A Streptococcus Quorum Sensing System Enables Suppression of Innate Immunity.

Some bacterial pathogens utilize cell-cell communication systems, such as quorum sensing (QS), to coordinate genetic programs during host colonization and infection. The human-restricted pathosymbiont Streptococcus pyogenes (group A streptococcus [GAS]) uses the Rgg2/Rgg3 QS system to modify the bacterial surface, enabling biofilm formation and lysozyme resistance. Here, we demonstrate that innate immune cell responses to GAS are substantially altered by the QS status of the bacteria. We found that macrophage activation, stimulated by multiple agonists and assessed by cytokine production and NF-κB activity, was substantially suppressed upon interaction with QS-active GAS but not QS-inactive bacteria. Neither macrophage viability nor bacterial adherence, internalization, or survival were altered by the QS activation status, yet tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interferon beta (IFN-ß) levels and NF-κB reporter activity were drastically lower following infection with QS-active GAS. Suppression required contact between viable bacteria and macrophages. A QS-regulated biosynthetic gene cluster (BGC) in the GAS genome, encoding several putative enzymes, was also required for macrophage modulation. Our findings suggest a model wherein upon contact with macrophages, QS-active GAS produce a BGC-derived factor capable of suppressing inflammatory responses. The suppressive capability of QS-active GAS is abolished after treatment with a specific QS inhibitor. These observations suggest that interfering with the ability of bacteria to collaborate via QS can serve as a strategy to counteract microbial efforts to manipulate host defenses.IMPORTANCEStreptococcus pyogenes is restricted to human hosts and commonly causes superficial diseases such as pharyngitis; it can also cause severe and deadly manifestations including necrotizing skin disease or severe postinfectious sequelae like rheumatic heart disease. Understanding the complex mechanisms used by this pathogen to manipulate host defenses could aid in developing new therapeutics to treat infections. Here, we examine the impact of a bacterial cell-cell communication system, which is highly conserved across S. pyogenes, on host innate immune responses. We find that S. pyogenes uses this system to suppress macrophage proinflammatory cytokine responses in vitro Interference with this communication system could serve as a strategy to disarm bacteria and maintain an effective immune response.

Micro-rheological properties of lung homogenates correlate with infection severity in a mouse model of Pseudomonas aeruginosa lung infection.

Lung infections caused by Pseudomonas aeruginosa pose a serious threat to patients suffering from, among others, cystic fibrosis, chronic obstructive pulmonary disease, or bronchiectasis, often leading to life-threatening complications. The establishment of a chronic infection is substantially related to communication between bacteria via quorum-sensing networks. In this study, we aimed to assess the role of quorum-sensing signaling molecules of the Pseudomonas quinolone signal (PQS) and to investigate the viscoelastic properties of lung tissue homogenates of PA-infected mice in a prolonged acute murine infection model. Therefore, a murine infection model was successfully established via intra-tracheal infection with alginate-supplemented Pseudomonas aeruginosa NH57388A. Rheological properties of lung homogenates were analyzed with multiple particle tracking (MPT) and quorum-sensing molecules were quantified with LC-MS/MS. Statistical analysis of bacterial load and quorum-sensing molecules showed a strong correlation between these biomarkers in infected lungs. This was accompanied by noticeable changes in the consistency of lung homogenates with increasing infection severity. Furthermore, viscoelastic properties of the lung homogenates strongly correlated with bacterial load and quorum sensing molecules. Considering the strong correlation between the viscoelasticity of lung homogenates and the aforementioned biomarkers, the viscoelastic properties of infected lungs might serve as reliable new biomarker for the evaluation of the severity of P. aeruginosa infections in murine models.

Intracellular survival and innate immune evasion of Burkholderia cepacia: Improved understanding of quorum sensing-controlled virulence factors, biofilm, and inhibitors.

Burkholderia cepacia complex (Bcc) are opportunistic pathogens implicated with nosocomial infections, and high rates of morbidity and mortality, especially in individuals with cystic fibrosis (CF). B. cepacia are naturally resistant to different classes of antibiotics, and can subvert the host innate immune responses by producing quorum sensing (QS) controlled virulence factors and biofilms. It still remains a conundrum as to how exactly the bacterium survives the intracellular environment within the host cells of CF patients and immunocompromised individuals although the bacterium can invade human lung epithelial cells, neutrophils, and murine macrophages. The mechanisms associated with intracellular survival in the airway epithelial cells and the role of QS and virulence factors in B. cepacia infections in cystic fibrosis remain largely unclear. The current review focuses on understanding the role of QS-controlled virulence factors and biofilms, and provides additional impetus to understanding the potentials of QS-inhibitory strategies against B. cepacia.

Pseudomonas aeruginosa quorum-sensing metabolite induces host immune cell death through cell surface lipid domain dissolution.

Bacterial quorum-sensing autoinducers are small chemicals released to control microbial community behaviours. N-(3-oxo-dodecanoyl) homoserine lactone, the autoinducer of the Pseudomonas aeruginosa LasI-LasR circuitry, triggers significant cell death in lymphocytes. We found that this molecule is incorporated into the mammalian plasma membrane and induces dissolution of eukaryotic lipid domains. This event expels tumour necrosis factor receptor 1 into the disordered lipid phase for its spontaneous trimerization without its ligand and drives caspase 3-caspase 8-mediated apoptosis. In vivo, P. aeruginosa releases N-(3-oxo-dodecanoyl) homoserine lactone to suppress host immunity for its own better survival; conversely, blockage of caspases strongly reduces the severity of the infection. This work reveals an unknown communication method between microorganisms and the mammalian host and suggests interventions of bacterial infections by intercepting quorum-sensing signalling.

A Metabolism-Based Quorum Sensing Mechanism Contributes to Termination of Inflammatory Responses.

Recruitment of immune cells with antimicrobial activities is essential to fight local infections but has the potential to trigger immunopathology. Whether the immune system has the ability to sense inflammation intensity and self-adjust accordingly to limit tissue damage remains to be fully established. During local infection with an intracellular pathogen, we have shown that nitric oxide (NO) produced by recruited monocyte-derived cells was essential to limit inflammation and cell recruitment. Mechanistically, we have provided evidence that NO dampened monocyte-derived cell cytokine and chemokine production by inhibiting cellular respiration and reducing cellular ATP:ADP ratio. Such metabolic control operated at the tissue level but only when a sufficient number of NO-producing cells reached the site of infection. Thus, NO production and activity act as a quorum sensing mechanism to help terminate the inflammatory response.

Induction of CD4 T cell memory by local cellular collectivity.

Cell differentiation is directed by signals driving progenitors into specialized cell types. This process can involve collective decision-making, when differentiating cells determine their lineage choice by interacting with each other. We used live-cell imaging in microwell arrays to study collective processes affecting differentiation of naïve CD4+ T cells into memory precursors. We found that differentiation of precursor memory T cells sharply increases above a threshold number of locally interacting cells. These homotypic interactions involve the cytokines interleukin-2 (IL-2) and IL-6, which affect memory differentiation orthogonal to their effect on proliferation and survival. Mathematical modeling suggests that the differentiation rate is continuously modulated by the instantaneous number of locally interacting cells. This cellular collectivity can prioritize allocation of immune memory to stronger responses.

Vibrio vulnificus quorum-sensing molecule cyclo(Phe-Pro) inhibits RIG-I-mediated antiviral innate immunity.

The recognition of pathogen-derived ligands by pattern recognition receptors activates the innate immune response, but the potential interaction of quorum-sensing (QS) signaling molecules with host anti-viral defenses remains largely unknown. Here we show that the Vibrio vulnificus QS molecule cyclo(Phe-Pro) (cFP) inhibits interferon (IFN)-ß production by interfering with retinoic-acid-inducible gene-I (RIG-I) activation. Binding of cFP to the RIG-I 2CARD domain induces a conformational change in RIG-I, preventing the TRIM25-mediated ubiquitination to abrogate IFN production. cFP enhances susceptibility to hepatitis C virus (HCV), as well as Sendai and influenza viruses, each known to be sensed by RIG-I but did not affect the melanoma-differentiation-associated gene 5 (MDA5)-recognition of norovirus. Our results reveal an inter-kingdom network between bacteria, viruses and host that dysregulates host innate responses via a microbial quorum-sensing molecule modulating the response to viral infection.

Quorum-sensing molecule dihydroxy-2,3-pentanedione and its analogs as regulators of epithelial integrity.

BACKGROUND AND OBJECTIVE: Quorum-sensing molecules regulate the behavior of bacteria within biofilms and at the same time elicit an immune response in host tissues. Our aim was to investigate the regulatory role of dihydroxy-2,3-pentanedione (DPD), the precursor of universal autoinducer-2 (AI-2), and its analogs (ethyl-DPD, butyl-DPD and isobutyl-DPD) in the integrity of gingival epithelial cells. MATERIAL AND METHODS: Human gingival keratinocytes were incubated with four concentrations (10 µmol L-1 , 1 µmol L-1 , 100 nmol L-1 and 10 nmol L-1 ) of DPD and its analogs for 24 hours. The numbers of viable cells were determined using a proliferation kit, matrix metalloproteinase (MMP)-2 and -9 activities were determined by gelatin zymography, and expression of occludin protein and occludin mRNA were determined by western blotting and RT-qPCR, respectively. RESULTS: Increased cell proliferation was observed in gingival keratinocytes incubated with 100 nmol L-1 of butyl-DPD. MMP-9 activity was elevated in cells incubated with 10 µmol L-1 of ethyl-DPD. On the other hand, MMP-2 activity did not show any significant change when gingival keratinocytes were incubated with or without DPD or analogs. Western blot analyses demonstrated five forms (105, 61, 52.2, 44 and 37 kDa) of occludin. Incubation with 1 µmol L-1 and 100 nmol L-1 of DPD and with 10 nmol L-1 of ethyl-DPD increased dimeric (105 kDa) forms of occludin, while incubation with 100 nmol L-1 of isobutyl-DPD increased monomeric (61 kDa) forms. DPD and ethyl-DPD decreased, and 100 nmol L-1 of isobutyl-DPD and 10 nmol L-1 of butyl-DPD increased, the monomeric (52.2 kDa and 44 kDa) forms of occludin, whereas ethyl-DPD decreased and isobutyl-DPD increased, the low-molecular-weight (37 kDa) forms. According to RT-qPCR analysis, the exposure of gingival keratinocytes to 10 µmol L-1 of isobutyl-DPD up-regulated expression of occludin. CONCLUSION: The results indicate that isobutyl-DPD has the potential to enhance the integrity of the epithelium by stimulating the formation of occluding, without affecting the proliferation or gelatinolytic enzyme activities of the exposed cells. The modulatory effect of an AI-2 analog on the epithelial cell response is shown for the first time.

Efeito de extratos orgânicos de variedades de cebola sobre o quorum sensing bacteriano / Effect of organic extracts of onion varieties on bacterial quorum sensing

Muitos genes bacterianos são regulados pelo mecanismo de comunicação denominado quorum sensing (QS). Neste sistema, moléculas sinalizadoras ativam um comportamento de grupo, conforme a densidade celular, permitindo o controle da expressão gênica. Estudos sugerem o potencial de compostos extraídos de plantas sobre o QS, a exemplo da quercetina, um flavonol presente em concentrações elevadas em algumas frutas e hortaliças. Este composto é o flavonoide majoritário presente em cebola (Allium cepa), mas não existem estudos que mostrem a atividade anti-QS de extratos orgânicos deste vegetal. Este trabalho avaliou o potencial antimicrobiano e anti-QS de extratos orgânicos de cebola branca e cebola roxa, assim como de alguns de seus componentes majoritários identificados, em fenótipos regulados pelo QS como a produção de violaceína em Chrormobacterium violaceum ATCC 12472, a motilidade tipo swarming e a formação de biofilmes em Pseudomonas aeruginosa PAO1 e Serratia marcescens MG1. Extratos de cebola branca e roxa foram obtidos por extração em fase sólida utilizando coluna de poliamida e seus compostos identificados e quantificados pelas técnicas de Cromatografia líquida- ionização por elétron spray-espectrometria de massas e cromatografia líquida de alta eficiência acoplada a detector de arranjo de diodo. A atividade antimicrobiana foi avaliada pelas curvas de multiplicação de cada micro-organismo. O efeito dos compostos quercetina aglicona (inibidor do QS já relatado na literatura e encontrado no extrato de cebola roxa) e quercetina-3-ß-D-glicosideo (um dos compostos majoritários encontrados em ambos extratos) sobre os micro-organismos utilizados neste estudo foi também avaliado. Foram obtidos três extratos: cebola branca em metanol (CB-MeOH), cebola branca em metanol amônia (CBMeOH/ NH4) e cebola roxa em metanol (CR-MeOH). Os compostos quercetina 3,4'- diglicosídeio, quercetina-4-glicosídeo, quercetina-3-ß-D-glicosideo e quercetina aglicona foram os predominantes nos extratos das duas variedades de cebola. Cianidina-3-O-glicosideo também foi identificada no extrato de cebola roxa. A concentração inibitória mínima (MIC) dos extratos foi igual ou superior a 125 µg/ml (p/v) de extrato seco. Não foi observada inibição significativa da produção de violaceína em C. violaceum pelos extratos orgânicos de cebola e nem pela quercetina-3-ß-D-glicosideo, nas concentrações sub-inibitórias avaliadas. No entanto, a quercetina aglicona inibiu significativamente a produção de violaceína em todas as concentrações. A glicosilação da quercetina pode ter afetado sua atividade sobre a inibição da produção de violaceina, já que estudos mostram menor atividade biológica deste composto quando glicosilado. Para a motilidade tipo swarming em P. aeruginosa PAO1 houve inibição significativa pelo extrato de cebola roxa, em todas as concentrações estudadas. Os demais extratos não apresentaram inibição contra este micro-organismo. Para S. marcescens MG1, foi observada inibição da motilidade swarming somente na concentração de 125 µg/ml de CBMeOH/ NH4. As análises de comparação entre os dois tipos de quercetina revelaram que, embora para as duas bactérias testadas os dois compostos apresentaram atividade inibitória sobre a motilidade tipo swarming, a quercetina-3-ß-D-glicosideo foi menos eficiente que a quercetina aglicona na concentração de 125 µg/ml. A formação de biofilmes não foi influenciada pelos extratos e, inesperadamente, não se detectou inibição da formação de biofilmes por ambos tipos de quercetina avaliados. De forma geral, os extratos orgânicos de cebola mostraram pouco efeito sobre os fenótipos controlados pelo quorum sensing e a glicosilação da quercetina provavelmente explica a baixa atividade antimicrobiana e anti-QS dos extratos

Many bacterial genes are regulated by a communication mechanism called quorum sensing (QS). In this system, signaling molecules activate a group behavior according to cell density, allowing the control of gene expression. Studies suggest the inhibitory potential of compounds extracted from plants on the QS system, like quercetin, a flavonol present in high concentrations in some fruits and vegetables. This compound is the main flavonoid found in onion (Allium cepa); however, there are no studies showing the anti-QS activity of organic extracts of this plant. The objective of this work was to evaluate the antimicrobial and anti-QS potential of organic extracts of white and red onions, and their major components studied in QS-regulated phenotypes such as violacein production in Chromobacterium violaceum, swarming motility and biofilm formation in Pseudomonas aeruginosa PAO1 and Serratia marcescens MG1.White and red onion extracts were obtained by solid phase extraction using a polyamide column and its compounds were identified and quantified by Liquid Chromatography - Electron Spray-Mass Spectrometry and high performance liquid chromatography coupled to diode array detector. O The antimicrobial activity was evaluated by growth curves of each microorganism. The effect of non-glycosylated quercetin (a QS inhibitor already reported in the literature and found in red onion extract) and quercetin-3-ß-D-glycoside (one of the major compounds found in both extracts) on the microorganisms used in this study was also evaluated. Three extracts were obtained: white onion in methanol (CB-MeOH), white onion in methanol ammonia (CB-MeOH / NH4) and red onion in methanol (CR-MeOH). Our results showed that quercetin 3,4'- diglycoside, quercetin-4-glycoside, quercetin-3-ß-D-glycoside and non-glycosylated quercetin were predominant in the extracts of the two onion varieties. Cyanidin-3-O-glycoside has also been identified in the purple onion extract. The minimum inhibitory concentration (MIC) of extracts was equal or greater than 125 µg / ml (w / v) of dry extract. There was no significant inhibition of violacein production in C. violaceum by organic onion extracts or by quercetin-3-ß- D-glycoside at the sub-inhibitory concentrations evaluated. However, non-glycosylated quercetin showed a significant inhibition of violacein production in all tested concentrations. The glycosylation of Quercetin could have altered its inhibition activity towards violacein production, and in fact, some studies have shown less biological activity of some phenolic compounds when they have been glycosylated. For swarming motility in P. aeruginosa PAO1 there was significant inhibition by red onion extract, in all studied concentrations. The other extracts did not present inhibition against this microorganism. For S. marcescens MG1, inhibition of swarming motility was observed only at the concentration of 125 µg / ml of CB-MeOH / NH4. Comparative analyses between the two types of quercetin showed that, although for the two bacteria tested the two compounds showed inhibitory activity on swarming motility, quercetin-3-ß-D-glycoside was less efficient than non-glycosylated quercetin in the concentration of 125 µg / ml. Biofilm formation was not influenced by the extracts and unexpectedly, both types of quercetin evaluated did not show inhibition towards biofilm formation. In general, organic onion extracts showed little effect on quorum sensing controlled phenotypes and glycosylation of quercetin probably explains the low antimicrobial and anti-QS activity of the extracts

Role of PON2 in innate immune response in an acute infection model.

N-(3-oxododecanoyl)-l-homoserine lactone (3OC(12)-HSL) is a quorum-sensing molecule produced by gram-negative microbial pathogens such as Pseudomonas aeruginosa (PAO1). 3OC(12)-HSL is involved in the regulation of bacterial virulence factors and also alters the function of the host immune cells. Others and we have previously shown that paraoxonase 2 (PON2), a member of the paraoxonase gene family expressed in immune cells, hydrolyzes 3OC(12)-HSL. In this study, we examined i) whether macrophage PON2 participates in 3OC(12)-HSL hydrolysis, ii) the effect of PON2 deficiency in acute PAO1 infection in mice and iii) the effect of 3OC(12)-HSL on PON2 deficient (PON2-def) macrophages. When compared to wild type macrophages, both intact cells and membrane-enriched protein lysates obtained from PON2-def macrophages show a marked impairment in their ability to hydrolyze 3OC(12)-HSL. PON2 expression (message and protein) is not altered in response to 3OC(12)-HSL in macrophages. 3OC(12)-HSL treated PON2-def macrophages showed i) an increase in ER stress and oxidative stress, ii) defective phosphatidylinositol 3-kinase (PI3 kinase)/AKT activation, and iii) reduced phagocytosis function. Moreover, the nitration to phosphorylation ratio of Tyr458 in p85 protein, the regulatory subunit of PI3-kinase that has been correlated with the phagocytosis function of macrophages, was increased in PON2-def macrophages. Antioxidant treatment reversed the effects of PON2 deficiency in macrophage phagocytosis function. Furthermore, following administration of 1.6 × 10(7) CFU of PAO1, bacterial clearance was significantly reduced in the lungs (5.7 fold), liver (2.5 fold), and spleen (14.8 fold) of PON2-def mice when compared to wild type mice. Our results suggest that PON2 plays an important role in innate immune defense against PAO1 infection.

The bacterial quorum-sensing signal molecule N-3-oxo-dodecanoyl-L-homoserine lactone reciprocally modulates pro- and anti-inflammatory cytokines in activated macrophages.

The bacterial molecule N-3-oxo-dodecanoyl-l-homoserine lactone (C12) has critical roles in both interbacterial communication and interkingdom signaling. The ability of C12 to downregulate production of the key proinflammatory cytokine TNF-α in stimulated macrophages was suggested to contribute to the establishment of chronic infections by opportunistic Gram-negative bacteria, such as Pseudomonas aeruginosa. We show that, in contrast to TNF-α suppression, C12 amplifies production of the major anti-inflammatory cytokine IL-10 in LPS-stimulated murine RAW264.7 macrophages, as well as peritoneal macrophages. Furthermore, C12 increased IL-10 mRNA levels and IL-10 promoter reporter activity in LPS-stimulated RAW264.7 macrophages, indicating that C12 modulates IL-10 expression at the transcriptional level. Finally, C12 substantially potentiated LPS-stimulated NF-κB DNA-binding levels and prolonged p38 MAPK phosphorylation in RAW264.7 macrophages, suggesting that increased transcriptional activity of NF-κB and/or p38-activated transcription factors serves to upregulate IL-10 production in macrophages exposed to both LPS and C12. These findings reveal another part of the complex array of host transitions through which opportunistic bacteria downregulate immune responses to flourish and establish a chronic infection.

Quorum sensing contributes to activated IgM-secreting B cell homeostasis.

Maintenance of plasma IgM levels is critical for immune system function and homeostasis in humans and mice. However, the mechanisms that control homeostasis of the activated IgM-secreting B cells are unknown. After adoptive transfer into immune-deficient hosts, B lymphocytes expand poorly, but fully reconstitute the pool of natural IgM-secreting cells and circulating IgM levels. By using sequential cell transfers and B cell populations from several mutant mice, we were able to identify novel mechanisms regulating the size of the IgM-secreting B cell pool. Contrary to previous mechanisms described regulating homeostasis, which involve competition for the same niche by cells having overlapping survival requirements, homeostasis of the innate IgM-secreting B cell pool is also achieved when B cell populations are able to monitor the number of activated B cells by detecting their secreted products. Notably, B cell populations are able to assess the density of activated B cells by sensing their secreted IgG. This process involves the FcγRIIB, a low-affinity IgG receptor that is expressed on B cells and acts as a negative regulator of B cell activation, and its intracellular effector the inositol phosphatase SHIP. As a result of the engagement of this inhibitory pathway, the number of activated IgM-secreting B cells is kept under control. We hypothesize that malfunction of this quorum-sensing mechanism may lead to uncontrolled B cell activation and autoimmunity.

Quorum sensing en la asociación beneficiosa de las bacterias con las plantas / Quorum sensing in beneficial plant-bacteria associations

Se conoce que el quorum sensing es un atributo común de muchas especies bacterianas y que puede ser un carácter universal de las bacterias. Actualmente se están describiendo a un paso más rápido nuevas señales y nuevos sistemas de regulación por quorum sensing y se han desarrollado las investigaciones acerca de la comunicación célula-célula en bacterias basada en el mecanismo de quorum sensing. En los ambientes naturales existen muchas bacterias que viven juntas y utilizan varias clases de moléculas señales. Dentro de las señales especie específicas predominan las acilhomoserín lactonas (AHLs), pero ya se han descrito una amplia diversidad de moléculas involucradas en la señalización célula-célula. Numerosos bioensayos y sistemas sensores se han desarrollado para la detección, caracterización y cuantificación de las AHLs. Se han obtenido evidencias de la acción de estas moléculas señales en la colonización de la rizosfera, el swarming, las interacciones simbióticas y la capacidad de interrumpir el proceso de señalización de otras bacterias que convivan en el mismo ambiente. Todas estas potencialidades de las bacterias que involucran el mecanismo de quorum sensing, pudieran ser utilizadas para fortalecerla acción estimuladora del crecimiento vegetal y el control biológico de patógenos en los agroecosistemas sostenibles.

The quorum sensing is a common attribute in some bacterial species. Currently, several signals and new regulation systems are describing and the researchers are very interested in the cell-cell communication based on quorum sensing mechanism. In the natural environments several bacteria are living together, then several types of signal molecules are using. The acylhomoserines lactones (AHLs), are predominant, but a wide range of molecules are involved in cell-cell communication. To detect, characterization and quantification of signals numerous bioassays and sensors systems were developed. It were demonstrated the action of signals molecules in the rhizosphere colonization, swarming, symbiotic interactions and the capacity to break the signaling process of another microorganism in the same environment. These potentialities of bacteria would be used to improve the plant growth stimulation and biological control of pathogens in sustainable agricultural.

Structural basis for ligand recognition and discrimination of a quorum-quenching antibody.

In the postantibiotic era, available treatment options for severe bacterial infections caused by methicillin-resistant Staphylococcus aureus have become limited. Therefore, new and innovative approaches are needed to combat such life-threatening infections. Virulence factor expression in S. aureus is regulated in a cell density-dependent manner using "quorum sensing," which involves generation and secretion of autoinducing peptides (AIPs) into the surrounding environment to activate a bacterial sensor kinase at a particular threshold concentration. Mouse monoclonal antibody AP4-24H11 was shown previously to blunt quorum sensing-mediated changes in gene expression in vitro and protect mice from a lethal dose of S. aureus by sequestering the AIP signal. We have elucidated the crystal structure of the AP4-24H11 Fab in complex with AIP-4 at 2.5 Å resolution to determine its mechanism of ligand recognition. A key Glu(H95) provides much of the binding specificity through formation of hydrogen bonds with each of the four amide nitrogens in the AIP-4 macrocyclic ring. Importantly, these structural data give clues as to the interactions between the cognate staphylococcal AIP receptors AgrC and the AIPs, as AP4-24H11·AIP-4 binding recapitulates features that have been proposed for AgrC-AIP recognition. Additionally, these structural insights may enable the engineering of AIP cross-reactive antibodies or quorum quenching vaccines for use in active or passive immunotherapy for prevention or treatment of S. aureus infections.

HHQ and PQS, two Pseudomonas aeruginosa quorum-sensing molecules, down-regulate the innate immune responses through the nuclear factor-kappaB pathway.

To explore whether bacterial secreted 4-hydroxy-2-alkylquinolines (HAQs) can regulate host innate immune responses, we used the extracts of bacterial culture supernatants from a wild-type (PA14) and two mutants of Pseudomonas aeruginosa that have defects in making HAQs. Surprisingly, the extract of supernatants from the P. aeruginosa pqsA mutant that does not make HAQs showed strong stimulating activity for the production of innate cytokines such as tumour necrosis factor-alpha and interleukin-6 in the J774A.1 mouse monocyte/macrophage cell line, whereas the extract from the wild-type did not. The addition of 4-hydroxy-2-heptylquinoline (HHQ) or 2-heptyl-3,4-dihydroxyquinoline (PQS, Pseudomonas quinolone signal) to mammalian cell culture media abolished this stimulating activity of the extracts of supernatants from the pqsA mutant on the expression of innate cytokines in J774A.1 cells and in the primary bronchoalveolar lavage cells from C57BL/6 mice, suggesting that HHQ and PQS can suppress the host innate immune responses. The pqsA mutant showed reduced dissemination in the lung tissue compared with the wild-type strain in a mouse in vivo intranasal infection model, suggesting that HHQ and PQS may play a role in the pathogenicity of P. aeruginosa. HHQ and PQS reduced the nuclear factor-kappaB (NF-kappaB) binding to its binding sites and the expression of NF-kappaB target genes, and PQS delayed inhibitor of kappaB degradation, indicating that the effect of HHQ and PQS was mediated through the NF-kappaB pathway. Our results suggest that HHQ and PQS produced by P. aeruginosa actively suppress host innate immune responses.

[Effect of the serum panel reactive antibody on proliferation and differentiation of cord blood CD34+ cells in vitro].

OBJECTIVE: The low rate of engraftment in children with beta-thalassemia has seriously restricted the popularity of the hematopoietic stem cell transplantation (HSCT). Panel reactive antibody (PRA) has been regarded as one of the important factors for the success of kidney transplantation. Poly-transfusion before transplantation is associated with the production of PRA. Also PRA is produced in the children with beta-thalassemia major who need poly-transfusion for life. PRA might be one of factors inducing the low rate of engraftment in children with beta-thalassemia. This study focused on observing the effect of PRA on the proliferation, differentiation, apoptosis and necrosis of cord blood CD34(+) cells in vitro by incubating the cord blood CD34(+) cells with serum containing PRA. METHOD: Seven samples of cord blood were collected and the HLA typing for every sample was done. Seven sera positive for PRA and seven negative sera were selected respectively. Mononuclear cells (MNCs) were obtained by Ficoll-Hypaque density gradient centrifugation. CD34(+) cells were isolated from MNCs by positive selection using an immunomagnetic separation (CD34(+) progenitor cell isolation kit and auto-MACS). The CD34(+) cells of umbilical cord blood were incubated with the serum and complement in the following groups: A (absence of serum), B (presence of PRA positive serum), C (presence of PRA positive serum and complement), D (presence of complement), and E (presence of PRA negative serum). After incubation the samples were centrifuged and the supernatant was collected for LDH detection. At the same time the CD34(+) cells were harvested for assessing the expression of Annexin V and CD95 of the CD34(+) cells by flow cytometry and also for the detection of the DNA synthesis by (3)H-TaR incorporation. Meanwhile the cells were inoculated into the methylcellulose cultural system. The proliferation and hematopoietic potential of the CD34(+) cell of cord blood by the colony formation assay were detected on the day 10. RESULT: The concentration of LDH in group A was (20.71 +/- 2.81) U/L, which was significantly lower than that in group B (64.28 +/- 5.12) U/L and group C (84.29 +/- 4.99) U/L. The concentration of LDH in group B was significantly lower than that in group C, while there were no significant differences in the concentration of LDH among groups A, D and E (P > 0.05). The cpm in group A was (22 629 +/- 3288), which was significantly higher than that in group B (4598 +/- 2178) and group C (1626 +/- 1192). And the cpm in group B was significantly higher than that in group C. There were no significant differences in the cpm among groups A, D and E (P > 0.05). On day 10 of culture, the total colonies, granulocyte-macrophage colony forming unit (CFU-GM), mixed colony forming unit (CFU-GEMM) and erythroid burst colony forming unit (BFU-E) in group A were significantly higher than that in group B and C. The total colonies, CFU-GM and CFU-GEMM in group B were significantly higher than those in group C. No significant differences were found in the total colonies, CFU-GM, CFU-GEMM and BFU-E among groups A, D and E (P > 0.05). There were no statistically significant differences in the CD95 and Annexin V expression among all the groups (P > 0.05). CONCLUSION: PRA could impair the membrane, decrease the DNA synthesis, and inhibit the colony formation of CD34(+) cord blood cells, which could be strengthened by the presence of the complement at the given concentration in our study. PRA had no significant influence on the apoptosis of CD34(+) cells in vitro.

Significant immunomodulatory effects of Pseudomonas aeruginosa quorum-sensing signal molecules: possible link in human sepsis.

Pathogenic bacteria use quorum-sensing signal molecules to co-ordinate the expression of virulence genes. Animal-based studies have demonstrated the immunomodulatory effects of quorum-sensing signal molecules. In the present study, we have examined the impact of these molecules on normal human immune function in vitro and compared this with immune changes in patients with sepsis where quorum-sensing signal molecules were detected in the sera of patients. Quorum-sensing signal molecules inhibited normal dendritic cell and T-cell activation and proliferation, and down-regulated the expression of co-stimulatory molecules on dendritic cells; in MLDCRs (mixed lymphocyte dendritic cell reactions), secretion of IL (interleukin)-4 and IL-10 was enhanced, but TNF-alpha (tumour necrosis factor-alpha), IFN-gamma (interferon-gamma) and IL-6 was reduced. Quorum-sensing signal molecules induced apoptosis in dendritic cells and CD4(+) cells, but not CD8(+) cells. Dendritic cells from patients with sepsis were depleted and ex vivo showed defective expression of co-stimulatory molecules and dysfunctional stimulation of allogeneic T-lymphocytes. Enhanced apoptosis of dendritic cells and differential CD4(+) Th1/Th2 (T-helper 1/2) cell apoptotic rate, and modified Th1/Th2 cell cytokine profiles in MLDCRs were also demonstrated in patients with sepsis. The pattern of immunological changes in patients with sepsis mirrors the effects of quorum-sensing signal molecules on responses of immune cells from normal individuals in vitro, suggesting that quorum-sensing signal molecules should be investigated further as a cause of immune dysfunction in sepsis.

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.