NtrBC Selectively Regulates Host-Pathogen Interactions, Virulence, and Ciprofloxacin Susceptibility of Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a metabolically versatile opportunistic pathogen capable of infecting distinct niches of the human body, including skin wounds and the lungs of cystic fibrosis patients. Eradication of P. aeruginosa infection is becoming increasingly difficult due to the numerous resistance mechanisms it employs. Adaptive resistance is characterized by a transient state of decreased susceptibility to antibiotic therapy that is distinct from acquired or intrinsic resistance, can be triggered by various environmental stimuli and reverted by removal of the stimulus. Further, adaptive resistance is intrinsically linked to lifestyles such as swarming motility and biofilm formation, both of which are important in infections and lead to multi-drug adaptive resistance. Here, we demonstrated that NtrBC, the master of nitrogen control, had a selective role in host colonization and a substantial role in determining intrinsic resistance to ciprofloxacin. P. aeruginosa mutant strains (ΔntrB, ΔntrC and ΔntrBC) colonized the skin but not the respiratory tract of mice as well as WT and, unlike WT, could be reduced or eradicated from the skin by ciprofloxacin. We hypothesized that nutrient availability contributed to these phenomena and found that susceptibility to ciprofloxacin was impacted by nitrogen source in laboratory media. P. aeruginosa ΔntrB, ΔntrC and ΔntrBC also exhibited distinct host interactions, including modestly increased cytotoxicity toward human bronchial epithelial cells, reduced virulence factor production and 10-fold increased uptake by macrophages. These data might explain why NtrBC mutants were less adept at colonizing the upper respiratory tract of mice. Thus, NtrBC represents a link between nitrogen metabolism, adaptation and virulence of the pathogen P. aeruginosa, and could represent a target for eradication of recalcitrant infections in situ.

Keywords: abscess; antibiotic resistance; nitrogen metabolism; respiratory infection; virulence.

Figure 1

Mutation of the nitrogen regulatory two-component system, NtrBC, increased P. aeruginosa susceptibility to ciprofloxacin. Kill curves were performed in MHB after addition of 25 µg/ml ciprofloxacin at t = 120 min. Data are reported as mean ± standard error of the mean (SEM) from three independent experiments performed in duplicate. ^a,b P < 0.05 at t = 150 min and t = 180 min according to Kruskal Wallis nonparametric test followed by Dunn’s post-hoc analysis.

Figure 2

Mutation of the nitrogen regulatory two-component system, NtrBC, sensitized P. aeruginosa LESB58 to ciprofloxacin treatment in vivo. Abscesses formed by LESB58 ΔntrBC following ciprofloxacin treatment (0.1 mg) were significantly smaller and contained fewer bacteria than those formed by WT. Briefly, mice were subcutaneously injected 5 x 10⁷ planktonic cells and treated 1 h later. After 72 h, abscesses were measured and harvested in phosphate buffered saline (PBS), homogenized and plated on LB for bacterial enumeration. Box and whiskers delineate interquartile range with geometric error from three independent experiments containing 2-3 biological replicates each (n = 8-10). Bacterial recovery data are reported as geometric mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significantly different compared to WT according to Two-Way ANOVA followed by Dunnett’s post-hoc analysis.

Figure 3

Mutation of the nitrogen regulatory two-component system, NtrBC, reduced bacterial load of P. aeruginosa in a murine model of sinusitis. Stationary-phase bacteria were inoculated dropwise in the left naris of C57Bl/6 mice (10⁶ CFU). 72 h later mice were euthanized, and lung tissue or nasal lavage fluid was collected for bacterial enumeration following serial dilution. Significantly less bacteria were recovered from the lungs and nasal cavities of mice infected with P. aeruginosa ΔntrB, ΔntrC or ΔntrBC mutants. Data are presented as geometric mean ± standard deviation for three independent experiments containing 3-4 biological replicates each (n = 10). ***P < 0.001 according to Two-Way ANOVA followed by Dunnett’s post-hoc analysis.

Figure 4

Biofilm formation of P. aeruginosa PA14 was influenced by nitrogen source. Biofilm was formed in BM2 medium containing casamino acids (CAA), nitrate $\left(\text{N}{\text{O}}_3^{-}\right)$, nitrite $\left(\text{N}{\text{O}}_2^{-}\right)$ glutamate (Glu), or urea in equimolar amounts. 96-well polypropylene plates were inoculated with bacteria suspended in basal medium (BM2) supplemented with 0.4% glucose. 18-20 h later, biomass was stained with crystal violet and measured by scanning its OD₅₉₅. Data reported as mean ± standard error of the mean (SEM) from three independent experiments containing 2-3 biological replicates each (n = 6-9). **P < 0.01, ***P < 0.001 according to Kruskal-Wallis nonparametric test followed by Dunn’s post-hoc analysis.

Figure 5

Mutation of the nitrogen regulatory two-component system, NtrBC, reduced production of virulence factors by P. aeruginosa. The supernatants of overnight cultures of similar density were assessed for pyoverdine (Pvd), pyocyanin (Pcn) and elastase (Las). Data were reported as mean ± standard error of the mean (SEM) from three independent experiments each containing three biological replicates (n = 9). ***P < 0.001 different from WT according to One-Way ANOVA followed by Dunn’s post-hoc analysis.

Figure 6

Mutation of the nitrogen regulatory two-component system, NtrBC, increased host-directed cytotoxicity of P. aeruginosa. Confluent human bronchial epithelial (HBE) cells (~7.5 x 10⁴ cells/well) were treated with mid-log phase bacteria at a multiplicity of infection (MOI) = 1 and incubated at 37°C for 16-18 h. Cell-free supernatants were collected and host-directed cytotoxicity (%) was estimated by LDH release from cells relative to cells lysed with Triton X-100. Significantly more cytotoxicity was caused after infection with P. aeruginosa ΔntrBC mutants. Data are presented as mean ± standard error of the mean from four independent experiments containing three biological replicates each (n = 12). **P < 0.01 according to One-Way ANOVA followed by Dunn’s post-hoc analysis.

Figure 7

Mutation of the nitrogen regulatory two-component system, NtrBC, increased uptake of P. aeruginosa by macrophages. Confluent THP-1 monocytes (~3.0 x 10⁵ cells/well) were differentiated and treated with mid-log phase bacteria at a multiplicity of infection (MOI) = 10 and incubated at 37°C for 30 min. Cells were washed, treated with gentamicin (500 µg/ml) for 30 min, and washed again to remove any residual bacteria in the supernatant. Cell lysates at t = 0, 60 min were plated for bacterial enumeration following serial dilution. Mutants were taken up and cleared from macrophages more efficiently than the WT. Data are presented as geometric mean ± standard deviation for three independent experiments containing 2 biological replicates each (n = 6). ns, not statistically different; ***P < 0.001 different according to Two-way ANOVA followed by Dunnett’s post-hoc analysis.