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Abstract
Quorum sensing (QS) signaling allows bacteria to control gene expression once a critical population density is achieved. The Gram-negative human pathogen Pseudomonas aeruginosa uses N-acylhomoserine lactones (AHL) as QS signals, which coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human cell behavior. Little is known about the mechanisms of the action of AHL on their eukaryotic targets. Here, we found that N-3-oxo-dodecanoyl-L-homoserine lactone 3O-C12-HSL modulates human intestinal epithelial Caco-2 cell migration in a dose- and time-dependent manner. Using new 3O-C12-HSL biotin and fluorescently-tagged probes for LC-MS/MS and confocal imaging, respectively, we demonstrated for the first time that 3O-C12-HSL interacts and co-localizes with the IQ-motif-containing GTPase-activating protein IQGAP1 in Caco-2 cells. The interaction between IQGAP1 and 3O-C12-HSL was further confirmed by pull-down assay using a GST-tagged protein with subsequent Western blot of IQGAP1 and by identifying 3O-C12-HSL with a sensor bioassay. Moreover, 3O-C12-HSL induced changes in the phosphorylation status of Rac1 and Cdc42 and the localization of IQGAP1 as evidenced by confocal and STED microscopy and Western blots. Our findings suggest that the IQGAP1 is a novel partner for P.aeruginosa 3O-C12-HSL and likely the integrator of Rac1 and Cdc42- dependent altered cell migration. We propose that the targeting of IQGAP1 by 3O-C12-HSL can trigger essential changes in the cytoskeleton network and be an essential component in bacterial – human cell communication.
Highlights
Quorum sensing (QS) is a population-density-dependent signaling system that primarily enables bacteria to control the expression of certain genes
We have previously shown that N-acylhomoserine lactones (AHL) alter epithelial barrier functions and increase chemotaxis in human neutrophils
Using newly designed and validated biotin- and fluorescein-based 3O-C12-HSL probes we identified the IQ-motifcontaining GTPase-activating protein IQGAP1 as a human target of 3O-C12-HSL
Summary
Quorum sensing (QS) is a population-density-dependent signaling system that primarily enables bacteria to control the expression of certain genes. Release and detect distinct low-molecular-weight QS signal molecules, which bind to intracellular receptors in the bacteria to coordinate transcription of QS-controlled genes [1]. In Gram-negative human pathogen Pseudomonas aeruginosa, there are two chemically distinct but subordinated QS systems that are N-acylhomoserine lactone(AHL) and 2-alkyl-4-quinolone-dependent, respectively. Two AHL molecules are produced by P.aeruginosa, N-3-oxo-dodecanoylL-homoserine lactone (3O-C12-HSL) (Figure 1A) and N-butyryl-Lhomoserine lactone (C4-HSL), which directly or indirectly control the expression of multiple virulence factors, secondary metabolites, swarming motility and biofilm development [2,3]. Rather little is known about the exact mechanisms of the action of AHL on eukaryotic cells and their direct target(s) or receptor(s), but it has been suggested that AHL acts through multiple signaling pathways [5]. There is evidence that lipophilic 3O-C12-HSL molecules with a long acyl chain and an intact homoserine lactone ring (Figure 1A)
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