanoyl)-homoserine lactone (AHL) is secreted by P. aeruginosa and governs the expression of numerous virulence factors. AHL can trigger intracellular calcium ([Ca²⁺]_i) fluctuations in many host cells. However, it is unclear whether AHL can regulate osteoblast metabolism by affecting [Ca²⁺]_i changes or its spatial correlation. We explored AHL-induced apoptosis and differentiation in pre-osteoblastic MC3T3-E1 cells and evaluated [Ca²⁺]_i mobilization using several extraction methods. The spatial distribution pattern of [Ca²⁺]_i among cells was investigated by Moran's I, an index of spatial autocorrelation. We found that 30 μM and 50 μM AHL triggered opposing osteoblast fates. At 50 μM, AHL inhibited osteoblast differentiation by promoting mitochondrial-dependent apoptosis and negatively regulating osteogenic marker genes, including Runx2, Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). In contrast, prolonged treatment with 30 μM AHL promoted osteoblast differentiation concomitantly with cell apoptosis. The elevation of [Ca²⁺]_i levels in osteoblasts treated with 50 μM AHL was spatially autocorrelated, while no such phenomenon was observed in 30 μM AHL-treated osteoblasts. The blocking of cell-to-cell spatial autocorrelation in the osteoblasts provoked by 50 μM AHL significantly inhibited apoptosis and partially restored differentiation. Our observations suggest that AHL affects the fate of osteoblasts (apoptosis and differentiation) by affecting the spatial correlation of [Ca²⁺]_i changes. Thus, AHL acts as a double-edged sword for osteoblast function.     

N-3-(oxododecanoyl)-L-homoserine lactone promotes the induction of regulatory T-cells by preventing human dendritic cell maturation

N-3-(Oxododecanoyl)-L-homoserine lactone (C12) is a small bacterial signaling molecule secreted by Pseudomonas aeruginosa (PA), which activates mammalian cells through TLR4-independent mechanisms. C12 acts as an immunosuppressant and it has been shown to modulate murine bone marrow-derived dendritic cell-mediated T-helper 2 (Th2) cell polarizations in vitro. In the present study, we initially examined the impact of C12 on the maturation of human monocyte-derived dendritic cells (Mo-DCs) and the induction of regulatory T-cells (iTregs) in culture. Our findings demonstrate that C12-treated Mo-DCs failed to undergo lipopolysaccharide (LPS)-induced maturation. At the molecular level, C12 blocked the upregulation of surface molecules, including CD11c, HLA-DR, CD40, and CD80, and it switched to an interleukin (IL)-10^high, IL-12p70^low phenotype. Moreover, C12 selectively inhibited the capacity of Mo-DCs to stimulate the proliferation of allogeneic CD4⁺ T-cells. Otherwise, the C12-treated Mo-DCs promoted the generation of CD4⁺CD25⁺Foxp3⁺-induced regulatory T-cells (iTregs) and enhanced their IL-10 and transforming growth factor (TGF)-β production associated with reduced interferon (IFN)-γ and IL-12p70 production. These findings provide new insights towards understanding the persistence of chronic inflammation in PA infection.     

N-butanoyl-L-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit

Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low.     

Induction of phenazine biosynthesis in cultures of Pseudomonas aeruginosa by L-N-(3-oxohexanoyl) homoserine lactone

Abstract A range of Pseudomonas spp. and other Gram-negative bacteria were screened for induction of antimicrobial activity in response to the autoregulatory factor l - N -(3-oxohexanoyl)homoserine lactone. In one of these, P. aeruginosa ATCC 10145, the production of phenazine metabolites was shown to be inducible in a dose-dependent manner. The production of phenazine-1-carboxamide increased over 50-fold compared to control cultures when supplemented with 200 μg/ml of the autoregulator. In addition, the production of an unidentified polar antibacterial substance by this strain increased with autoregulator concentration.     

Caspase-1-dependent mechanism mediating the harmful impacts of the quorum-sensing molecule N-(3-oxo-dodecanoyl)-l-homoserine lactone on the intestinal cells

N-(3-oxododecanoyl)-l -homoserine lactone (3-oxo-C12-HSL), a quorum-sensing (QS) molecule produced by Gram-negative bacteria in the gastrointestinal tract, adversly impacts host cells. Our previous study demonstrated that 3-oxo-C12-HSL induced a decrease in cell viability via cell apoptosis and eventually disrupted mucin synthesis from LS174T goblet cells. However, the molecular mechanism underlying cell apoptosis and whether pyroptosis was involved in this process are still unknown. In this study, we emphasized on the caspases signal pathway and sterile inflammation to reveal the harmful effects of 3-oxo-C12-HSL on LS174T goblet cells. Our data showed that 3-oxo-C12-HSL is a major inducer of oxidative stress indicated by a high level of intracellular reactive oxygen species (ROS). However, TQ416, an inhibitor of paraoxonase 2, can effectively block oxidative stress. A higher ROS level is the trigger for activating the caspase-1 and 3 cascade signal pathways. Blockade of ROS synthesis and caspase-1 and 3 cascades can obviously rescue the viability of LS174T cells after 3-oxo-C12-HSL treatment. We also found that paralleled with a higher level of ROS and caspases activation, an abnormal expression of proinflammatory cytokines was induced by 3-oxo-C12-HSL treatment; however, the blockage of TLRs-NF-κB pathway cannot restore cell viability and secretary function. These data collectively indicate that 3-oxo-C12-HSL exposure induces damages to cell viability and secretary function of LS174T goblet cells, which is mediated by oxidative stress, cell apoptosis, and sterile inflammation. Overall, the data in this study will provide a better understanding of the harmful impacts of some QS molecules on host cells and their underlying mechanism.     

The Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)Homoserine Lactone Contributes to Virulence and Induces Inflammation In Vivo

Pseudomonas aeruginosa has two well-characterized quorum-sensing systems, Las and Rhl. These systems are composed of LuxR-type proteins, LasR and RhlR, and two acyl homoserine lactone (AHL) synthases, LasI and RhlI. LasI catalyzes the synthesis of N-(3-oxododecanoyl)homoserine lactone (3O-C12-HSL), whereas RhlI catalyzes the synthesis of N-butyryl-homoserine lactone. There is little known about the importance of AHLs in vivo and what effects these molecules have on eukaryotic cells. In order to understand the role of AHLs in vivo, we first tested the effects that deletions of the synthase genes in P. aeruginosa had on colonization of the lung. We demonstrate that in an adult mouse acute-pneumonia model, deletion of the lasI gene or both the lasI and rhlI genes greatly diminished the ability of P. aeruginosa to colonize the lung. To determine whether AHLs have a direct effect on the host, we examined the effects of 3O-C12-HSL injected into the skin of mice. In this model, 3O-C(12)-HSL stimulated a significant induction of mRNAs for the cytokines interleukin-1alpha (IL-1alpha) and IL-6 and the chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemotactic protein 1, MIP-1beta, inducible protein 10, and T-cell activation gene 3. Additionally, dermal injections of 3O-C12-HSL also induced cyclooxygenase 2 (Cox-2) expression. The Cox-2 enzyme is important for the conversion of arachidonic acid to prostaglandins and is associated with edema, inflammatory infiltrate, fever, and pain. We also demonstrate that 3O-C12-HSL activates T cells to produce the inflammatory cytokine gamma interferon and therefore potentially promotes a Th1 environment. Induction of these inflammatory mediators in vivo is potentially responsible for the significant influx of white blood cells and subsequent tissue destruction associated with 3O-C12-HSL dermal injections. Therefore, the quorum-sensing systems of P. aeruginosa contribute to its pathogenesis both by regulating expression of virulence factors (exoenzymes and toxins) and by inducing inflammation.     

The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone

Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).