Interconnections of Pseudomonas aeruginosa Quorum-Sensing Systems in Intestinal Permeability and Inflammation

Abstract

Quorum sensing (QS) is a highly conserved microbial communication mechanism based on the production and sensing of secreted signaling molecules. The recalcitrant pathogen Pseudomonas aeruginosa is a problematic nosocomial pathogen with complex interconnected QS systems controlling multiple virulence functions. The relevance of QS in P. aeruginosa pathogenesis is well established; however, the regulatory interrelationships of the three major QS systems, LasR/LasI, MvfR (PqsR)/PqsABCD, and RhlR/RhlI, have been studied primarily in vitro. It is, therefore, unclear how these relationships translate to the host environment during infection. Here, we use a collection of P. aeruginosa QS mutants of the three major QS systems to assess the interconnections and contributions in intestinal inflammation and barrier function in vivo. This work reveals that MvfR, not LasR or RhlR, promotes intestinal inflammation during infection. In contrast, we find that P. aeruginosa-driven murine intestinal permeability is controlled by an interconnected QS network involving all three regulators, with MvfR situated upstream of LasR and RhlR. This study demonstrates the importance of understanding the interrelationships of the QS systems during infection and provides critical insights for developing successful antivirulence strategies. Moreover, this work provides a framework to interrogate QS systems in physiologically relevant settings. IMPORTANCE Pseudomonas aeruginosa is a common multidrug-resistant bacterial pathogen that seriously threatens critically ill and immunocompromised patients. Intestinal colonization by this pathogen is associated with elevated mortality rates. Disrupting bacterial communication is a desirable anti-infective approach since these systems coordinate multiple acute and chronic virulence functions in P. aeruginosa. Here, we investigate the role of each of the three major communication systems in the host intestinal functions. This work reveals that P. aeruginosa influences intestinal inflammation and permeability through distinct mechanisms.

Keywords: LasR; MvfR; PqsR; Pseudomonas aeruginosa; RhlR; antivirulence; intestinal inflammation; intestinal permeability; quorum sensing; virulence.

FIG 1

Dissemination of P. aeruginosa from the site of infection to the ileum (A) and colon (B). The inflammatory response in the ileum TNF-α levels (C) increased when mice were infected with ΔlasR, ΔrhlR, and WT strains, whereas the TNF-α level was similar to burn alone in ΔmvfR, double, and triple mutants. (D) The flow of the FITC-dextran from the intestinal lumen to the systemic circulation was not increased significantly in the ΔmvfR single mutant in comparison to burn alone, whereas the WT, ΔlasR, ΔrhlR, and double and triple mutants increased intestinal permeability. Each dot represents data from one mouse. The error bars denote ± SEM. Significance was assessed by Kruskal-Wallis nonparametric test with Dunnett's posttest applied. Exact P values are provided. A portion of the underlying data for the WT, burn alone, and ΔmvfR were previously published in reference . These data were reused and reanalyzed, as these experiments were performed in parallel to experiments with the other single, double, and triple mutants.

FIG 2

Immunofluorescence staining of the tight junction Caco-2 cells. (A) The integrity of the tight junction protein claudin-1 was compromised when cells were infected with WT, ΔlasR, ΔrhlR, and double and triple mutants, whereas it was unaffected in the cells infected with ΔmvfR mutants. Following a 2-h infection, Caco-2 cells were fixed in 4% paraformaldehyde for 10 min at 37°C, washed with PBS three times, and blocked with 2% bovine serum albumin (BSA) in PBS for 60 min at room temperature. Cells were incubated with primary antibody (anti-claudin-1, catalog no. 71-7800, and anti-ZO1, catalog no. 339100; Invitrogen, USA), with final concentration of 1:1,000, overnight at 4°C, washed three times, and incubated with secondary antibody and DAPI (4′,6-diamidino-2-phenylindole) for 1 h at room temperature in dark conditions. The samples were washed three times with PBS and mounted, and images were acquired using a confocal microscope (Nikon Eclipse Ti2; Japan) and analyzed using ImageJ. Scale bar, 20 μm. (B) Quantification of claudin-1 fluorescence. The error bars denote ± SEM. One-way analysis of variance (ANOVA) followed by Dunnett’s posttest was applied. Exact P values are provided. (C) Model of QS network in intestinal inflammation and permeability. Increased intestinal inflammation is driven by MvfR (solid arrow). MvfR triggers tight junction disruption and intestinal permeability indirectly (dashed arrow) at least through the P. aeruginosa QS network, which consists of three interconnected QS regulators, MvfR, LasR, and RhlR (color-coded interconnecting lines). Specifically, each regulator contributes to intestinal permeability, with MvfR functioning upstream of LasR and RhlR through a series of negative interactions (solid stop arrow), Solid stop arrow indicates inhibition. The specific nature of the regulatory relationships and feedback between these regulators in vivo is currently unknown.