The Pseudomonas aeruginosa N-Acylhomoserine Lactone Quorum Sensing Molecules Target IQGAP1 and Modulate Epithelial Cell Migration

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-C ₁₂-HSL modulates human intestinal epithelial Caco-2 cell migration in a dose- and time-dependent manner. Using new 3O-C ₁₂-HSL biotin and fluorescently-tagged probes for LC-MS/MS and confocal imaging, respectively, we demonstrated for the first time that 3O-C ₁₂-HSL interacts and co-localizes with the IQ-motif-containing GTPase-activating protein IQGAP1 in Caco-2 cells. The interaction between IQGAP1 and 3O-C ₁₂-HSL was further confirmed by pull-down assay using a GST-tagged protein with subsequent Western blot of IQGAP1 and by identifying 3O-C ₁₂-HSL with a sensor bioassay. Moreover, 3O-C ₁₂-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-C ₁₂-HSL and likely the integrator of Rac1 and Cdc42- dependent altered cell migration. We propose that the targeting of IQGAP1 by 3O-C ₁₂-HSL can trigger essential changes in the cytoskeleton network and be an essential component in bacterial – human cell communication.

Author Summary

The human pathogen Pseudomonas aeruginosa and other bacteria communicate with each other using quorum sensing (QS). This is important for their growth, virulence, motility and the formation of biofilms. Furthermore, eukaryotic cells “listen and respond” to QS signaling, but the exact mechanisms and receptors on mammalian cells have not been identified. We have previously shown that N-acylhomoserine lactones (AHL) alter epithelial barrier functions and increase chemotaxis in human neutrophils. We show here that 3O-C ₁₂-HSL modulates the migration of epithelial cells in a dose- and time-dependent manner. Using newly designed and validated biotin- and fluorescein-based 3O-C ₁₂-HSL probes we identified the IQ-motif-containing GTPase-activating protein IQGAP1 as a human target of 3O-C ₁₂-HSL. We propose that the interaction between IQGAP1 and 3O-C ₁₂-HSL provides a novel mechanism for its mode of action on eukaryotic cells, and by affecting the distribution of IQGAP1 and phosphorylation of Rac1 and Cdc42, upstream effectors of filamentous actin remodeling, also cell migration. We suggest that recognition of IQGAP1 by 3O-C ₁₂-HSL is a very early event in the communication between bacteria and human epithelial cells.