cUMP elicits interendothelial gap formation during Pseudomonas aeruginosa infection

Abstract

Pseudomonas aeruginosa utilizes a type 3 secretion system to intoxicate host cells with the nucleotidyl cyclase ExoY. After activation by its host cell cofactor, filamentous actin, ExoY produces purine and pyrimidine cyclic nucleotides, including cAMP, cGMP, and cUMP. ExoY-generated cyclic nucleotides promote interendothelial gap formation, impair motility, and arrest cell growth. The disruptive activities of cAMP and cGMP during the P. aeruginosa infection are established; however, little is known about the function of cUMP. Here, we tested the hypothesis that cUMP contributes to endothelial cell barrier disruption during P. aeruginosa infection. Using a membrane permeable cUMP analog, cUMP-AM, we revealed that during infection with catalytically inactive ExoY, cUMP promotes interendothelial gap formation in cultured pulmonary microvascular endothelial cells (PMVECs) and contributes to increased filtration coefficient in the isolated perfused lung. These findings indicate that cUMP contributes to endothelial permeability during P. aeruginosa lung infection.NEW & NOTEWORTHY During pneumonia, bacteria utilize a virulence arsenal to communicate with host cells. The Pseudomonas aeruginosa T3SS directly introduces virulence molecules into the host cell cytoplasm. These molecules are enzymes that trigger interkingdom communication. One of the exoenzymes is a nucleotidyl cyclase that produces noncanonical cyclic nucleotides like cUMP. Little is known about how cUMP acts in the cell. Here we found that cUMP instigates pulmonary edema during Pseudomonas aeruginosa infection of the lung.

Keywords: cyclic nucleotide monophosphate; exoenzyme Y; permeability; pneumonia; type 3 secretion system.

Graphical abstract

Figure 1.

cUMP-AM stimulates interendothelial gap formation during ExoY^K81M infection. A: pulmonary microvascular endothelial cells were treated with HBSS with 0.1% DMSO as a control, cUMP-AM alone (1, 10, or 100 µM), or infected for 6 h at a 20:1 multiplicity of infection (MOI) with ExoY⁺ or ExoY^K81M with or without cUMP-AM (1, 10, or 100 µM). B: gap analyses of images taken after infection were analyzed by a custom macro in ImageJ. Kruskal-Wallis with an uncorrected Dunn’s post hoc test, means ± SD; *P ≤ 0.05; ns, not significant. C: gap analyses are represented with each data point as a percentage of the average ExoY⁺ gap formation. Kruskal–Wallis with Benjamini–Hochberg post hoc test, means ± SD, *P ≤ 0.05. Images are representative of 3–7 experiments, taken with ×10 objective, scale bar: 200 μm, inset: black-white gap analysis map where black regions indicate gapping.

Figure 2.

Cyclic UMP promotes endothelial permeability during ExoY^K81M infection. A: gross inspection of the isolated perfused lung infected with ExoY^K81M in the presence and absence of cUMP-AM (25 µM), before and after infection. Note edematous patches in the presence of cUMP-AM. Images are representative of five separate experiments. B: comparison of filtration coefficient (K_f) values as an indicator of permeability, taken at 0, 2, and 4 h. Two-way ANOVA with Tukey post hoc test, means ± SE, *P ≤ 0.05.

Figure 3.

ExoY⁺-infected cells do not reseal infection-induced gaps 24 h postinfection. A: pulmonary microvascular endothelial cells (PMVECs) were infected with either ExoY⁺, ExoY^K81M, ExoY^K81M with 25 µM cUMP-AM, all at a multiplicity of infection (MOI) of 20:1, or alternatively treated with 0.025% DMSO or 25 µM cUMP-AM without bacteria. Treatments were for 4 h. Following the treatment, cells were washed and a fresh medium with additional antibiotics was added. Cells were allowed to recover for 24 h and then imaged to assess their recovery. B: in total, 4-h infection is sufficient to promote interendothelial cell gap formation following ExoY⁺ and ExoY^K81M + 25 µM cUMP-AM infection. C: ExoY⁺-infected cells did not recover postinfection, whereas the ExoY^K81M + 25 µM cUMP-AM-treated cells did reseal. Images are representative of 5–10 experiments of each experimental group. Images were taken using a ×10 objective. The scale bar represents 200 µm. [Figure created with Biorender.com.]

Figure 4.

cUMP-AM does not recapitulate the stunted proliferation that is induced by ExoY⁺ infection. Pulmonary microvascular endothelial cells (PMVECs) were infected with ExoY⁺, ExoY^K81M, ExoY^K81M + 25 µM cUMP-AM, 25 µM cUMP-AM, or treated 0.025% DMSO in HBSS, washed extensively with PBS, then treated with medium in the presence of 10% serum and antibiotics to remove bacteria. After 24 h PMVECs were seeded at 1 × 10⁵ cells and allowed to grow to confluence. A: images of cells were taken every 24 h until control HBSS-DMSO-treated PMVECs were confluent as seen on day 4. All treatments reached near confluency by day 4 except for ExoY⁺. Cyclic UMP-AM treatment did not stunt growth but surprisingly rescued proliferation post-ExoY^K81M infection. B: cells were counted every day after 24 h of growth, Two-way ANOVA with Tukey’s post hoc test, means ± SE, *P ≤ 0.05 vs. ExoY⁺-infected cells. Mixed effects analysis with Tukey’s post hoc test, ^P ≤ 0.05 vs. day 1 of growth curve. Images are representative of five separate experiments, taken with ×10 objective, scale bar: 200 μm.

Figure 5.

Cell motility is reduced following ExoY⁺ infection and cUMP-AM treatment. Time-lapse microscopy was performed using a Nikon T2-eclipse microscope, where images were captured every 5 min for 12 h while cells were maintained at 37°C, 21% O₂, 5% CO₂, and 74% N₂ using an environmental chamber. Images taken from the first and last 2 h of the 12-h video were analyzed; 225 cells from each experiment were compared for analysis. A: ExoY⁺-treated cells exhibited decreased, whereas ExoY^K81M-treated cells exhibited increased, cellular speed. Kruskal–Wallis test with Dunn’s post hoc, means ± SD, *P ≤ 0.05. Frequency histograms fitted with Gaussian curve, goodness of fit, r² = 0.93–0.97 B: area of individual cells postinfection. Spread area was greatest in ExoY^K81M-treated cells, followed by ExoY⁺-treated cells. cUMP-AM treated cells exhibited the smallest spread area. Kruskal–Wallis test with Dunn’s post hoc, means ± SD, *P ≤ 0.05. Frequency histogram fitted with Gaussian curve, goodness of fit r² = 0.98–0.99. Five videos of each treatment were analyzed and compared. C: circularity of cells postinfection, where 1 represents a perfect circle and lower values indicate elongation. ExoY⁺-treated cells were more circular than ExoY^K81M-treated cells. Kruskal–Wallis test with Dunn’s post hoc, means ± SD, *P ≤ 0.05. Frequency histogram fitted with Gaussian curve, goodness of fit r² = 0.94–0.98.