Pseudomonas aeruginosa Utilizes Host-Derived Itaconate to Redirect Its Metabolism to Promote Biofilm Formation

Abstract

The bacterium Pseudomonas aeruginosa is especially pathogenic, often being associated with intractable pneumonia and high mortality. How P. aeruginosa avoids immune clearance and persists in the inflamed human airway remains poorly understood. In this study, we show that P. aeruginosa can exploit the host immune response to maintain infection. Notably, unlike other opportunistic bacteria, we found that P. aeruginosa alters its metabolic and immunostimulatory properties in response to itaconate, an abundant host-derived immunometabolite in the infected lung. Itaconate induces bacterial membrane stress, resulting in downregulation of lipopolysaccharides (LPS) and upregulation of extracellular polysaccharides (EPS). These itaconate-adapted P. aeruginosa accumulate lptD mutations, which favor itaconate assimilation and biofilm formation. EPS, in turn, induces itaconate production by myeloid cells, both in the airway and systemically, skewing the host immune response to one permissive of chronic infection. Thus, the metabolic versatility of P. aeruginosa needs to be taken into account when designing therapies.

Keywords: bacterial metabolism; biofilm; cystic fibrosis; extracellular polysaccharide; immunometabolism; inflammation; itaconate; lipopolysaccharide; pneumonia; pseudomonas aeruginosa.

Figure 1.

Metabolic preferences of P. aeruginosa in the airway. A) Scheme showing how metabolism regulates extracellular polysaccharides (EPS) (red box) and lipopolysaccharide (LPS) (blue box) synthesis and transport in P. aeruginosa. Itaconate metabolism (gray box), TCA cycle activity (yellow circle) and glucose catabolism (orange-green boxes) are also shown. Gluconeogenic flux is shown as red arrows. B) Compared with their respective controls (fold increase), expression of different genes involved in EPS and LPS production by P. aeruginosa isolates from mouse and human subjects. C) Airway abundance of succinate, itaconate and glucose by Mass-Spec in mice infected with P. aeruginosa PAO1 or a collection of 17 CF host-adapted P. aeruginosa isolates. PBS-treated mice are controls. 4–5 mice pooled from n=2 independent experiments. D) Fold overnight growth of each P. aeruginosa strain with respect to succinate: laboratory strains, ICU and CF isolates are from different subjects. E) P. aeruginosa membrane potential as measured by flow cytometry with baclight DiOC₂(3) dye after overnight glucose incubation with or without itaconate (1:1). Membrane potential uncoupler CCCP was used for 30min as positive control for dye specificity. F) LPS extracts from PAO1 grown overnight in glucose or glucose and itaconate (1:1) and stained for O-antigen and core. E.coli LPS: positive control. Respect to glucose only, core, core + 1 and O-antigen LPS band intensities for PAO1 grown in glucose and itaconate were quantified with FIJI. G) Fold increase of genes involved in EPS and LPS synthesis in PAO1 growth in glucose and itaconate, with respect to growth only in glucose. C, E: One-Way ANOVA; F: Student’s t-test. Data are shown as average +/− SEM. D-G represent at least n = 3. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns: non-significant. See also Figure S1.

Figure 2.

ΔlptD4213 PAO1 induces macrophage itaconate metabolism in the airway. A-C) Extracellular acidification rates (ECAR) by Seahorse (A), mitochondrial ROS (O₂*⁻) as determined by Mitosox and flow cytometry (B), and oxygen consumption rates (OCR) by Seahorse (C) in mouse BMDMs either uninfected (PBS) or infected with PAO1 or ΔlptD4213 PAO1. D) Levels of the inflammatory cytokines IL-1β (left), IL-6 (middle) and TNFα (right) in the BAL from uninfected (PBS) or 16h-intranasally infected mice with PAO1 or ΔlptD4213 PAO1. E-F) The OCR (E) and the ECAR (F) for Irg1^+/+ and Irg1^−/− BMDMs that were either uninfected (PBS) or infected with ΔlptD4213 PAO1. G) Mice were infected as in D and itaconate was quantified by Mass Spec in BAL. H) Respect with WT PAO1, ΔlptD4213 PAO1 fold overnight growth in different carbon sources. I-J) Crystal violet biofilms produced by PAO1 and ΔlptD4213 PAO1 strains growth in M9 supplemented either with itaconate or succinate. K) Bacterial burden (CFUs) measured in Irg1^+/+ and Irg1^−/− lungs of mice infected with ΔlptD4213 PAO1. L) Fold increase of genes involved in EPS and LPS synthesis in ΔlptD4213 PAO1, in respect to PAO1. Data are shown as average +/− SEM. B, D, G: One-Way ANOVA; K: t-Student; A, C, E-F, I-J: Two-Ways ANOVA. In vivo data are from n = 2 (6–7 mice total). Seahorse were a minimal of n = 3. ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns: non-significant. See also Figure S2.

Figure 3.

P. aeruginosa EPS alginate induces anti-oxidant itaconate metabolism. A-D) Extracellular acidification rate (ECAR) (A) and oxygen consumption rate (OCR) (B) by Seahorse, and mitochondrial membrane potential ΔΨ (C) and mitochondrial ROS (O₂*⁻) (D) by flow cytometry in mouse BMDMs either untreated (PBS) or treated with alginate or LPS. E-H) The ECAR (E), the OCR (F), the ΔΨ (G), and the O₂*⁻ levels (H) in mouse BMDMs treated with PBS, WT PAO1 or ΔalgD PAO1. Data are shown as average +/− SEM. C-D, G-H: One-Way ANOVA. Seahorse and flow cytometry experiments were a minimal of n=3. ****: P < 0.0001; ***: P < 0.001; **: P< 0.01; *: P < 0.05; ns: non-significant. See also Figure S3.

Figure 4.

P. aeruginosa EPS alginate induces airway itaconate and prevents from cell death. A-D) Percentage of DAPI⁺ alveolar macrophages (death cells) by flow cytometry (A), number of alveolar macrophages (left), Ly6C^HighLy6G⁻ monocytes (middle) and neutrophils (right) by flow cytometry (B), pro-inflammatory IL-1β (left), IL-6 (middle) and TNFα (right) cytokines (C) by ELISA and itaconate by Mass Spec (D) in BAL of mice left untreated (PBS) or infected with PAO1 WT or ΔalgD PAO1. E) Pro-inflammatory IL-1β (left), IL-6 (middle) and TNFα (right) cytokines in BAL of Irg1^+/+ and Irg1^−/− mice treated with PBS or infected with PAO1. Data are shown as average +/− SEM. A-E: One-Way ANOVA; Data are from n=2 (7–8 mice total). ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns: non-significant. See also Figure S3.

Figure 5.

Reduced glycolytic flux facilitates itaconate assimilation by P. aeruginosa. A) Compared with their respective controls (fold increase), expression of different genes involved in glucose catabolism in P. aeruginosa isolates from mouse and human subjects. B) Zwf mRNA levels by qRT-PCR in M9 + glucose-fed PAO1 exposed or not to itaconate (n=3). C-E) Energy production by Biolog plates (C), bacterial growth under different itaconate concentrations in LB (D) and zwf, ict, ich and ccl mRNA levels by qRT-PCR (E) in Δzwf PAO1 and its complement Δzwf PAO1 Comp control, respect with WT PAO1. F-G) Pro-inflammatory IL-1β (left), IL-6 (middle) and TNFα (right) cytokines by ELISA (F), and alveolar macrophages (left), Ly6C^highLy6G⁻ monocytes (middle) and Ly6C^high/lowLy6G⁺ neutrophils (right) numbers by flow cytometry (G) in BAL of mice untreated (PBS) or 16h-infected with Δzwf PAO1 or its Δzwf PAO1 complement control. Data are shown as average +/− SEM. B, E, F-G: t-Student. D: Two-Way ANOVA. In vivo data are from n=2 (8 mice total). ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; ns: non-significant. See also Figure S4.

Figure 6.

Host-adapted P. aeruginosa isolates exploit host itaconate to colonize the airway. A) Numbers of non-synonymous SNPs found in a collection of 17 P. aeruginosa isolates from an individual with CF, as compared with PAO1 genome. Pathways analyzed: LPS-EPS biosynthesis-trafficking and itaconate catabolism. B) Fold increase gene expression by qRT-PCR respect with PAO1 in two representative P. aeruginosa isolates from A: mucoid 605 and small colony variant 686. C) Ict-ich-ccl locus expression by qRT-PCR in different ICU and CF P. aeruginosa isolates, in respect with PAO1. D) Antibiogram of PA605 mucoid strain. E-H) Itaconate-mediated both biofilm and growth in presence of glucose (E-F) or succinate (G-H) by mucoid 605 P. aeruginosa and its isogenic Δict mutant. I-J) Bacterial burden (CFUs) found in Irg1^+/+ and Irg1^−/− lungs and BALs from mice left untreated (PBS) or infected with CF isolates (I) and PAO1 (J). K) IL-1β levels in 17 CF isolates-infected Irg1^+/+ and Irg1^−/− BALs. Data are shown as average +/− SEM. B: One-Way ANOVA; I-K: t-Student. In vivo data are from n=2 (7–8 mice total). ****: P < 0.0001; ***: P < 0.001; **: P < 0.01; *: P < 0.05; ns: non-significant. See also Figure S5 and Figure S6.

Figure 7.

IRG1 producing airway myeloid cells feed P. aeruginosa isolates infection. A-D) Representative density plots for Ly6C^highLy6G⁻ and Ly6C^lowLy6G⁻ monocyte populations (A), their respective percentage in the total Ly6C⁺Ly6G⁻ population (B) and the percentage of Irg1⁺Ly6C^highLy6G⁻ population (C-D) in BAL of mice treated with PBS, PAO1 or a collection of 17 CF P. aeruginosa isolates. E-F) Representative density plots (E) and frequency of cells (F) expressing CD14 and/or CD16 surface markers in sputum from healthy subjects (HS) and individuals with CF by flow cytometry. G) Number of alveolar macrophages (CD14⁺CD16⁺ in E) found in sputum and their IRG1 expression. H-I) Number of CD14⁺CD16⁻ monocytes (shown in E) found in sputum and their IRG1 expression. J) Sputum metabolomics from healthy subjects (HS) and individuals with CF. Data are shown as average +/− SEM. B, D, G, H, I: t-Student. In vivo data are from n=2 (5 mice total). In G, H and J each point represents a single patient. ****: P < 0.0001; ***: P < 0.001; *: P < 0.05; ns: non-significant. See also Figure S7.