The Small RNA ErsA Plays a Role in the Regulatory Network of Pseudomonas aeruginosa Pathogenicity in Airway Infections

Abstract

Bacterial small RNAs play a remarkable role in the regulation of functions involved in host-pathogen interaction. ErsA is a small RNA of Pseudomonas aeruginosa that contributes to the regulation of bacterial virulence traits such as biofilm formation and motility. Shown to take part in a regulatory circuit under the control of the envelope stress response sigma factor σ²², ErsA targets posttranscriptionally the key virulence-associated gene algC Moreover, ErsA contributes to biofilm development and motility through the posttranscriptional modulation of the transcription factor AmrZ. Intending to evaluate the regulatory relevance of ErsA in the pathogenesis of respiratory infections, we analyzed the impact of ErsA-mediated regulation on the virulence potential of P. aeruginosa and the stimulation of the inflammatory response during the infection of bronchial epithelial cells and a murine model. Furthermore, we assessed ErsA expression in a collection of P. aeruginosa clinical pulmonary isolates and investigated the link of ErsA with acquired antibiotic resistance by generating an ersA gene deletion mutant in a multidrug-resistant P. aeruginosa strain which has long been adapted in the airways of a cystic fibrosis (CF) patient. Our results show that the ErsA-mediated regulation is relevant for the P. aeruginosa pathogenicity during acute infection and contributes to the stimulation of the host inflammatory response. Besides, ErsA was able to be subjected to selective pressure for P. aeruginosa pathoadaptation and acquirement of resistance to antibiotics commonly used in clinical practice during chronic CF infections. Our findings establish the role of ErsA as an important regulatory element in the host-pathogen interaction.IMPORTANCEPseudomonas aeruginosa is one of the most critical multidrug-resistant opportunistic pathogens in humans, able to cause both lethal acute and chronic lung infections. Thorough knowledge of the regulatory mechanisms involved in the establishment and persistence of the airways infections by P. aeruginosa remains elusive. Emerging candidates as molecular regulators of pathogenesis in P. aeruginosa are small RNAs, which act posttranscriptionally as signal transducers of host cues. Known for being involved in the regulation of biofilm formation and responsive to envelope stress response, we show that the small RNA ErsA can play regulatory roles in acute infection, stimulation of host inflammatory response, and mechanisms of acquirement of antibiotic resistance and adaptation during the chronic lung infections of cystic fibrosis patients. Elucidating the complexity of the networks regulating host-pathogen interactions is crucial to identify novel targets for future therapeutic applications.

Keywords: ErsA; Pseudomonas aeruginosa; antibiotic resistance; clinical isolates; cystic fibrosis; mouse model of infection; opportunistic infections; pathogenicity; respiratory infections; small RNAs; virulence.

FIG 1

Deletion of ErsA results in decreased P. aeruginosa-induced cytotoxicity of pulmonary cells. (A) Time course of cell death of CF bronchial epithelial cells after bacterial infection with P. aeruginosa PA14 wild-type and ΔersA. Viability of IB3-1 cells uninfected (no infection) or infected with an MOI of 100 (PA14 wild-type and PA14 ΔersA) was analyzed by MTS assay. At each time point, results are plotted as the ratio of the average values for infected (blank subtracted) cells to that for the uninfected cells. The data are pooled from three independent experiments and are represented as means ± standard errors of the means (SEMs). Significance by one-way analysis of variance (ANOVA) with post hoc Tukey’s honestly significant difference (HSD) is indicated as follows. PA14 wild-type versus ΔersA: *, P < 0.05; **, P < 0.01. PA14 wild-type versus no infection: #, P < 0.05; ##, P < 0.01; ###, P < 0.001. PA14 ΔersA versus no infection: §, P < 0.05; §§, P < 0.01. (B) Relative viability percentages of IB3-1 cells after bacterial infection as measured by MTS assay. Cytotoxicity attenuation (%) of PAO1 wild-type, PAO1 ΔersA, and PA14 ΔersA is shown with respect to the PA14 wild-type strain during infection of IB3-1 cells with an MOI of 100. At each time point after infection, results are plotted as the ratio of the average values for infected (blank subtracted) cells to that for the uninfected cells. Results are shown as the difference of the ratios between each strain and PA14 wild type. Data are pooled from three independent experiments and are represented as means ± SEMs. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 in the one-way ANOVA with post hoc Tukey’s HSD. Significance of each strain versus PA14 wild-type is indicated above single histograms.

FIG 2

ErsA levels influence the proinflammatory response in pulmonary cells. Inflammatory response of CF bronchial epithelial cells after stimulation with P. aeruginosa PAO1 wild-type and PAO1 ΔersA deleted mutant strains (A) and P. aeruginosa PAO1 strain harboring the empty vector pGM931 or the sRNA-overexpressing vector pGM-ersA (B). IL-8 was evaluated by ELISA in supernatants of IB3-1 cells 24 h postinfection (MOI = 0.1). Uninfected IB3-1 cells were used as control (Ctrl). Data are represented as means ± SEMs. The data are pooled from three independent experiments. **, P < 0.01; ***, P < 0.001 in the Student’s t test.

FIG 3

Survival, the incidence of chronic colonization, bacterial burden, and leukocyte recruitment after chronic lung infection by wild-type and ΔersA P. aeruginosa PAO1. C57BL/6NCrlBR mice were infected with 1 × 10⁶ CFU/lung embedded in agar beads. At day 13 postinfection, mice were sacrificed, bronchoalveolar lavage fluid (BALF) was collected, and lungs were excised and homogenized. (A) Survival was evaluated on challenged mice. (B) Clearance (<1,000 CFU of P. aeruginosa from lung plus BALF cultures) and capacity to establish chronic airways infection (≥1,000 CFUs of P. aeruginosa from lung plus BALF cultures) were determined on surviving mice. (C) CFUs were evaluated in the lungs and BALF after plating onto tryptic soy agar. Dots represent values for individual mice, and horizontal lines represent median values. (D) Neutrophils, macrophages, and total cells were measured in the BALF. Values represent the means ± SEMs. The data were pooled from at least three independent experiments (n = 20 to 28). ***, P < 0.001 in the Mantel-Cox test.

FIG 4

Chemokine levels after chronic lung infection by wild-type and ΔersA P. aeruginosa PAO1. C57BL/6NCrlBR mice were infected with 1 × 10⁶ CFU/lung embedded in agar beads. At day 13 postinfection, mice were sacrificed and lungs were excised and homogenized. KC (A) and JE (B) levels were measured by ELISA in the supernatant fluids of lung homogenates. Values represent the means ± SEMs. The data were pooled from at least three independent experiments (n = 14 to 20). *, P < 0.05; **, P < 0.01 in the nonparametric two-tailed Mann-Whitney U test.

FIG 5

Dissemination of the ersA gene and its expression levels in a collection of clinical isolates. Bacterial strains from CF (gray pentagons), COPD patients (white circles), and environmental isolates (black squares) are indicated at the top. After overnight growth at 37°C on BHI agar plates, culture samples were processed for genomic DNA extraction and total RNA purification and analysis by Northern blotting. PAO1 and PA14 were used as control strains. The presence (+) or absence (−) of the ersA gene is indicated below each Northern blot lane. The relative abundance of ErsA in each isolate was calculated to the reference strain PAO1 after normalization to 5S RNA.