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.     

Endogenous interferon-gamma impairs bacterial clearance from lungs during Pseudomonas aeruginosa pneumonia

Interferon (IFN-)gamma is thought to play a role in the resistance to various pathogens. To study the role of IFN-gamma in the pathogenesis of Pseudomonas pneumonia, IFN-gamma receptor (R) alpha-subunit-deficient [IFN-gammaR(-/-)] mice and wild type mice were intranasally inoculated with Pseudomonas aeruginosa (10(5) CFU). IFN-gammaR(-/-) mice demonstrated an enhanced clearance of P. aeruginosa from their lungs when compared to normal wild type mice (P < 0.05 at 24 hours after the infection), which was associated with a tendency towards an improved survival. These findings were not accompanied by a more effective activation of several components of the innate immune system known to contribute to host defense against pneumonia, i.e. the lung concentrations of cytokines and chemokines were similar in IFN-gammaR(-/-) and wild type mice, while the influx of neutrophils in bronchoalveolar lavage fluid (BALF) was even higher in wild type mice than in IFN-gammaR(-/-) mice. Remarkably, IFN-gammaR(-/-) mice had higher nitric oxide levels in the BALF at 24 hours after infection (P < 0.05). Endogenous IFN-gamma impairs rather than augments host defense during pneumonia caused by P. aeruginosa.     

The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN-gamma-mediated macrophage killing

The ability of Pseudomonas aeruginosa to form biofilms and cause chronic infections in the lungs of cystic fibrosis patients is well documented. Numerous studies have revealed that P. aeruginosa biofilms are highly refractory to antibiotics. However, dramatically fewer studies have addressed P. aeruginosa biofilm resistance to the host's immune system. In planktonic, unattached (nonbiofilm) P. aeruginosa, the exopolysaccharide alginate provides protection against a variety of host factors yet the role of alginate in protection of biofilm bacteria is unclear. To address this issue, we tested wild-type strains PAO1, PA14, the mucoid cystic fibrosis isolate, FRD1 (mucA22+), and the respective isogenic mutants which lacked the ability to produce alginate, for their susceptibility to human leukocytes in the presence and absence of IFN-gamma. Human leukocytes, in the presence of recombinant human IFN-gamma, killed biofilm bacteria lacking alginate after a 4-h challenge at 37 degrees C. Bacterial killing was dependent on the presence of IFN-gamma. Killing of the alginate-negative biofilm bacteria was mediated through mononuclear cell phagocytosis since treatment with cytochalasin B, which prevents actin polymerization, inhibited leukocyte-specific bacterial killing. By direct microscopic observation, phagocytosis of alginate-negative biofilm bacteria was significantly increased in the presence of IFN-gamma vs all other treatments. Addition of exogenous, purified alginate to the alginate-negative biofilms restored resistance to human leukocyte killing. Our results suggest that although alginate may not play a significant role in bacterial attachment, biofilm development, and formation, it may play an important role in protecting mucoid P. aeruginosa biofilm bacteria from the human immune system.     

植物精油对铜绿假单胞菌群体感应系统的抑制作用研究进展

群体感应（quorum sensing,QS）是细菌个体与个体之间的一种交流机制,广泛存在于细菌中。铜绿假单胞菌是人类的一种条件致病菌,它具有至少3种QS系统,即las、rhl和pqs系统,且各系统之间存在着级联调控关系,它们共同作用调控着该菌众多毒力基因的表达和毒力因子的产生。近年来,通过抑制铜绿假单胞菌的QS系统以控制其毒力和致病力,成为一种新型的铜绿假单胞菌感染防控策略。植物精油是一种天然的群体感应抑制剂（quorum sensing inhibitors, QSI）,多种精油活性化合物都能抑制铜绿假单胞菌的QS系统,而且尚未发现细菌对其产生耐药性。基于此,梳理了铜绿假单胞菌QS系统的组成及其级联调控关系,简要介绍了植物精油的QS抑制机制和抑制活性,并重点综述了萜烯类化合物、芳香族化合物、脂肪族化合物、含硫含氮化合物4类精油化合物对铜绿假单胞菌QS系统抑制作用的研究进展,以期为从天然化合物中发现和筛选安全、高效的细菌QSI的相关研究提供参考,并为致病菌的防控奠定理论基础。     

Quantitative real-time PCR study of the influence of probiotic culture soluble fraction on the expression of Pseudomonas aeruginosa quorum sensing genes

The opportunistic pathogen Pseudomonas (Ps.) aeruginosa causes severe infections, particularly in immunocompromised individuals and patients with cystic fibrosis (CF). A serious side effect of antibiotic therapy in Ps. aeruginosa infections is the development of resistance to antibiotics. During the infection process Ps. aeruginosa forms biofilms, rendering bacterial cells more resistant to disinfectants, antibiotics and the action of host immune defense effectors. Pseudomonas aeruginosa employs the intercellular communication system, known as quorum sensing (QS) to coordinate the expression of tissue-damaging factors. Since the QS systems controls the production of different virulence factors, it is possible that the inhibition of its regulatory activity to severely compromise the ability of Ps. aeruginosa to cause infections in humans. Many studies have shown that some probiotic strains exhibit inhibitory activity on different virulence properties of pathogenic bacteria (adherence to cellular or inert substrate, soluble virulence factors expression). The aim of the present study was to investigate by real-time RT-qPCR the influence of probiotic culture soluble factors on the QS genes expression in 30 Ps. aeruginosa strains isolated from patients hospitalized in the National Institute for Cardiovascular Infections, Prof. C.C. Iliescu Fundeni Hospital, Bucharest. The results of the real time RT-qPCR have shown that in all Ps. aeruginosa strains grown in the presence of probiotic culture sterile filtrates, the level of QS genes expression was reduced comparatively with those from control cultures. In conclusion, these results proved that the inhibition of virulence factors regulation mechanisms by soluble molecules secreted by probiotics could represent an interesting way pathogenicity and virulence attenuation in Ps. aeruginosa nosocomial strains.     

Neutrophil elastase mediates innate host protection against Pseudomonas aeruginosa

According to the widely accepted view, neutrophil elastase (NE), a neutrophil-specific serine protease, is a major contributor to Pseudomonas aeruginosa infection-associated host tissue inflammation and damage, which in severe cases can lead to death. Herein, we provide for the first time compelling evidence that the host rather employs NE to protect itself against P. aeruginosa infection. Using a clinically relevant model of pneumonia, targeted deficiency in NE increased the susceptibility of mice to P. aeruginosa. We found that NE was required for maximal intracellular killing of P. aeruginosa by neutrophils. In investigating the mechanism of NE-mediated killing of P. aeruginosa, we found that NE degraded the major outer membrane protein F, a protein with important functions, including porin activity, maintenance of structural integrity, and sensing of host immune system activation. Consistent with this, the use of an isogenic mutant deficient in outer membrane protein F negated the role of NE in host defense against P. aeruginosa infection.     

Regulation on Expression of Toll-like Receptors on Monocytes After Stimulation with the 3-o-C12-HSL Molecule from Pseudomonas aeruginosa

Quorum sensing (QS) is a type of cell-to-cell communication. The Pseudomonas aeruginosa QS molecule N-3-(oxododecanoyl)-L: -homoserine lactone (3-o-C12-HSL) has the potential to modulate the immune system of its host. However, the mechanism of that activity is yet to be fully characterized. To be able to understand this activity, we determined whether 3-o-C12-HSL has a direct effect on the immune function and the expression of toll-like receptors (TLRs) in monocytes. Monocytes were cultured with 3-o-C12-HSL at different concentrations (0, 10, 25, 50, and 100?μmol/L) for 12?h; upon exposure to 3-o-C12-HSL, IL-12 production in monocytes was inhibited, monocyte proliferation was blocked, TLR2- and 4-mRNA expressions were reduced, and TLR5-mRNA expression was increased in a dose-dependent manner. Strikingly, 3-o-C12-HSL was able to significantly induce mRNA changes in the monocytes even at the lowest concentration (10?μmol/L, P?相似文献   

GacA对假单胞菌株M18两个phz基因簇和菌群传感的调控

摘要:【目的】假单胞菌株M18（Pseudomonas sp. M18）是从甜瓜根际土壤中分离获得的一株对多种植物病原菌具有显著拮抗作用的菌株,在菌群传感（quorum sensing）系统的调控下,能分泌吩嗪-1-羧酸（PCA）以及多种吩嗪（phz）类衍生物的抗真菌物质。全局性因子GacA是M18菌株吩嗪类物质的合成与菌群传感系统的重要调控因子,本文将就GacA对上述两者的调控做进一步研究。【方法】PCR基因扩增和测序研究M18菌株中PCA合成基因簇,运用RT-PCR及构建phzA-lacZ转录融合手段     

Quorum-sensing blockade as a strategy for enhancing host defences against bacterial pathogens

Conventional antibiotics target the growth and the basal life processes of bacteria leading to growth arrest and cell death. The selective force that is inherently linked to this mode of action eventually selects out antibiotic-resistant variants. The most obvious alternative to antibiotic-mediated killing or growth inhibition would be to attenuate the bacteria with respect to pathogenicity. The realization that Pseudomonas aeruginosa, and a number of other pathogens, controls much of their virulence arsenal by means of extracellular signal molecules in a process denoted quorum sensing (QS) gave rise to a new 'drug target rush'. Recently, QS has been shown to be involved in the development of tolerance to various antimicrobial treatments and immune modulation. The regulation of virulence via QS confers a strategic advantage over host defences. Consequently, a drug capable of blocking QS is likely to increase the susceptibility of the infecting organism to host defences and its clearance from the host. The use of QS signal blockers to attenuate bacterial pathogenicity, rather than bacterial growth, is therefore highly attractive, particularly with respect to the emergence of multi-antibiotic resistant bacteria.     

Characterization of biofilm-like structures formed by Pseudomonas aeruginosa in a synthetic mucus medium

ABSTRACT: BACKGROUND: The accumulation of thick stagnant mucus provides a suitable environment for the growth of Pseudomonas aeruginosa and Staphylococcus aureus within the lung alveoli of cystic fibrosis (CF) patients. These infections cause significant lung damage, leading to respiratory failure and death. In an artificial mucin containing medium ASM+, P. aeruginosa forms structures that resemble typical biofilms but are not attached to any surface. We refer to these structures as biofilm like structures (BLS). Using ASM+ in a static microtiter plate culture system, we examined the roles of mucin, extracellular DNA, environmental oxygen (EO2), and quorum sensing (QS) in the development of biofilm-like structures (BLS) by P. aeruginosa; and the effect of EO2 and P. aeruginosa on S. aureus BLS. RESULTS: Under 20% EO2, P. aeruginosa strain PAO1 produced BLS that resemble typical biofilms but are confined to the ASM+ and not attached to the surface. Levels of mucin and extracellular DNA within the ASM+ were optimized to produce robust well developed BLS. At 10% EO2, PAO1 produced thicker, more developed BLS, while under 0% EO2, BLS production was diminished. In contrast, the S. aureus strain AH133 produced well-developed BLS only under 20% EO2. In PAO1, loss of the QS system genes rhlI and rhlR affected the formation of BLS in ASM+ in terms of both structure and architecture. Whether co-inoculated into ASM+ with AH133, or added to established AH133 BLS, PAO1 eliminated AH133 within 48--56 h. CONCLUSIONS: The thick, viscous ASM+, which contains mucin and extracellular DNA levels similar to those found in the CF lung, supports the formation of biofilm-like structures similar to the aggregates described within CF airways. Alterations in environmental conditions or in the QS genes of P. aeruginosa, as occurs naturally during the progression of CF lung infection, affect the architecture and quantitative structural features of these BLS. Thus, ASM+ provides an in vitro medium in which the effect of changing levels of substances produced by the host and the bacteria can be analyzed to determine the effect on such structures and on the susceptibility of the bacteria within the BLS to various treatments.     

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.     

Quorum sensing system lactones do not increase invasiveness of a MexAB-OprM efflux mutant but do play a partial role in Pseudomonas aeruginosa invasion

We studied the quorum sensing (QS) system and the related homoserine lactones (HSLs) observing Pseudomonas aeruginosa invasion using the epithelial cell monolayer penetration assay model. Compared to the PAO1 wild-type, the QS mutants, DeltalasI and DeltarhlI, were compromised in their capacity to invade. The decreased invasiveness of DeltarhlI was restored by adding 100 microM exogenous C(4)-HSL. However, the decreased invasiveness of an efflux mutant, DeltamexAB-oprM, was not restored in the presence of exogenous HSLs. The QS system partially plays a role in P. aeruginosa invasion; however, C(4)-HSL and 3-O-C(12)-HSL are not the essential determinants for invasiveness for P. aeruginosa.     

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。     

Caenorhabditis elegans senses bacterial autoinducers

Pseudomonas aeruginosa uses virulence factors controlled by quorum sensing (QS) to kill Caenorhabditis elegans. Here we show that C. elegans is attracted to the acylated homoserine lactones (AHSLs) that mediate QS in P. aeruginosa. Our data also indicate that C. elegans can distinguish AHSLs and may use them to mediate aversive or attractive learning.     

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。     

Mutation of pfm affects the adherence of Pseudomonas aeruginosa to host cells and the quorum sensing system

The Pseudomonas aeruginosa quorum sensing (QS) system is controlled by the signal molecules acyl homoserine lactones (AHLs) that are synthesized from acyl enoyl-acyl carrier proteins (acyl-ACPs) provided by the fatty acid biosynthesis cycle. Pfm (PA2950), an enoyl-CoA reductase, has previously been shown to affect swimming mobility and fatty acid biosynthesis. In this report, we further show that pfm influences bacterial adherence to human cells. Microarray assay results suggest that pfm affects bacterial adherence through its influence on the QS system. Further experiments confirmed that the pfm mutant strain produces significantly less QS signal molecules than the corresponding wild-type strain. Using strains Escherichia coli DH5α(pECP64, lasB'-lacZ) and E.?coli DH5α(pECP61.5, rhlA'-lacZ), biosensors for N-(3-oxododecanoyl) homoserine lactone (3O-C(12) -HSL) and N-butyryl homoserine lactone (C(4) -HSL), respectively, we found that pfm mutant strain produces decreased amounts of both signal molecules. Elastase activity and pyocyanin measurements further confirmed the reduced levels of 3O-C(12) -HSL and C(4) -HSL in the pfm mutant. Finally, bacterial virulence, as assessed by the Caenorhabditis elegans worm killing assay, is decreased in the pfm mutant. Taken together, these data indicate that pfm can be an important target for the control of P.?aeruginosa infectivity.     

Biofilm and Quorum sensing mediated pathogenicity in Pseudomonas aeruginosa

The emergence of multidrug resistance has become an alarming and lifethreatening phenomenon for humans. Various mechanisms are involved in the development of resistance in bacteria towards antimicrobial compounds and immune system. Bacterial biofilm is a complicated, selfdefensive, rigid structure of bacteria crowded together to develop a selfrecessive nature, which enhances the ability to cause infections much easier in the living host. P. aeruginosa biofilm formation is supported by extracellular polymeric substances (EPS) such as exopolysaccharides, extracellular DNA (eDNA), proteins and biomolecules. Published evidences suggest that biofilm formation can also be the result of several other mechanisms such as cell signaling or communication. Bacterial biofilm is also regulated by strong intercellular communication known as Quorum Sensing (QS). It is a cellular communication mechanism involving autoinducers and regulators. In P. aeruginosa, Acyl Homoserine Lactone, the prime signaling molecule, controls approximately 300 genes responsible for various cellular functions, including its pathogenesis. The surrounding environment and metabolism have a specific effect on the biofilm and QS, thus, understanding the involvement of QS in the biofilm developing mechanism is still complicated and complex to understand. Therefore, this review will include basic knowledge of the biofilmforming mechanism and other regulatory factors involved in causing infections and diseases in the host organisms.