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Pseudomonas aeruginosa is a pathogen responsible for severe opportunistic pulmonary infections and a considerable source of complications in the burn wards. Quorum sensing is a chemical-based communication mechanism in prokaryotes. In the basic mode, an autoinducer released by select individuals is sensed by intracellular receptors in other members of the community, leading to the collective isogenic autoinducer synthesis and synchronized activities. In P. aeruginosa, there are two quorum-sensing circuits: LasR–LasI and RhlR–RhlI; the former uses N-(3-oxo-dodecanoyl) homoserine lactone (3OC12 HSL or 3oc) as the autoinducer, and the latter uses C4 (butanoyl) HSL. 3oc is the most studied quorum-sensing autoinducer and it has widespread affect in mammalian hosts. HSLs have been reported to regulate neutrophil phagocytosis, inhibit dendritic cell antigen presentation, induce pro-inflammatory cytokines, trigger lymphocyte cell death.
An unexpected mechanism that ordered lipid domains that are unique in the eukaryotic membrane are dissolvable in the presence of HSLs. This in turn results in a forced expulsion of tumour necrosis factor receptor 1 (TNFR1) into the disordered phase of the membrane, leading to a higher degree of spontaneous trimerization and TNFR1 signalling without external ligand. This distribution shift alone drives the entire process of caspase 8–caspase 3 axis activation and apoptotic cell death and is used by P. aeruginosa to suppress host immunity. These findings suggest a previously unknown mechanism for how eukaryotic cells sense microbial metabolic products.
3oc can suppress T cell activation, at modest concentrations, 3oc triggered significant T cell death. This cell death was detectable at the nanomolar range and was blocked by pan-caspase and caspase-8-specific inhibitors. Similar levels of 3-oxo-C10 HSL and 3-oxo-C14 HSL were also effective. In 3oc-treated CD4 T cells, caspase 8 and caspase 3 were cleaved. Among the analogues, 3-oxo-C10 HSL, 3oc and 3-oxoC14 HSLs induced the caspase 8 digestion. Fas-associated protein with death domain (FADD) was found to be in complex with caspase 8 following the treatment in reciprocal immunoprecipitation assays. A dominant negative version of FADD also reduced 3oc-induced cell death. As FADD links caspase 8 to cell surface death receptors, the overall results suggest that the initiation signal comes from one of the TNFR family members.
The eukaryotic cell plasma membrane contains regions rich in cholesterol and glycosphingolipids. As 3oc seemed to mediate a signal from a cell surface receptor, researchers wondered whether HSLs were retained in the plasma membrane. The results indicate that HSL retention in the plasma membrane is dependent on lipid domains and this membrane entrapment effect echoes the apoptosis induction.
In the death-inducing signalling complex, the signalling cascade becomes active upon the trimerization of TNFR family receptors. For TNFR1, recruitment to the lipid rafts is required for NF-κB activation; however, the signal is turned apoptotic if lipid domains are disrupted. To see the effect of 3oc on the membrane directly, single-molecule photobleaching step-counting (SMPSC) assay was performed to analyse TNFR1 in its native state. Through a mutagenesis study, scientist determined that the entire extracellular cysteine-rich domain 1 (CRD1) domain of TNFR1 was required for the 3oc-induced trimerization. 3oc and its active analogues specifically target domains formed in the presence of cholesterol and sphingolipids. Thus, it is possible that liquid ordered components remained in the membrane following 3oc treatment, in a phase other than the liquid ordered domains.
A new proposal suggests that lipid disordered phases are more intertwined with the cortical cytoskeleton, creating a physical barrier to confine the ordered domains. Transmembrane proteins under this constrain display motion trajectories characterized as simple, directed, stationary and restricted. Extracellular TNFR1-specific antibody was digested by papain to yield the Fab fragment, which was coupled to a quantum dot. The experimental results show that TNFR1 time to trimerization is inversely correlated with its diffusion rate; in other words, a fast-moving TNFR1 will have better odds to form trimers.
The LasI–LasR circuit mediates a large number of factors that may contribute to the infectivity of P. aeruginosa in addition to the cytotoxic effect of 3oc. It was still important to ascertain that 3oc mediated cell death can counter host immunity as a factor independent of other gene regulations. Research shows that 3oc-mediated host cell death seems to operate as an independent factor to facilitate P. aeruginosa infection in the lung, and 3oc indeed triggered neutrophil apoptotic death. Collectively, these results suggest a non-antibiotic bacterial infection control via intercepting the neutrophil apoptosis that results from autoinducer release by invading pathogens.
A surprising finding of this work is that P. aeruginosa uses 3oc to disarm the immunity via directly triggering the host's own TNFR1 signalling. This result exposes an immune regulatory mechanism by the autoinducer that is directly coupled to the signalling in host cell defence. This finding and the revelation that bacterial autoinducers use a previously unknown inter-kingdom communication method to regulate host innate immunity may provide important clues for our future investigations into this class of microbial metabolites.
| Target | Cat. No. | Product Name | Expression System | Tag/Conjugate | Application | Inquiry |
| P. aeruginosa | DAGC014 | Recombinant Pseudomonas aeruginosa Pseudolysin (lasB) protein [His-SUMO] | E. coli | His-SUMO | N/A | Inquiry |
| P. aeruginosa toxA | DAG4062 | P. aeruginosa Exotoxin A | Pseudomonas aeruginosa | Unconjugated | N/A | Inquiry |
| Target | Cat. No. | Product Name | Size | Species Reactivity | Application | Detection Sample | Inquiry |
| P. aeruginosa toxA | DEIA-LL250 | Pseudomonas Exotoxin A ELISA Kit | Universal | 96T | Quantitative | Serum, plasma, tissue homogenates and other biological fluids | Inquiry |
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