Sulfated vizantin suppresses mucin layer penetration dependent on the flagella motility of Pseudomonas aeruginosa PAO1

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections, such as pneumonia and bacteremia. Several studies demonstrated that flagellar motility is an important virulence factor for P. aeruginosa infection. In this study, we determined whether sulfated vizantin affects P. aeruginosa flagellar motility in the absence of direct antimicrobial activity. We found that 100 μM sulfated vizantin suppressed P. aeruginosa PAO1 from penetrating through an artificial mucin layer by affecting flagellar motility, although it did not influence growth nor bacterial protease activity. To further clarify the mechanism in which sulfated vizantin suppresses the flagellar motility of P. aeruginosa PAO1, we examined the effects of sulfated vizantin on the composition of the flagellar filament and mRNA expression of several flagella-related genes, finding that sulfated vizantin did not influence the composition of the flagellar complex (fliC, motA, and motB) in P. aeruginosa PAO1, but significantly decreased mRNA expression of the chemotaxis-related genes cheR1, cheW, and cheZ. These results indicated that sulfated vizantin is an effective inhibitor of flagellar motility in P. aeruginosa.

Fig 1. Sulfated vizantin suppresses P. aeruginosa mucin penetration without influencing P. aeruginosa growth.

(A) Transwell chambers were filled with the indicated concentrations of sulfated vizantin (SV). After addition of P. aeruginosa PAO1 or ΔfliC to the top chamber, the number of bacteria in the bottom chamber was counted. The graph shows penetration relative to that of PAO1 in the absence of SV, with the data representative of five separate experiments. Error bars indicate standard error (n = 5). *P < 0.05 as compared with PAO1 in the absence of SV. (B) P. aeruginosa PAO1 was incubated with 100 μM SV or without SV, and after incubation for the indicated times, the OD₆₀₀ of the culture was measured. The data are representative of three separate experiments. Error bars indicate standard error (n = 3). (C) P. aeruginosa PAO1 was incubated with 100 μM SV, and after incubation for 3 h, CFUs were counted to quantify the viable bacteria. The graph shows viable cells relative to that of PAO1 in the absence of SV, with the data representative of five separate experiments. Error bars indicate standard error (n = 5).

Fig 2. Sulfated vizantin inhibits the flagellar-dependent motility of P. aeruginosa.

(A) After incubation of P. aeruginosa PAO1 with 100 μM sulfated vizantin (SV) or without SV, as well as PAO1 with protease inhibitors or LB broth only (control), azocasein-degradation activity was measured. The data are representative of three separate experiments, and the graph shows degradation activity relative to that of PAO1 in the absence of SV. Error bars indicate standard error (n = 3). *P < 0.05 as compared with PAO1 in the absence of SV. (B) P. aeruginosa PAO1 or ΔfliC was spotted on swimming agar plates with 100 μM SV or without SV, and after incubation for 12 h, the plates were observed and photographed. Typical plates are shown. (C) Measurement of the diameter distance of the swimming zone. The graph shows the distance of the swimming zone relative to that of PAO1 in the absence of SV, and the data are representative of three separate experiments. Error bars indicate standard error (n = 3). *P < 0.05 as compared with PAO1 in the absence of SV. (D) After incubation of P. aeruginosa PAO1 or ΔfliC with 100 μM SV or without SV for 3 h, bacterial cultures were mixed with 0.5% GTG agarose gel and loaded onto a glass slide. Bacterial cells were recorded as a movie using an EVOS microscope, and moving speed was calculated from the distance that the bacterial moved over 20 s, with the number of bacteria moving >1 μm/s determined using the MTrackJ plugin in ImageJ software. Cell movement >1 μm/s was measured in five fields, with the number of bacteria per field ~30 cells. The graph shows the number of bacteria moving >1 μm/s relative to PAO1 in the absence of SV, and the data are representative of five separate experiments. Error bars indicate standard error (n = 5). *P < 0.05 as compared with PAO1 in the absence of SV.

Fig 3. Influence of a flagellar filament in P. aeruginosa treated with sulfated vizantin.

(A) After incubation of P. aeruginosa PAO1 or ΔfliC with 100 μM sulfated vizantin (SV) or without SV for 3 h, total FliC protein in P. aeruginosa was determined by western blot using an anti-FliC antibody. Typical images are shown. (B) Band intensities were measured using ImageJ software. The graph shows band intensity relative to that of PAO1 in the absence of SV, and the data are representative of three separate experiments. Error bars indicate standard error (n = 3). *P < 0.05 as compared with PAO1 in the absence of SV. (C) After incubation of P. aeruginosa PAO1 or ΔfliC with 100 μM SV or without SV for 3 h, total surface FliC protein in P. aeruginosa was determined by ELISA using an anti-FliC antibody. The graph shows absorbance relative to that of PAO1 in the absence of SV, and the data are representative of three separate experiments. Error bars indicate standard error (n = 3). *P < 0.05 as compared with PAO1 in the absence of SV. (D) After incubation of P. aeruginosa PAO1 or ΔfliC with 100 μM SV or without SV for 3 h, bacterial cultures were loaded onto a glass slide, and bacterial cells underwent Leifson staining. Visualization of the flagellar filament in treated and untreated P. aeruginosa was performed using light microscopy, with cells measured in five fields. Number of bacteria per field was ~50 cells. The graph shows the number of bacteria with a single polar flagellum relative to that of PAO1 in the absence of SV, and the data are representative of five separate experiments, and the error bars indicate standard error (n = 5). *P < 0.05 as compared with PAO1 in the absence of SV. (E) After incubation of P. aeruginosa PAO1 or ΔfliC with 100 μM SV or without SV for 3 h, visualization of the flagellar filament in treated and untreated P. aeruginosa was performed using TEM. Scale bars, 1 μm. Typical images are shown.

Fig 4. Effects of sulfated vizantin on the mRNA expression of flagellar genes in P. aeruginosa.

(A) After incubation of P. aeruginosa PAO1 with 100 μM sulfated vizantin (SV) or without SV for 3 h, mRNA expression of the indicated genes was determined by RT-PCR. Typical gel images are shown. (B) Band intensities were measured using ImageJ software, and the gyrB gene was used as an internal control. The graph shows band intensity relative to gyrB expression in each cell, and the data are representative of five separate experiments. Error bars indicate standard error (n = 5). *P < 0.05 as compared with PAO1 in the absence of SV.