NtrBC Regulates Invasiveness and Virulence of Pseudomonas aeruginosa During High-Density Infection

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of nosocomial and chronic infections contributing to morbidity and mortality in cystic fibrosis patients. One of the reasons for its success as a pathogen is its ability to adapt to a broad range of circumstances. Here, we show the involvement of the general nitrogen regulator NtrBC, which is structurally conserved but functionally diverse across species, in pathogenic and adaptive states of P. aeruginosa. The role of NtrB and NtrC was examined in progressive or chronic infections, which revealed that mutants (ΔntrB, ΔntrC, and ΔntrBC) were reduced in their ability to invade and cause damage in a high-density abscess model in vivo. Progressive infections were established with mutants in the highly virulent PA14 genetic background, whereas chronic infections were established with mutants in the less virulent clinical isolate LESB58 genetic background. Characterization of adaptive lifestyles in vitro confirmed that the double ΔntrBC mutant demonstrated >40% inhibition of biofilm formation, a nearly complete inhibition of swarming motility, and a modest decrease and altered surfing motility colony appearance; with the exception of swarming, single mutants generally had more subtle or no changes. Transcriptional profiles of deletion mutants under swarming conditions were defined using RNA-Seq and unveiled dysregulated expression of hundreds of genes implicated in virulence in PA14 and LESB58 chronic lung infections, as well as carbon and nitrogen metabolism. Thus, transcriptional profiles were validated by testing responsiveness of mutants to several key intermediates of central metabolic pathways. These results indicate that NtrBC is a global regulatory system involved in both pathological and physiological processes relevant to the success of Pseudomonas in high-density infection.

Keywords: NtrC; Pseudomonas aeruginosa; abscess; adaptive lifestyles; high-density infection; invasiveness; nitrogen metabolism; virulence.

FIGURE 1

Virulence was reduced in an LESB58 mutant strain ΔntrBC compared to the wild-type (WT) in a chronic model of CD-1 murine infection. Abscess size was significantly reduced in LESB58 ΔntrBC compared to the WT (A), but no different than WT control when transformed with plasmid carrying the ntrBC gene (B). In contrast, bacterial recovery from abscesses formed by LESB58 mutants or WT were similar (C,D). Briefly, mice were subcutaneously injected 5 ± 3 × 10⁷ planktonic cells and abscesses were formed for 72 h. At experimental endpoint, 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 four independent experiments containing 3–4 biological replicates each (n = 10–12) (A,B). Otherwise, data reported as mean ± standard error of the mean (SEM) (C,D). **P < 0.01 compared to WT according to Kruskal–Wallis nonparametric test followed by Dunn’s post hoc analysis.

FIGURE 2

Biofilm formation was reduced in PA14 mutant strain ΔntrBC compared to the wild-type (WT). (A) Biofilm formation was significantly reduced in PA14 ΔntrBC compared to the WT. (B) Biofilm formation was similar in PA14 ΔntrBC transformed with plasmid containing ntrBC and WT transformed with plasmid. Briefly, bacteria were seeded from overnight cultures into 96-well microtiter plates at low density (OD₆₀₀ = 0.1) and incubated at 37°C for 24 h statically. Biomass formed in wells was washed and stained with 0.1% crystal violet (CV) prior to dissolution of aggregates with 70% ethanol. Biomass was measured (OD₅₉₅) using a BioTek SynergyH1 microplate reader and taken relative to the WT. Data reported as mean ± standard error of the mean (SEM) from three independent experiments containing three biological replicates each (n = 9). **P < 0.01 according to Welch’s t-test.

FIGURE 3

Growth of PA14 ntrBC mutant strains was influenced by nitrogen source and significantly reduced in the presence of nitrate or nitrite as well as casamino acids for the double mutant. Briefly, bacteria were seeded from overnight cultures into batch cultures at low density (OD₆₀₀ = 0.1) and incubated at 37°C for 10 h with shaking in (A) basal medium (BM2) in which (NH₄)₂SO₄ was replaced with (B) 0.1% casamino acids (CAA) (C) 14 mM NaNO₂ or (D) 14 mM NaNO₃. OD₆₀₀ values were measured using an Eppendorf BioSpectrometer corrected for background absorbance. The mean logarithmic OD₆₀₀ ± standard error of the mean (SEM) from three independent experiments is shown (n = 3). Complemented mutants were also tested and grew like the WT.

FIGURE 4

Swarming motility was dependent on both ntrB and ntrC. Shown are representative images of mutants and complemented strains. (A) Swarming motility was reduced or completely inhibited in PA14 mutant strains ΔntrB,ΔntrC, and ΔntrBC compared to WT. Swarming motility in PA14 mutant strains transformed with plasmid containing ntrB, ntrC, or ntrBC genes was similar to WT transformed with plasmid. Swarm plates were inoculated with 5 μl of planktonic cells suspended at an OD₆₀₀ = 0.4–0.6 in basal medium (BM2) supplemented with 0.1% casamino acids (CAA) and 0.4% glucose, then incubated for 18–24 h at 37°C. Images captured using a BioRad ChemiDoc. (B) Raw surface area coverage (%) of swarming colonies was assessed using ImageJ software. Data reported as mean ± standard error of the mean (SEM) from three independent experiments containing three biological replicates each (n = 9). *P < 0.05, **P < 0.01 according to Kruskal–Wallis nonparametric test followed by Dunn’s post hoc analysis.

FIGURE 5

Surfing motility of PA14 was modestly reduced in mutants with ntrB deleted. (A) Surfing motility was reduced in PA14 mutant strains ΔntrB and ΔntrBC compared to WT. Surfing motility in PA14 mutant strains transformed with plasmid containing ntrB, ntrC, or ntrBC genes was similar to WT transformed with plasmid. Surf plates supplemented with 0.4% mucin were inoculated with 5 μl of planktonic cells suspended at an OD₆₀₀ = 0.4–0.6 in MSCFM, then incubated for 18–24 h at 37°C. Images captured using a BioRad ChemiDoc. (B) Raw surface area coverage (%) of surfing colonies was assessed using ImageJ software. Data reported as mean ± standard error of the mean (SEM) from three independent experiments containing three biological replicates each (n = 9). *P < 0.05, **P < 0.01 according to Kruskal-Wallis nonparametric test followed by Dunn’s post hoc analysis.

FIGURE 6

Rhamnolipid precursor production was significantly reduced in the ΔntrBC double mutant when compared to the WT. Diameter of halo (mm) was measured following 120 h static incubation at room temperature (RT) on iron-limited salt medium. Data reported as mean ± standard error of the mean (SEM) from three independent experiments containing two biological replicates each (n = 6). **P < 0.05 according to Kruskal–Wallis nonparametric test followed by Dunn’s post hoc analysis.

FIGURE 7

NtrBC was a global regulator that influenced expression of genes involved in physiological processes other than nitrogen metabolism. Heatmaps are shown for differentially expressed (DE) genes implicated in (A) carbon or nitrogen metabolism and (B) virulence in LESB58 or PA14 infection. Briefly, swarm plates were inoculated with 5 μl of planktonic cells suspended at an OD₆₀₀ = 0.4–0.6 in basal medium (BM2) supplemented with 0.1% casamino acids (CAA) and 0.4% glucose, then incubated for 18–24 h at 37°C. Swarming cells were harvested from the tip of the swarm tendrils and RNA was isolated using Qiagen RNEasy MiniPrep kit.