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.     

P. aeruginosa quorum-sensing systems and virulence

Quorum sensing is an important mechanism for the regulation of genes in many Gram-negative and Gram-positive bacteria. In the opportunistic pathogen Pseudomonas aeruginosa, the absence of one or more components of the quorum-sensing system results in a significant reduction in virulence. Recent advances in the past year have demonstrated that the quorum-sensing signal molecule 3O-C(12)-HSL is also a potent stimulator of multiple eukaryotic cells and thus may alter the host response during P. aeruginosa infections. Therefore, via the regulation of multiple factors and the production of 3O-C(12)-HSL, quorum-sensing systems have a significant effect on the virulence of the bacteria and also on how the host responds to P. aeruginosa infections.     

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。     

Modeling the effect of acylated homoserine lactone antagonists in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a gram-negative bacterium that causes serious illnesses, particularly in immunocompromised individuals, often with a fatal outcome. The finding that the acylated homoserine lactone quorum sensing (QS) system controls the production of virulence factors in P. aeruginosa makes this system a possible target for antimicrobial therapy. It has been suggested that an N-(3-oxododecanoyl)-homoserine lactone (3O-C12-HSL) antagonist, a QS blocker (QSB), would interfere efficiently with the quorum sensing system in P. aeruginosa and thus reduce the virulence of this pathogen. In this work, a mathematical model of the QS system in P. aeruginosa has been developed. The model was used to virtually add 3O-C12-HSL antagonists that differed in their affinity for the receptor protein and for their ability to mediate degradation of the receptor. The model suggests that very small differences in these parameters for different 3O-C12-HSL antagonists can greatly affect the success of QSB based inhibition of the QS system in P. aeruginosa. Most importantly, it is proposed that the ability of the 3O-C12-HSL antagonist to mediate degradation of LasR is the core parameter for successful QSB based inhibition of the QS system in P. aeruginosa. Finally, this study demonstrates that QSBs can shift the system to a low steady state, corresponding to an uninduced state and thus, suggests that the use of 3O-C12-HSL antagonists may constitute a promising therapeutic approach against P. aeruginosa involved infections.     

Human and murine paraoxonase 1 are host modulators of Pseudomonas aeruginosa quorum-sensing

The pathogenic bacterium Pseudomonas aeruginosa uses acyl-HSL quorum-sensing signals to regulate genes controlling virulence and biofilm formation. We found that paraoxonase 1 (PON1), a mammalian lactonase with an unknown natural substrate, hydrolyzed the P. aeruginosa acyl-HSL 3OC12-HSL. In in vitro assays, mouse serum-PON1 was required and sufficient to degrade 3OC12-HSL. Furthermore, PON2 and PON3 also degraded 3OC12-HSL effectively. Serum-PON1 prevented P. aeruginosa quorum-sensing and biofilm formation in vitro by inactivating the quorum-sensing signal. Although 3OC12-HSL production by P. aeruginosa was important for virulence in a mouse sepsis model, Pon1-knock-out mice were paradoxically protected. These mice showed increased levels of PON2 and PON3 mRNA in epithelial tissues suggesting a possible compensatory mechanism. Thus, paraoxonase interruption of bacterial communication represents a novel mechanism to modulate quorum-sensing by bacteria. The consequences for host immunity are yet to be determined.     

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.