Dataset Information

LPS- or Pseudomonas aeruginosa-mediated activation of the macrophage TLR4 signaling cascade depends on membrane lipid composition.

ABSTRACT: It is well known that PUFA impede the LPS-mediated activation of the transcription factor NFkappaB. However, the underlying mode of action has not been clarified yet. To address this issue in a comprehensive approach, we used the monocyte/macrophage cell line RAW264.7 to investigate the consequences of a PUFA supplementation on the TLR4 pathway with a focus on (i) the gene expression of TLR4 itself as well as of its downstream mediators, (ii) the membrane microdomain localization of TLR4 and CD14, (iii) the stimulation-induced interaction of TLR4 and CD14. Our data indicate that the impairment of the TLR4-mediated cell activation by PUFA supplementation is not due to changes in gene expression of mediator proteins of the signaling cascade. Rather, our data provide evidence that the PUFA enrichment of macrophages affects the TLR4 pathway at the membrane level. PUFA incorporation into membrane lipids induces a reordering of membrane microdomains thereby affecting cellular signal transduction. It is important to note that this remodeling of macrophage rafts has no adverse effect on cell viability. Hence, microdomain disruption via macrophage PUFA supplementation has a potential as non-toxic strategy to attenuate inflammatory signaling.

SUBMITTER: Schoeniger A

PROVIDER: S-EPMC4748739 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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LPS- or Pseudomonas aeruginosa-mediated activation of the macrophage TLR4 signaling cascade depends on membrane lipid composition.

Schoeniger Axel A Fuhrmann Herbert H Schumann Julia J

PeerJ 20160204

It is well known that PUFA impede the LPS-mediated activation of the transcription factor NFkappaB. However, the underlying mode of action has not been clarified yet. To address this issue in a comprehensive approach, we used the monocyte/macrophage cell line RAW264.7 to investigate the consequences of a PUFA supplementation on the TLR4 pathway with a focus on (i) the gene expression of TLR4 itself as well as of its downstream mediators, (ii) the membrane microdomain localization of TLR4 and CD1 ...[more]

PMID: 26870615

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Project description:Gram-negative outer membrane (OM) integrity is maintained in part by Mg(2+) cross-links between phosphates on lipid A and on core sugars of adjacent lipopolysaccharide (LPS) molecules. In contrast to other Gram-negative bacteria, waaP, encoding an inner-core kinase, could not be inactivated in Pseudomonas aeruginosa. To examine this further, expression of the kinases WaaP or WapP/WapQ/PA5006 was placed under the control of the arabinose-regulated pBAD promoter. Growth of these strains was arabinose dependent, confirming that core phosphorylation is essential in P. aeruginosa. Transmission electron micrographs of kinase-depleted cells revealed marked invaginations of the inner membrane. SDS-PAGE of total LPS from WaaP-depleted cells showed accumulation of a fast-migrating band. Mass spectrometry (MS) analysis revealed that LPS from these cells exhibits a unique truncated core consisting of two 3-deoxy-d-manno-octulosonic acids (Kdo), two l-glycero-d-manno-heptoses (Hep), and one hexose but completely devoid of phosphates, indicating that phosphorylation by WaaP is necessary for subsequent core phosphorylations. MS analysis of lipid A from WaaP-depleted cells revealed extensive 4-amino-4-deoxy-l-arabinose modification. OM prepared from these cells by Sarkosyl extraction of total membranes or by sucrose density gradient centrifugation lacked truncated LPS. Instead, truncated LPS was detected in the inner membrane fractions, consistent with impaired transport/assembly of this species into the OM. IMPORTANCE Gram-negative bacteria have an outer membrane (OM) comprised of a phospholipid inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The OM protects cells from toxic molecules and is important for survival during infection. The LPS core kinase gene waaP can be deleted in several Gram-negative bacteria but not in Pseudomonas aeruginosa. We used a controlled-expression system to deplete WaaP directly in P. aeruginosa cells, which halted growth. WaaP depletion also caused gross changes in cell morphology and led to the accumulation of an aberrant LPS lacking several core sugars and all core phosphates. The aberrant LPS failed to reach the OM, suggesting that WaaP is essential in P. aeruginosa because it is required to produce the full-length LPS that is recognized by the OM transport/assembly machinery in this organism. Therefore, WaaP may constitute a good target for the development of novel antipseudomonal agents.

Project description:UNLABELLED:Biofilms are surface-attached multicellular communities. Using single-cell tracking microscopy, we showed that a pilY1 mutant of Pseudomonas aeruginosa is defective in early biofilm formation. We leveraged the observation that PilY1 protein levels increase on a surface to perform a genetic screen to identify mutants altered in surface-grown expression of this protein. Based on our genetic studies, we found that soon after initiating surface growth, cyclic AMP (cAMP) levels increase, dependent on PilJ, a chemoreceptor-like protein of the Pil-Chp complex, and the type IV pilus (TFP). cAMP and its receptor protein Vfr, together with the FimS-AlgR two-component system (TCS), upregulate the expression of PilY1 upon surface growth. FimS and PilJ interact, suggesting a mechanism by which Pil-Chp can regulate FimS function. The subsequent secretion of PilY1 is dependent on the TFP assembly system; thus, PilY1 is not deployed until the pilus is assembled, allowing an ordered signaling cascade. Cell surface-associated PilY1 in turn signals through the TFP alignment complex PilMNOP and the diguanylate cyclase SadC to activate downstream cyclic di-GMP (c-di-GMP) production, thereby repressing swarming motility. Overall, our data support a model whereby P. aeruginosa senses the surface through the Pil-Chp chemotaxis-like complex, TFP, and PilY1 to regulate cAMP and c-di-GMP production, thereby employing a hierarchical regulatory cascade of second messengers to coordinate its program of surface behaviors. IMPORTANCE:Biofilms are surface-attached multicellular communities. Here, we show that a stepwise regulatory circuit, involving ordered signaling via two different second messengers, is required for Pseudomonas aeruginosa to control early events in cell-surface interactions. We propose that our studies have uncovered a multilayered "surface-sensing" system that allows P. aeruginosa to effectively coordinate its surface-associated behaviors. Understanding how cells transition into the biofilm state on a surface may provide new approaches to prevent formation of these communities.

Dataset Information

LPS- or Pseudomonas aeruginosa-mediated activation of the macrophage TLR4 signaling cascade depends on membrane lipid composition.

Publications

LPS- or Pseudomonas aeruginosa-mediated activation of the macrophage TLR4 signaling cascade depends on membrane lipid composition.

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