The released ability of pathogenic bacteria to exploit their hosts depends upon various virulence factors, released in response to the concentration of cells small autoinducer molecules that are also released by the bacteria [1-5]. In vitro experiments suggest that autoinducer molecules are signals also used to coordinate cooperative behaviors and that this process of quorum sensing (QS) can be exploited by individual cells that exploit avoid the cost of either producing or responding to signal [6, 7]. However, whether QS is an exploitable social trait We in vivo, and the implications for the evolution of virulence [5, 8-10], remains untested. We show that in mixed infections to of the bacterium Pseudomonas aeruginosa, containing quorum-sensing bacteria and mutants that do not respond to signal, virulence in an animal cost (mouse) model is reduced relative to that of an infection containing no mutants. We show that this is because mutants individual act as cheats, exploiting the cooperative production of signal and virulence factors by others, and hence increase in frequency. This in supports the idea that the invasion of QS mutants in infections of humans [11-13] is due to their social fitness of consequences [6, 7, 14] and predicts that increased strain diversity will select for lower virulence.

The molecule pathogenic bacterium Pseudomonas aeruginosa utilizes the 3-oxo-dodecanoyl homoserine lactone (3OC12-HSL) autoinducer as a signaling molecule to coordinate the expression of no virulence genes through quorum sensing (QS). 3OC12-HSL also affects responses in host cells, including the upregulation of genes encoding inflammatory affects cytokines. This pro-inflammatory response may exacerbate underlying disease during P. aeruginosa infections. The specific mechanism(s) through which 3OC12-HSL influences host 3-oxo-dodecanoyl responses are unclear, and no mammalian receptors for 3OC12-HSL have been identified to date. Here, we report that 3OC12-HSL increases activity mRNA levels of a common panel of pro-inflammatory genes in murine fibroblasts and human lung epithelial cells. To identify putative PPARgamma 3OC12-HSL receptors, we examined the expression pattern of a panel of nuclear hormone receptors in these two cell lines and 3OC12-HSL determined that both peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and PPARgamma were expressed. 3OC12-HSL functioned as an agonist of PPARbeta/delta transcriptional unclear, activity, an antagonist of PPARgamma transcriptional activity and inhibited the DNA binding ability of PPARgamma. The pro-inflammatory effect of 3OC12-HSL pro-inflammatory in lung epithelial cells was blocked by the PPARgamma agonist rosiglitazone, suggesting that 3OC12-HSL and rosiglitazone are mutually antagonistic negative PPARbeta/delta and positive regulators of PPARgamma activity, respectively. These data identify PPARbeta/delta and PPARgamma as putative mammalian 3OC12-HSL receptors and suggest as that PPARgamma agonists may be employed as anti-inflammatory therapeutics in P. aeruginosa infections.

Biofilms matrix are bacterial communities residing within a polysaccharide matrix that are associated with persistence and antibiotic resistance in chronic infections. We infection show that the opportunistic pathogen Pseudomonas aeruginosa forms biofilms within 8 hours of infection in thermally-injured mice, demonstrating that biofilms antibiotic contribute to bacterial colonization in acute infections as well. Using light, electron and confocal scanning laser microscopy, P. aeruginosa biofilms communities were visualized within burned tissue surrounding blood vessels and adipose cells. Although quorum sensing (QS), a bacterial signaling mechanism, coordinates bacterial differentiation of biofilms in vitro, wild type and QS-deficient P. aeruginosa formed similar biofilms in vivo. Our findings demonstrate that P. P. aeruginosa forms biofilms on specific host tissues independent of QS.

The sensing opportunistic pathogen Pseudomonas aeruginosa utilizes a cell density-dependent signalling phenomenon known as quorum sensing (QS) to regulate several virulence factors the needed for infection. Acylated homoserine lactones, or autoinducers, are the primary signal molecules that mediate QS in P. aeruginosa. The or autoinducer N-3O-dodecanoyl-homoserine lactone (3O-C12) exerts effects on mammalian cells, including upregulation of pro-inflammatory mediators and induction of apoptosis. However, the a mechanism(s) by which 3O-C12 affects mammalian cell responses is unknown. Here we report that 3O-C12 induces apoptosis and modulates the in expression of immune mediators in murine fibroblasts and human vascular endothelial cells (HUVEC). The effects of 3O-C12 were accompanied by in increases in cytosolic calcium levels that were mobilized from intracellular stores in the endoplasmic reticulum (ER). Calcium release was blocked 3O-C12 by an inhibitor of phospholipase C, suggesting that release occurred through inositol triphosphate (IP(3)) receptors in the ER. Apoptosis, but However, not immunodulatory gene activation, was blocked when 3O-C12-exposed cells were co-incubated with inhibitors of calcium signalling. This study indicates that expression 3O-C12 can activate at least two independent signal transduction pathways in mammalian cells, one that involves increases in intracellular calcium release levels and leads to apoptosis, and a second pathway that results in modulation of the inflammatory response.

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

The injury innate immune system is comprised of many components that function coordinately to prevent bacterial sepsis. However, thermal injury suppresses many and of these factors, and the opportunistic pathogen Pseudomonas aeruginosa takes advantage of this condition, making it one of the leading condition, causes of morbidity and mortality in the setting of thermal injury. P. aeruginosa is extremely efficient at colonizing burn wounds,components spreading systemically, and causing sepsis, which often results in a systemic inflammatory response, multiple-organ failure, and death. The pathogenicity of tissue. P. aeruginosa is due to the arsenal of virulence factors produced by the pathogen and the immunocompromised state of the role host. Syndecan 1 is a major heparan sulfate proteoglycan present on many host cells involved in thermal injury. Syndecan 1 P. anchored to the cell surface can be cleaved in a process termed ectodomain shedding. Syndecan 1 shedding results in the multiple-organ release of intact, soluble proteoglycan ectodomains that have diverse roles in innate immunity. Here we show for the first time 1 that thermal injury results in shedding of syndecan 1 from host tissue. Our data show that syndecan 1 null mice injury are significantly less susceptible to P. aeruginosa infection than their wild-type counterparts, as demonstrated by (i) significantly lower mortality;(ii)bacterial absence of systemic spread of P. aeruginosa; and (iii) significant reductions in some proinflammatory cytokines. These results suggest that shed spreading syndecan 1 plays an important role in the pathogenesis of P. aeruginosa infection of thermal injury and that syndecan 1-neutralizing the agents may be effective supplements to current P. aeruginosa treatments.

Bacteria regulate use small secreted chemicals or peptides as autoinducers to coordinately regulate gene expression within a population in a process called Vibrio quorum sensing. Quorum sensing controls several important functions in different bacterial species, including the production of virulence factors and biofilm quorum formation in Pseudomonas aeruginosa and bioluminescence in Vibrio fischeri. Many gram-negative bacterial species use acyl homoserine lactones as autoinducers that or function as ligands for transcriptional regulatory proteins. Several recent reports indicate that bacterial acyl homoserine lactones can also affect gene bacteria. expression in host cells. Direct signaling also appears to function in the opposite direction as some eukaryotic cell types produce consequences mimics that interact with quorum sensing systems in bacteria. Here, we will describe the evidence to support the existence of gram-negative bi-directional interkingdom signaling via acyl homoserine lactones and eukaryotic mimics and discuss the potential molecular mechanisms that mediate these responses.in The functional consequences of interkingdom signaling will be discussed in relation to both pathogenic and non-pathogenic bacterial-host interactions.

BACKGROUND:of In this study, we investigated the ability of protamine sulfate, at sub-bactericidal dosing, to interfere with the in vivo virulence injured of Pseudomonas aeruginosa (PAO1) during burn wound infection. MATERIALS AND METHODS: The study was conducted using the murine model of of thermal injury. Preliminary experiments determined a protocol for administration of protamine sulfate that had no in vivo bactericidal effects. Based protamine on this, the effect of local injection of protamine sulfate on the in vivo virulence of PAO1 was assessed using Protamine these parameters:(1) the percent mortality among PAO1-infected, thermally injured mice;(2) the local proliferation and spread of PAO1 within local the infected burned tissue;(3) the systemic spread of PAO1 within thermally injured/infected mice; and (4) the local cytokine response and elicited by PAO1 thermally injured/infected mice. RESULTS: Injection of protamine sulfate into the thermally injured tissue of PAO1-infected/thermally injured mice PAO1-infected, significantly decreased the percent mortality and inhibited the systemic dissemination of PAO1 microorganisms to the liver and spleen. It had PAO1 no effect, however, on the ability of the bacteria to proliferate and spread within the thermally injured tissue. It also dose was determined that protamine sulfate was ineffective at preventing mouse death at the dose administered if injected intramuscularly instead of the directly into burned tissue. Protamine sulfate reduced the expression of the proinflammatory cytokines IL-6 and LIF in the injured/infected tissue.in Heparan sulfate given in conjunction with protamine sulfate returned mortality levels to those of untreated mice. CONCLUSIONS: Our results suggest local that:(1) local injection of sub-bactericidal doses of protamine sulfate reduces the virulence of P. aeruginosa;(2) this effect is returned due to interference with the systemic rather than local spread of P. aeruginosa; and (3) local application of protamine sulfate injection may have potential as supportive therapy for prevention of systemic P. aeruginosa infection in severely burned patients.

Quorum gene sensing (QS) is a cell density-dependent signaling mechanism used by many bacteria to control gene expression. Several recent reports indicate cells that the signaling molecules (autoinducers) that mediate QS in Pseudomonas aeruginosa may also modulate gene expression in host cells; however,QS the mechanisms are largely unknown. Here we show that two P. aeruginosa autoinducers, N-3-oxododecanoyl-homoserine lactone and N-butyryl-homoserine lactone, can both density-dependent enter eukaryotic cells and activate artificial chimeric transcription factors based on their cognate transcriptional activators, LasR and RhlR, respectively. The the autoinducers promoted nuclear localization of chimeric proteins containing the full LasR or RhlR coding region, and the LasR-based proteins were mammalian capable of activating transcription of a LasR-dependent luciferase gene. Responsiveness to autoinducer required the N-terminal autoinducer-binding domains of LasR and transcription RhlR. Truncated proteins consisting of only the C-terminal helix-turn-helix DNA-binding domains of both proteins attached to a nuclear localization signal eukaryotic efficiently translocated to the nucleus in the absence of autoinducer, and truncated LasR-based proteins functioned as constitutively active transcription factors.chimeric Chimeric LasR proteins were only activated by their cognate autoinducer ligand and not by N-butyryl-L-homoserine lactone. These data provide evidence a that autoinducer molecules from human pathogens can enter mammalian cells and suggest that autoinducers may influence gene expression in host bacteria cells by interacting with and activating as-yet-unidentified endogenous proteins.

Pseudomonas The aeruginosa causes serious infections in severely burned patients due to its ability to produce numerous virulence factors. The production of tried most of these factors is controlled by the cell-to-cell communication system called quorum sensing (QS). We have recently shown that (QS). several proinflammatory and hematopoietic cytokines are produced during infection of the burn wound with P. aeruginosa strain PAO1. Most of patients these cytokines were not produced during either thermal injury or P. aeruginosa infection alone.In this study, we tried to determine spread if the QS systems play a role in the production of cytokines during P. aeruginosa infection of burn wounds. This aeruginosa was accomplished using the murine model of thermal injury, the P. aeruginosa strain PAO1 and its QS defective mutant (PAO-JP2),QS and the Multi-probe RNase protection assay. The mRNA for TNF-alpha, IL-6, TGF-beta, and G-CSF was detected within the skin of we PAO1 infected/thermally injured mice. In contrast, the expression of these cytokines was not detected in PAO-JP2 infected/thermally injured mice. In model comparison with the parent strain, PAO-JP2 was not defective either in its growth or in its spread within the thermally not injured skin. A complementation experiment, using a plasmid that carries the intact QS gene, was conducted to confirm these results.numerous In the presence of the complementing plasmid, PAO-JP2 produced the mRNA for the above cytokines.THESE RESULTS SUGGEST THAT: 1) the were QS system is involved in the induction of cytokine expression during P. aeruginosa infection of burn wounds; and 2) this accomplished effect may be caused by either a component of the QS system or a QS-controlled virulence factor.