Targeting bacterial quorum sensing shows promise in improving intestinal barrier function following burn‑site infection

Abstract

Burn‑site infections, commonly due to Pseudomonas aeruginosa, have been associated with deranged intestinal integrity, allowing bacteria and their products to translocate from the gut to the circulatory system. The P. aeruginosa quorum sensing (QS) transcription factor MvfR (PqsR) controls the expression of numerous virulence factors, and the synthesis of several toxic products. However, the role of QS in intestinal integrity alterations, to the best of our knowledge, has not been previously investigated. Using a proven anti‑MvfR, anti‑virulence agent, the in vivo results of the present study revealed that inhibition of MvfR function significantly decreased Fluorescein Isothiocyanate‑Dextran (FITC‑Dextran) flow from the intestine to the systemic circulation, diminished bacterial translocation from the intestine to mesenteric lymph nodes (MLNs), and improved tight junction integrity in thermally injured and infected mice. In addition, the MvfR antagonist administration alleviates the intestinal inflammation, as demonstrated by reduced ileal TNF‑α and fecal lipocalin‑2 concentrations. In addition, it is associated with lower levels of circulating endotoxin and decreased P. aeruginosa dissemination from the burn wound to the ileum. Collectively, these results hold great promise that the inhibition of this QS system mitigates gut hyperpermeability by attenuating the derangement of morphological and immune aspects of the intestinal barrier, suggesting that MvfR function is crucial in the deterioration of intestinal integrity following P. aeruginosa burn‑site infection. Therefore, an anti‑virulence approach targeting MvfR, could potentially offer a novel therapeutic approach against multi‑drug resistant P. aeruginosa infections following thermal injuries. Since this approach is targeting virulence pathways that are non‑essential for growth or viability, our strategy is hypothesized to minimize the development of bacterial resistance, and preserve the beneficial enteric microbes, while improving intestinal integrity that is deranged as a result of burn and infection.

Figure 1.

P. aeruginosa infection dramatically increases intestinal permeability following burn and infection. Mice were burnt (orange line) or burnt and infected with P. aeruginosa strain PA14 (red line). FITC-dextran 3–5 kDa flow from the intestinal lumen to the systemic circulation increases following burn alone, with a peak at 4 h, reaching 1,700 ng/ml and a gradual drop thereafter. FITC levels following BI show a continuous rise, reaching a concentration of over 17,000 ng/ml at 18 h following insult. The difference between the two groups becomes statistically significant at 10 h (P<0.001) and further rises at 18 h (P<0.001). Mice at 0 h demonstrate the sham FITC levels. PA14 burn-site infection was induced by intradermal administration of 10⁵ CFUs/animal. FITC-dextran 3–5 kDa levels were assessed in the serum with fluorescent spectrophotometry (excitation, 480 nm and emission, 520 nm). Data show the average +/- SEM (n=5). Statistical significance was assessed using two-way ANOVA + Bonferroni correction. FITC-dextran, Fluorescein Isothiocyanate-Dextran. ***P<0.001.

Figure 2.

MvfR antagonists reduce the flow from the intestinal lumen to the systemic circulation, as well as the bacterial translocation to the MLNs. (A) FITC-dextran 3–5 kDa flow, and (B) bacterial translocation from the intestinal lumen to the systemic circulation at 18 h following burn and infection are considerably elevated in the BI group, while MvfR antagonist administration significantly reduces both FITC and bacterial flux out of the gut. FITC levels were assessed in the serum with fluorescent spectrophotometry (excitation, 480 nm and emission, 520 nm). LB agar plates were used for the bacterial CFUs assessment in the MLNs. Black bars, sham; Orange bars, burn; Red bars, PA14 burn-site infection (intradermal administration of 10⁵ CFUs/animal); Blue bars, MvfR antagonist intravenous administration at 2, 4, 8 and 16 h following burn and infection. Data show the average +/- SEM (n=5). Statistical significance was assessed using one-way ANOVA + Tukey's post-hoc test. FITC-dextran, Fluorescein Isothiocyanate-Dextran; MLNs, mesenteric lymph nodes; BI, burn plus infection; CFU, colony forming units; LB, Luria Bertani; ANOVA, one-way analysis of variance. *P<0.05; ***P<0.001.

Figure 3.

MvfR antagonists ameliorate the tight junction downregulation in BI mice. (A) Confocal microscopy images of distal ileum immunostaining for claudin-1 at 18 h following BI; (Aa). Representative image from sham animal depicting a uniform, organized pattern of claudin-1 staining at the periphery of the cells; (Ab). PA14 burn-site infection dysregulates the smooth, continuous distribution of claudin-1 and eliminates the cell periphery delineation; (Ac). Treatment with MvfR antagonist exhibits a more even localization of claudin-1 at the sites of cell-cell interaction, with less areas of claudin-1 breakdown. Size bar, 50 µm. (B) Quantification of confocal microscopy images. (C) RT-qPCR analysis of claudin-1 mRNA expression levels at 18 h post BI validates that PA14 burn wound infection downregulates claudin-1 expression, while MvfR inhibition alleviates this effect. Normalization of mRNA expression was performed for the reference gene TBP. Black bar, sham; Orange bar, burn; Red bar, PA14 burn-site infection (intradermal administration of 10⁵ CFUs/animal); Blue bar, MvfR antagonist intravenous administration at 2, 4, 8 and 16 h following burn and infection. Data show the average +/- SEM (n=5). Statistical significance was assessed using one-way ANOVA + Tukey's post-hoc test. TBP, TATA-box Binding Protein; BI, burn plus infection; CFU, colony forming units; RT-qPCR, reverse transcription-quantitative polymerase chain reaction. *P<0.05; **P<0.01; ***P<0.001.

Figure 4.

The role of MvfR in augmenting the intestinal inflammation post BI. (A) shows TNF-α concentration from distal ileal tissue at 18 h post BI. Animals that received MvfR antagonist exhibit significantly reduced TNF-α levels in the ileum. (B) shows Lcn-2 concentrations from animal feces at 4, 10 and 18 h following BI. Lcn-2 levels increase with time in both BI and MvfR antagonist groups, however there is a disproportionate rise in the Lcn-2 levels compared to the MvfR inhibition group at 18 h. TNF-α and Lcn-2 levels were assessed using ELISA. Black bars, sham; Orange bars, burn; Red bars, PA14 burn-site infection (intradermal administration of 10⁵ CFUs/animal); Blue bars, MvfR antagonist intravenous administration at 2, 4, 8 and 16 h following burn and infection. Data show the average +/- SEM (n=5). Statistical significance was assessed using one-way ANOVA + Tukey's post-hoc test in Fig. 5A and B, and two-way ANOVA + Bonferroni correction in Fig. 5C. TNF-α, tumor necrosis factor α; Lcn-2, lipocalin-2; BI, burn plus infection; ANOVA, one-way analysis of variance; CFU, colony formation unit. ***P<0.001.

Figure 5.

MvfR inactivation ameliorates the post-BI elevated systemic inflammation and PA14 systemic dissemination. (A) Systemic serum endotoxin levels, (B) bacterial CFUs number, and (C) TNF-α concentration respectively, at 18 h following BI. A marked elevation of all three systemic inflammation indicators is demonstrated in the BI group. MvfR inhibition achieves a significant decrease of endotoxin levels and CFUs number in the serum. TNF-α concentration also follows the same downward trend in the MvfR inhibition group, even though it does not reach statistical significance. (D) Treatment with MvfR antagonist results in a significant decrease in PA14 CFUs in the ileum, compared to the BI group, thus representing the respective trends in PA14 dissemination from the burn wound to the intestine. Endotoxin levels were determined using a LAL assay; TNF-α was assessed using ELISA; LB agar plates were used for the bacterial CFUs assessment in the serum; LB agar plates containing 100 µg/ml rifampicin were used for the PA14 CFUs assessment in the ileal tissue. Black bars, sham; Orange bars, burn; Red bars, PA14 burn-site infection (intradermal administration of 10⁵ CFUs/animal); Blue bars, MvfR antagonist intravenous administration at 2, 4, 8 and 16 h following burn and infection. Data show the average +/- SEM (n=5). Statistical significance was assessed using one-way ANOVA + Tukey's post-hoc test. EU, endotoxin units; CFU, colony formation unit; TNF-α, tumor necrosis factor α; LAL, Limulus Amebocyte Lysate; ANOVA, analysis of variance. **P<0.01; ***P<0.001.