Sequestration of the phagocyte metabolite itaconate by Pseudomonas aeruginosa RpoN promotes successful pulmonary infection

Domain: biology
Published: 2025-12-13
Editorial outcome: minor_revision

Abstract
Inhaled opportunistic pathogens such as Pseudomonas aeruginosa actively modify gene expression to meet the challenges of a new environment. In the infected airway the bacteria must respond to the immunometabolite itaconate, which is abundantly produced by macrophages and has anti-inflammatory and anti-oxidant functions that protect the host from airway damage and causes toxicity to bacteria. As a dicarboxylate that targets cysteine residues, itaconate can modify both bacterial and host proteins often altering metabolic activity. We demonstrate that itaconate promotes a global metabolic response in P. aeruginosa by enhancing the activity of the major alternative transcription factor RpoN. Itaconate is actively transported into the bacteria, induces σ54 rpoN expression and covalently binds cysteine residues 218 and 275 on RpoN helping to neutralize its toxicity. The S-itaconated RpoN exhibits a gain of function driving increased glucose catabolism and enhanced utilization of the bioenergetically efficient Entner–Doudoroff pathway. Thus, the accumulation of itaconate in the infected airway promotes the adaptation of P. aeruginosa to the lung by optimizing its metabolic activity and ability to cause pneumonia.

Introduction
ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens, such as P. aeruginosa, whether antibiotic resistant or not, are a major cause of healthcare associated pneumonia worldwide1. They are typically aspirated into the lung from abiotic sites such as puddles, showers, or sinks and must rapidly adapt or risk immune clearance. To establish a nidus of infection, successful opportunists evade a variety of host antibacterial effectors. They must optimize metabolic activity, selecting from the abundant carbon sources available, but without fueling excessive growth that would elicit an immune response toxic to both host and pathogen2. We wanted to understand how these bacteria sense they are in a human airway and coordinate gene expression to enable proliferation in response to this specific environmental challenge.

Both classical and alternative sigma factors coordinate bacterial sensing and adaptation to diverse environments. The alternative σ54 factor RpoN is among the over 400 P. aeruginosa transcriptional regulators that control bacterial gene expression in response to local cues. RpoN is involved in both the positive and negative regulation of genes that influence bacterial persistence in the respiratory tract, including multiple quorum sensing systems, biofilm formation, motility, and especially metabolism3–6. As RpoN directs pathways involved in the uptake and assimilation of desirable carbon sources to generate energy, it seemed likely that this transcription factor plays a major role in bacterial adaptation to the environment imposed by the infected airway.

Itaconate, synthesized by immunoresponsive gene 1 (Irg1), is a major airway metabolite released by phagocytes in response to infection7. It modifies both host and bacterial proteins through covalent interactions with specific cysteine residues, often altering metabolic activities8–10. In the host, Itaconate shapes immunoregulatory processes via S-itaconation of proteins involved in pro-inflammatory signaling, such as the NLRP3 inflammasome, the Nrf2-sequestrator Keap1, and succinate dehydrogenase. In bacteria Itaconate is toxic11,12 and acts as an electrophile, inducing substantial membrane stress as well as blocking TCA cycle function and inhibiting carbohydrate catabolism, particularly glycolysis13,14. As one of the most abundant metabolites in the infected airway, we postulated that itaconate serves as a host-specific signal that stimulates P. aeruginosa to adapt to the lung environment. In the experiments detailed in this report, we demonstrate how the binding of itaconate at two highly conserved cysteine residues in RpoN sequesters this electrophile and modifies RpoN function to promote P. aeruginosa carbohydrate catabolism, fueling bacterial adaptation to the lung.

Results
RpoN directs P. aeruginosa responses to itaconate in the lung
P. aeruginosa pulmonary infection elicits a brisk phagocytic response, dominated by their abundant release of itaconate14. To directly test the impact of RpoN-dependent transcription on P. aeruginosa in vivo, we compared the outcomes of pulmonary infection in mice exposed to the WT PAO1 strain or a ΔrpoN mutant, appreciating that over 400 genes are under either positive or negative RpoN regulation15. The ΔrpoN strains achieved a higher bacterial load than the WT PAO1 (Fig. 1a, b) and stimulated slightly greater levels of IL-1a, IL-1b, MIG, and MCP-1, but not IL-6 or TNFa (Supplementary Fig. 1a), consistent with the increased bacterial load. Mice infected with WT PAO1 exhibited decreased amounts of itaconate in the airway (Fig. 1c) yet had similar numbers of phagocytes, the cells that produce itaconate (Fig. 1d, e). The apparent ability of WT bacteria to limit the accumulation of itaconate in the airways could be either a bacterial or host effect. Taken together, these results suggested to us that RpoN may interact with itaconate specifically in its response to the airway metabolome.

We observed that the organisms respond to itaconate by increasing rpoN mRNA expression, as well as expression of the itaconate transporters trapB, trapC, and ict involved in itaconate assimilation16 (Fig. 1f, g); suggesting that the pathogen employs rpoN as both sensor and defense mechanism in response to the immunometabolite. In vitro in the presence of itaconate rpoN expression protects P. aeruginosa proliferation (Fig. 1h). Whereas, the ΔrpoN mutant exhibits decreased growth rate (Fig. 1i) but increased production of biofilm in response to itaconate stress (Fig. 1j). Transcriptional studies confirmed that the ΔrpoN mutant responded to itaconate with increased expression of the families of genes involved in quorum sensing (las, rhl) and biofilm formation (psl, pel, alginate), mechanisms to protect against oxidant stress (Supplementary Fig. 1b). These studies indicate the physiological relevance of RpoN for P. aeruginosa adaptation to an itaconate-rich environment like the lung. We next addressed how RpoN and itaconate might interact to regulate P. aeruginosa adaptation to the lung.

RpoN S-itaconation sequesters itaconate and enhances P. aeruginosa persistence
As an electrophile, itaconate can function as a non-specific oxidant, but it also has a major role in post-translational modification of specific targets by covalently binding available cysteine residues—i.e., S-itaconation; modifications that can result in either loss or gain of function17. The conserved cysteine residues in P. aeruginosa PAO1 RpoN at 218 and 275 are sites of S-itaconation identified using a biorthogonal probe (C3A) validated for itaconate (Fig. 2a and Supplementary Fig. 2a)10. We postulated that S-itaconation of RpoN at these two cysteine residues is a key factor responsible for P. aeruginosa to thrive in the host lung.

We compared the outcomes of infection using P. aeruginosa strains with mutated rpoN lacking expression of the key cysteine residues that are itaconated versus the native rpoN. We complemented the PAO1 ΔrpoN mutant strain with a plasmid expressing either WT rpoN or an rpoN gene in which the cysteine residues at 218 and 275 were replaced with alanines and thus unable to be S-itaconated18 (Fig. 2b). For simplicity of notation, PAO1 ΔrpoN complemented with plasmid encoding WT rpoN is referred to as “WT rpoN” in contrast to PAO1 ΔrpoN expressing plasmid encoding C218/275A mutant rpoN, referred to as “C218/275A”. P. aeruginosa harboring RpoN available for S-itaconation, whether chromosomal or plasmid mediated, had equivalent ability to sense the immunometabolite pressure in vivo and in vitro, as shown by similar levels of murine infection (Supplementary Fig. 2b) and biofilm formation (Supplementary Fig. 2c). We predicted that C218/275A, lacking the cysteine residues essential for itaconate binding, would exhibit biologically relevant phenotypes specifically in the presence of itaconate, but appreciated that other RpoN functions that are itaconate independent might be affected by the cysteine to alanine mutations. Phenotypic comparison revealed a slight growth advantage of the C218/275A mutant over WT rpoN when itaconate or succinate were used as sole carbon sources, whereas growth in glucose was inhibited by itaconate (Supplementary Fig. 2d–f). Aside from these differences, both WT rpoN and C218/275A had equivalent rates of oxygen consumption and swarming motility, which are under RpoN regulation (Supplementary Fig. 2g, h).

In vivo, RpoN S-itaconation supported P. aeruginosa survival in the respiratory tract. Compared with animals exposed to the C218/275A mutant, lungs of mice infected with the WT rpoN strain exhibited increased bacterial burden (Fig. 2c, d). This increased infection was dependent upon the presence of itaconate as both WT rpoN and the C218/275A mutant strains achieved similar levels of infection in Irg1−/− animals lacking itaconate (Fig. 2c, d). In vitro, the C218/275A mutant was significantly impaired in biofilm production in response to itaconate (Fig. 2e, f). Importantly, the bacteria expressing RpoN capable of sensing the itaconate via cysteine modification as well as those with an alanine substitution, did not have significant alterations in other host effector responses against P. aeruginosa, such as their induction of itaconate production (Fig. 2g), stimulation of infiltrating phagocytes (Fig. 2h–j), or release of inflammatory cytokines (Supplementary Fig. 2i). Together, these findings strongly suggest that itaconate sequestration by RpoN via S-itaconation confers P. aeruginosa with major survival advantages in a phagocyte-dominated setting.

RpoN S-itaconation promotes glucose catabolism via the ED pathway
We anticipated that the protection conferred to P. aeruginosa by RpoN S-itaconation would involve mechanisms that promote the establishment of the bacterial biomass in the lung. Transcriptomic profiles of the WT rpoN and C218/275A mutant (Supplementary data 1) indicated several changes attributable to the C-A mutations under control conditions (Supplementary Fig. 3a, c), as well as marked differences in key pathways in the presence of itaconate (Fig. 3a and Supplementary Fig. 3b). While itaconate itself can be assimilated by P. aeruginosa14, its preferred mechanism to generate ATP and other precursor molecules under conditions of oxidant stress is via the energetically efficient Entner–Doudoroff (ED) pathway (Fig. 3a)19–22. This prioritizes the allocation of glucose to a range of metabolic networks involved in the synthesis of antioxidants, such as NADP+, as well as anthranilate metabolism, quorum sensing, iron scavenging, and nucleotide synthesis all important for biomass expansion. We observed that itaconated RpoN was associated with significantly increased expression of gcd, which converts glucose to gluconate, as compared with the C218/275A mutant. Additionally, RpoN S-itaconation favored gluconate oxidation to ketogluconates and its utilisation via the ED pathway, particularly by preventing mRNA repression of kguDET, zwf, pgl, and eda (Fig. 3c–e). There was also increased expression of the antranilates, antABC, which participate in the degradation of tryptophan via the kyrenurine pathway, which generates NAD+ and NADP23,24, cofactors needed to accept electrons in the production of gluconate and glycerol-3 phosphate via DHAP (Fig. 3a, b).

Using carbon tracing assays, we corroborated how RpoN S-itaconation facilitated P. aeruginosa [13C]-glucose catabolism comparing the effects of itaconate on WT rpoN and C218/275A mutants (Supplementary data 2). In the presence of itaconate, more of the [13C] signature was integrated into specific isotopologues of NADP+, NAD+ and glycerol-3-phosphate (Fig. 3f–h) core elements on the ED pathway. There was significant [13C] enrichment in nucleic acid precursors; uridine monopohosphate (UMP), 5-thymidylic acid (dTMP), and cytidine monophosphate (CMP) in WT rpoN as compared to C218/C275A as well (Fig. 3i–k). WT rpoN incorporated significantly more [13C] into lipid and rhamnolipid precursors derived from acetylCoA, such as 2-hydroxycaproic acid and D-2-hydroxyglutaric acid (Supplementary Fig. 3g) and into the glycerolipid precursors glycerol-3 phosphate and O-phosphoethanolamine (Fig. 3h and Supplementary Fig. 3g). WT rpoN also incorporated more [13C]-glucose into N-acetyl-glucosamine-1-phosphate (GlcNAc-1-P) and UDP-glucose (UDP-Glc) (Fig. 3l, m), essential components of peptidoglycan and biofilm.

We also observed changes in 13C glucose flux attributed solely to C-A mutations (Supplementary Fig. 3e, f); namely decreased 13C enrichment into guanine diphosphate, ornithine, alanine, asparagine and allantoin (Supplementary Fig. 3h), but increased 13C incorporation into L-cystathione and niacinamide (Supplementary Fig. 3i). The 13C incorporation into GlcNAc-1-P and UMP was slightly reduced under basal conditions in WT rpoN organisms decreased significantly upon itaconate exposure (Supplementary Fig. 3j). Overall the transcriptomic and carbon tracing studies demonstrate that the availability of itaconate to modify RpoN provides a coordinated enhancement of the fundamental metabolic activities required for bacterial proliferation, promoting successful P. aeruginosa infection in a setting of inflammation and oxidant stress.

RpoN 218/275 cysteines are conserved in clinical isolates
Our findings thus far indicate that post-translational modification of RpoN by itaconate enhances the ability of the organisms to establish pulmonary infection by optimizing bacterial metabolic activity, as modeled by the laboratory standard strain PAO1. Although rpoN is an essential gene15, it is a site of mutation in clinical isolates25. We postulated that the cysteine residues at 218 and 275 in RpoN would be highly conserved in P. aeruginosa from patients with pneumonia if they are biologically important to enable infection amidst phagocytes and itaconate stress. We analyzed rpoN sequences in publically available data sets from clinical isolates of P. aeruginosa, known to associated with either acute ICU or chronic (cystic fibrosis (CF) pulmonary infection (Fig. 4a). We identified several mutations in the clinical isolates, mostly synonymous variants, a few missense mutations, or deletions. The frequency of mutations was significantly greater in isolates from long standing CF infection than in the ICU isolates (Fig. 4b). A comparison of the rpoN genomic sequences and predicted proteins from these different datasets indicated conservation of the cysteine residues in the same location as in the reference PAO1 sequence (Fig. 4c). This is a region of RpoN expected to be involved in RNA polymerase binding18, outside of the canonical DNA binding domain associated with the σ54 −12 and −24 regions of target genes (Fig. 4d, e). As depicted in the ribbon diagrams, mutations in the clinical strains did not appear likely to involve critical regions of RpoN, those involved in RNAP interactions or DNA recognition sites (Fig. 4d), although they could affect interactions with enhancer binding proteins26. The conservation of cysteines at positions 218 and 275 across the P. aeruginosa isolates from pneumonia patients worldwide is consistent with the physiologic role of S-itaconation of RpoN to promote the pathogenesis of P. aeruginosa pulmonary infection.

Methods
Mouse experiments
All animal experiments were conducted in accordance with institutional guidelines at Columbia University Irving Medical Center and approved under IACUC protocol AABD5602. Wild-type (WT) C57BL/6NJ mice (7–8 weeks old, 20–25 × g; Jax#005304) were obtained from the Jackson Laboratory. Irg1−/− (Acod1−/−) mice (Jax#029340) were also obtained from the Jackson Laboratory and bred in-house at Columbia University Irving Medical Center. Both WT and Irg1−/− mice were immunocompetent and did not receive any medical or drug treatments prior to infection. Each in vivo experiment included an equal number of male and female mice (50:50 ratio), and no sex-based differences were anticipated. Animals were randomly assigned to cages and housed in barrier facilities under standard conditions (12-h light/dark cycle, temperature 18–23°C, 30–50% humidity). Mice were fed a regular irradiated chow diet (Purina Cat#5053, distributed by Fisher).

Key reagents
Dream Taq PCR mastermix (K1081, Thermo Scientific™), EcoRI-HF (NEB R0101S) and HindIII-HF (NEB, R3104S), QIAprep Spin Miniprep Kit (QIAGEN, 27104), T4 DNA Ligase (5U/μL) (Thermo Scientific™, EL0011), ReliaPrep™ DNA Clean-Up and Concentration System (Promega A2893).
Tetracycline hydrochloride (Sigma, T7660), Itaconate (Sigma# I29204), D-(+)-Glucose (Sigma-Aldrich, G8270), M9 minimal media (Gibco™ A1374401), Seahorse calibrant (Agilent# 100840-000).
RNAprotect Bacteria Reagent (QIAGEN, 76506), Proteinase K (QIAGEN, 19131), TRK lysis buffer (Omega Bio-tek, R6834-02), Multiscribe Reverse Transcriptase (Applied Biosystems, 43-688-14), PowerUp SYBR Green Master Mix (Applied Biosystems, 25742), DNA-free™ DNA Removal Kit (Invitrogen, AM1906).
Antibodies and staining reagents: Anti-CD45-AF700 (BioLegend, 103127), anti-CD11b-AF594 (BioLegend, 101254), anti-CD11c-BV605 (BioLegend, 117334), anti-SiglecF-AF647 (BD, 562680), anti-MHCII-APC-Cy7 (BioLegend, 107628), anti-Ly6C-BV421 (BioLegend, 128032), and anti-Ly6G-PerCP-Cy5.5 (BioLegend, 127616), LIVE/DEAD viability dye (Invitrogen, L23105A), 15.45μm DragonGreen, Bangs Laboratories Inc., FS07F).

Growth condition of bacterial strains
P. aeruginosa strains—PAO1, ΔrpoN, ΔrpoN:prpoN, ΔrpoN:pC218/C275, ΔrpoN empty vector control (EVC) were used pHERD26T. In the experiments, laboratory Pseudomonas strains were grown in LB for overnight and subcultured until exponential phase. All strains were streaked and maintained on LB agar plates, while ΔrpoN:prpoN, ΔrpoN:pC218/C275, ΔrpoN (EVC) were maintained on LB agar plates supplemented with 50 µg/mL of tetracycline.

Strains and plasmids
Strains, plasmid, and primers used in this study are listed in Supplementary data 3. We constructed an rpoN mutant in which the codons for cysteines at positions 218 and 275 were substituted with alanine using the overlap extension PCR method31. The rpoN sequence was PCR-amplified using DreamTaq PCR mastermix (Thermo Scientific™, K1081) from genomic DNA of PAO1 WT using primer pairs, RpoN_F and RpoN_R. The mutation at 218 position was introduced using primer sets RpoN_F/218_R and 218_F/RpoN_R, followed by fusion of resultant PCR products using RpoN_F and RpoN_R. Similarly, substitution mutation was introduced in C218A*rpoN at 275 position using primer set RpoN_F/275_R and 275_F/RpoN_R, followed by fusion of resultant PCR products using RpoN_F and RpoN_R. The amplicons, rpoN and C218/275A*rpoN were cloned into vector pHerd26T using EcoRI-HF (NEB R0101S) and HindIII-HF (NEB, R3104S), yielding plasmids pHerd26T-WT rpoN and pHerd26T-C218/C275A. Verified plasmids were electroporated into ΔrpoN PAO1, and Transformants were selected on LB agar plates supplemented with 50 µg/mL of tetracycline, as previously described32. Clones were screened by PCR using flanking primers RpoN_F and RpoN_R, followed by Sanger sequencing of the amplicons.

Mouse pneumonia model
Mouse pneumonia infection was carried out with 7–8 weeks old male and female (C57BL/NJ6 WT and Irg1−/− (Acod1−/−) mice as previously described14. After anesthesia, animals were exposed to either PBS, WT PAO1, ΔrpoN, ΔrpoN:prpoN, or ΔrpoN:pC218/C275. A 50 µL volume of ∼106 CFU of either P. aeruginosa strain or PBS alone (non-infected) was intranasally inoculated into the mice. Mice were euthanized at 16 h post-infection; whole lungs and BALF were collected aseptically. The lung was homogenized through 40 µm cell strainers (Falcon, 352340). Aliquots of BALF and lung homogenates were serially diluted and plated on LB agar plates (supplemented with 50 µg/mL tetracycline when needed) to determine the bacterial burden. The BALF and lung homogenates were spun down and the BALF supernatant was collected for cytokine and untargeted metabolomic analysis. After hypotonic lysis of the red blood cells, the remaining BALF and lung cells were prepared for fluorescence-activated cell sorting (FACS) analysis as described below. No calculation was used to determine the number of mice required. No data blinding was performed.

Flow cytometry for immune cell recruitment in infection studies
Immune cell profiling in BALF and lung tissue was performed by staining isolated cells with a LIVE/DEAD viability dye (Invitrogen, L23105A) and a fluorescent antibody cocktail, along with 10 µL of counting beads (15.45μm DragonGreen, Bangs Laboratories Inc., FS07F). The antibody panel included: anti-CD45-AF700 (BioLegend, 103127), anti-CD11b-AF594 (BioLegend, 101254), anti-CD11c-BV605 (BioLegend, 117334), anti-SiglecF-AF647 (BD, 562680), anti-MHCII-APC-Cy7 (BioLegend, 107628), anti-Ly6C-BV421 (BioLegend, 128032), and anti-Ly6G-PerCP-Cy5.5 (BioLegend, 127616), each diluted 1:200 in PBS. Staining was carried out for 1 h at 4°C. After washing, cells were fixed in 2% paraformaldehyde (Electron Microscopy Sciences, 15714-S) and analyzed on a BD LSRII flow cytometer using FACSDiva v9 software. Data were processed with FlowJo v10. Cell populations were identified based on the following markers: Alveolar macrophages: CD45⁺ CD11b⁺/− SiglecF⁺ CD11c⁺; Neutrophils: CD45⁺ CD11b⁺ SiglecF− MHCII− CD11c− Ly6G⁺ Ly6C⁺/−; Monocytes: CD45⁺ CD11b⁺ SiglecF− MHCII− CD11c− Ly6G− Ly6C⁺/−. Gating strategy by FACS is provided in the Supplementary Fig. 4.

Cytokine analysis
Cytokine levels in mouse BALF supernatants were measured by Eve Technologies (Calgary, Canada) using bead-based multiplex technology.

Untargeted metabolomic analysis
Metabolite profiling of BALF samples was conducted using high-resolution LC-MS at the Calgary Metabolomics Research Facility (Calgary, Canada). Metabolite extraction was performed using a 50% methanol (Supelco# 106018) and water (v/v) solution. LC-MS analysis was carried out on a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to a Vanquish UHPLC system (Thermo Fisher Scientific). Chromatographic separation was conducted on a Syncronis HILIC UHPLC column (2.1 mm × 100 mm × 1.7 μm, Thermo Fisher Scientific) using a binary solvent system at a flow rate of 600 µL/min. Solvent A was 20 mM ammonium formate at pH 3 in mass spectrometry-grade water, and solvent B was mass spectrometry-grade acetonitrile containing 0.1% formic acid (v/v). The gradient program was as follows: 0–2 min, 100% B; 2–7 min, 100%–80% B; 7–10 min, 80%–5% B; 10–12 min, 5% B; 12–13 min, 5%–100% B; 13–15 min, 100% B. A 2 ml of sample was injected. The mass spectrometer operated in negative full-scan mode at a resolution of 240,000, scanning from 50 to 750 m/z. Metabolites were identified by matching observed m/z values (±10 ppm) and retention times with those of commercial metabolite standards (Sigma-Aldrich). Data analysis was conducted using E-Maven v0.10.0.

Isolation of bacterial RNA
WT rpoN and C218/C275 strains were grown overnight in LB media (+50 µg/mL tetracycline). Overnight cultures were inoculated (1/100) into LB (+50 µg/mL tetracycline) with or without itaconate (Sigma# I29204) and were grown at 37°C to late exponential phase. Approximately 2 × 10⁸ P. aeruginosa cells were harvested by centrifugation, and the resulting pellet was resuspended in RNAprotect Bacteria Reagent (QIAGEN, 76506). The suspension was vortexed briefly and incubated at room temperature (RT) for 10 min before centrifugation. The bacterial pellets were then lysed in a buffer pH 8.0 (containing 30 mM Tris (Corning, 46-030-CM), 1 mM EDTA (Thermo Fisher Scientific, 1861283), 15 mg/mL lysozyme (Sigma, L6876), and 200 mg/mL proteinase K (QIAGEN, 19131)), with incubation at RT for 10 min. Following lysis, TRK lysis buffer (Omega Bio-tek, R6834-02) and 70% ethanol (v/v) were added to the mixture. The lysates were then applied to E.Z.N.A. RNA isolation columns (Omega Bio-tek), and total RNA was extracted according to the manufacturer’s protocol. Residual genomic DNA was removed by DNase treatment using the DNA-free™ DNA Removal Kit (Invitrogen, AM1906).

Complementary DNA (cDNA) synthesis and qRT–PCR
Complementary DNA (cDNA) was synthesized from RNA using Multiscribe Reverse Transcriptase (Applied Biosystems, 43-688-14). Quantitative real-time PCR was carried out with gene-specific or housekeeping primers as listed in Supplementary data 3, PowerUp SYBR Green Master Mix (Applied Biosystems, 25742), and the StepOnePlus Real-Time PCR System (Applied Biosystems). Relative gene expression levels were calculated using the ΔΔCt (delta-delta Ct) method.

Bulk RNA sequencing
Bacterial RNA was extracted as previously described. A ribosomal RNA (rRNA)-depleted cDNA library was prepared according to the manufacturer’s instructions using the Universal Prokaryotic RNA-Seq Prokaryotic AnyDeplete kit (NuGEN #0363-32) and sequenced with Illumina HiSeq. Raw base call files were converted to fastq format using Bcl2fastq. Quality-filtered reads were then aligned to the P. aeruginosa PAO1 reference genome (RefSeq: GCF_000006765.1) using STAR Aligner v2.7.3a. Aligned reads were annotated with read group information and duplicate reads were identified using Picard Tools v2.22.3. Quantification of raw counts was performed using FeatureCounts from the Subread package v1.6.3. Differential gene expression analysis was conducted using DESeq2 in R v3.5.3 and low-abundant transcript having threshold of 3 reads per gene were excluded in the analysis. The bacterial transcriptional data were plotted as a volcano plot heatmap plot using GraphPad Prism (v10.0c).

13C glucose labeling and stable isotope tracing
WT rpoN and C218/C275 strains were grown overnight in LB media (+50 µg/mL tetracycline) with or without 20 mM of itaconate. The overnight cultures were washed and resuspended in the same volume of M9 minimal media (Gibco™ A1374401). The cultures were inoculated (1/50) into M9 minimal media supplemented with 7.5 mM of 13C glucose (Sigma# 389374) (+50 µg/mL tetracycline) and grown at 37°C to late exponential phase. For metabolite extraction, each culture was pelleted and washed with PBS twice centrifuged in 2000 × g for 10 min at 1°C. The pellets were resuspended in a 3:1 methanol:water extraction solution and lysed with 10 freeze-thaw cycles by alternating emersion in liquid nitrogen and a dry-ice/ethanol bath. The debris was removed by centrifugation at 14,000 × g for 5 min at 1°C and the supernatant was stored for analysis. Targeted LC/MS analysis was performed on a Q Exactive Orbitrap mass spectrometer (Thermo Scientific) coupled to a Vanquish UPLC system (Thermo Scientific). The Q Exactive operated in polarity-switching mode. A Sequant ZIC-HILIC column (2.1 mm i.d. × 150 mm, Merck) was used for separation of metabolites. Flow rate was set at 150 μL/min. Buffers consisted of 100% acetonitrile for mobile A, and 0.1% NH4OH/20 mM CH3COONH4 in water for mobile B. Gradient ran from 85 to 30% A in 20 min followed by a wash with 30% A and re-equilibration at 85% A. Metabolites were identified based on exact mass within 5 ppm and standard retention times. Relative quantitation was performed based on peak area for each isotopologue. All data analysis was done using MAVEN 2011.6.17.

In situ itaconation profiling of cysteines in P. aeruginosa
Cysteines in proteome subjected to S-itaconation were identified using the workflow described here10 with n=3 samples. P. aeruginosa PAO1 were grown in LB media at 37°C to stationary phase. Cultures were washed and resuspended in pre-chilled PBS. Where 800 μL of P. aeruginosa PAO1 suspension in PBS was treated without or with 80 μL pH-adjusted itaconate, was incubates on the ThermoMixer (950 rpm, 1 h, 37°C, 30 min). Both groups were then incubated with 8 μL of the C3A probe (950 rpm, 37°C, 1 h). Cells were pelleted, washed, resuspended in 1 mL of 0.1% PBST, and lysed. Lysates were clarified by centrifugation, and the supernatant was transferred to fresh tubes. After competition, both groups were incubated with 8 mL of the C3A probe on the ThermoMixer (950 rpm, 37°C, 1 h). Bacteria were pelleted, washed, and resuspended in 1 mL of 0.1% PBST for lysis. Lysates was centrifuged (20,000 × g, 10 min, RT) to remove the debris and the supernatants was transferred into a new tubes. Click reaction was carried out at 29°C for 1 h (1200 rpm) on a Thermomixer with 106 μL of Click reagent mix comprising of: 60 μL TBTA ligand, 20 μL 50 mM CuSO4, 20 μL freshly prepared 50 mM TCEP, and 6 μL 20 mM acid-cleavable azide-biotin tag (Confluore). The click-labeled lysates were precipitated by 5 mL of MeOH/chloroform (4:1) and 3 mL of ddH2O. Precipitates were washed twice with pre-chilled MeOH, resuspended in 1 mL of 1.2% SDS in PBS, followed by sonication, heating for 10 min at 90°C, and centrifuged (20,000 × g, 10 min, RT) to remove excessive copper. Supernatants were combined with the pre-washed streptavidin beads (Thermo Fisher) and incubated for 4 h at 29°C. Beads were washed PBS three times and ddH2O three times, followed by resuspension in 500 mL of 8 M urea (Sigma)/100 mM TEAB (Sigma). The samples were reduced on ThermoMixer (1200 rpm, 30 min, 37°C) by 25 mL of 200 mM DTT (Shanghai Yuanye Bio-Technology Co., Ltd) and alkylated on the ThermoMixer (1200 rpm, 30 min, 35°C) by 25 mL of 400 mM 2-iodoacetamide (Sigma). The beads were then resuspended in resuspended in 200 mL of 2 M urea/100 mM TEAB containing 1 mM CaCl2 and 10 ng mL−1 trypsin (Promega) for digestion on ThermoMixer (1200 rpm, 30 min, 35°C). Post-digestion, the supernatants were carefully transferred into a new 1.5 mL Protein LoBind Tubes (Eppendorf). The beads were washed, and post-washed supernatants were pooled with primary digest. The digested peptides were subjected to mass spectrometry for identification of itaconation of cysteines.

Genomic analysis of clinical isolates
Publicly available whole genome sequences for Pseudomonas CF isolates (Bioproject accession number: PRJEB5438, PRJNA528628, PRJNA1023362, PRJNA1023843 on NCBI) and ICU isolates (ENA accession number: PRJNA629475) were used for mutation analysis with respect to the reference genome PAO1 (Genomics accession no.: AE004091.2). SNPs in rpoN were identified in CF and ICU isolates of P. aeruginosa using Snippy v4.6.0 (https://github.com/tseemann/snippy), with the PAO1 genome as the reference. The detected variants, including SNPs and indels, were visualized using Jalview for comparative sequence analysis to determine the conservation of cysteines residues at the codon and protein sequence level. Representative sequences of rpoN from each bioprojects with respect to reference PAO1 were used to represent conservation of cysteine residues.

Extracellular flux analysis
WT rpoN, C218/C275, and ΔrpoN strains were grown in LB overnight, then washed twice with filtered PBS before inoculating (1 × 107) into 450 μL of 1 × of M9 minimal media supplemented with 2 mM of MgSO4 and 0.1 mM of CaCl2 in Seahorse XFe24 cell culture plate (Agilent #102334-000B). 50 μL of 75 mM glucose or 200 mM itaconate in M9 media was loaded onto the Seahorse XFe24 cartridge, which was previously hydrated in Seahorse calibrant (Agilent #100840-000) overnight at 37°C. After 60 min of stabilization at the basal state, glucose or itaconate was acutely injected into the plate to achieve the final concentration of 7.5 mM or 20 mM, respectively. Bacterial extracellular acidification and oxygen consumption rates were captured with Seahorse XFe24 Analyzer (Agilent #1002238-100) using Seahorse Wave Desktop v2.6.0.

Biofilm quantification
A clear, flat-bottom 96-well plate (Greiner, #M2936) was prepared using either LB or M9 minimal medium supplemented with 0.5% (w/v) glucose, with or without 20 mM itaconate. Each well was inoculated with 1 × 108 CFU of P. aeruginosa strains (WT rpoN, C218/275A mutant, or ΔrpoN) and incubated statically at 37°C for 24 h (or 48 h for M9+ glucose conditions). Bacterial growth was monitored by measuring optical density at 600 nm (OD₆₀₀) using SkanIt Software 7.0 RE. For biofilm staining, the supernatant was carefully removed, wells were washed and air-dried, and biofilms were fixed with 100% methanol. Biofilms were then stained with 1% crystal violet. After removing the stain, plates were washed, dried, and the biofilm was solubilized in 33% acetic acid. Absorbance at 540 nm was measured using a Varioskan Lux plate reader (Thermo Scientific, #3020-82355).

Growth curves
Growth assays for bacterial growth curves, a U-bottomed, clear 96-well plate (Greiner Bio-One, 650161) was prepared with 198 µL of M9 minimal media supplemented with glucose and/or succinate, itaconate. Each well was inoculated with 2 µL overnight bacterial culture grown in LB with or without 20 mM itaconate, standardized to an OD600nm of 4. Absorbance at 600 nm was read every 10 min for 18 h on the SpectraMax M2 plate reader (Molecular Devices), as the plate incubated at 37°C with shaking.

Swarming motility
Swarming assays were conducted following a previously established protocol33, using LB medium supplemented with 0.5% (w/v) agar. Once solidified, the plates were briefly air-dried at room temperature and spot-inoculated with 2 μL of overnight LB cultures. Plates were incubated face-up in stacks of no more than two at 37°C for 18 h. Swarming motility was assessed by measuring the diameter of the swarming zone.

Protein structure
The three-dimensional structure of the P. aeruginosa RpoN protein was predicted using AlphaFold 3. PyMOL™ version 3.0.4 (Schrödinger, LLC) software was used to visualize and perform the structural mapping of domains, conserved cysteine residues, and mutations.

Statistical analysis
Experiments in this study were not conducted in a blinded manner. All statistical analyses and graphing were performed using GraphPad Prism 9. Graphical data are presented as mean ± SEM, under the assumption of a normal distribution. For comparisons involving more than two groups, one-way ANOVA followed by post hoc multiple comparisons was used. When analyzing two or more groups over time, two-way ANOVA with post hoc testing was applied. Differences between two groups were assessed using parametric tests (Student’s t-test or ANOVA) when data were normally distributed, or nonparametric tests (Mann–Whitney or Kruskal–Wallis) when normality could not be assumed. A two-tailed P value of < 0.05 was considered statistically significant. Specific P values, along with the number of independent experiments and replicates, are provided in the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Beg et al. investigate itaconate’s role as a host-specific signal for P. aeruginosa adaptation to the lungs, as well as S-itaconated RpoN’s role in the metabolic shift required for such adaptation. The authors observed that in vivo RpoN is important for P. aeruginosa in establishing infection in the lung and shifting the bacteria’s metabolic program. The authors also identified two conserved cysteine residues in RpoN required for S-itaconation, and these modifications promote the metabolic shift observed in the presence of itaconate.\n\nThe article presents a well-written look into the role RpoN in regulating gene expression in the itaconate-rich lung environment. To improve its readability, I suggest minor revisions:\n- Combine the methods’ references section with the main text’s references.\n- Keep consistency for temperature notation as it should be, for example, 37 ºC.\n- In Extended data Fig. 2. the diagram in b is not necessary, as cloning, mutagenesis, and complementation with plasmids are standard protocols; it just seems to use space in an already busy figure.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "Beg and colleagues provide an experimental study showing that sequestration of the phagocyte metabolite itaconate by P. aeruginosa RpoN promotes successful pulmonary infection. The data show the importance of rpoN, a bacterial gene not yet considered a virulence factor in the pathogenesis of pulmonary infection. The expression of rpoN contributes to the exceptional metabolic versatility of P. aeruginosa supporting pathogen persistence in the oxidant-rich lung. This finding is of particular importance beyond P. aeruginosa infections as other Gram-negative bacteria and members of the ESKAPE family express rpoN with conserved cysteine residues in a similar configuration as P. aeruginosa. Thus, strategies to prevent bacterial adaptation to the itaconate dominated milieu of the infected lung by interfering with RpoN might prove more useful than the development of vaccines targeting individual gene products.\n\nThe study is well conducted and the manuscript is nicely written. Particularly, the combination of in vitro and in vivo models with rpoN sequence analyses in data sets from clinical isolates of P. aeruginosa is convincing. However, the following issues need to be raised.\nMajor:\n1) P. aeruginosa is frequently the cause of pneumonia in pre-injured lungs (bronchiectasis, mechanical ventilation, etc.), and rarely in healthy lungs. Are the current results from very young, healthy mice translatable into the human clinical situation? This reviewer would suggest to add data from murine CF-model and VAP model and/or discuss thoroughly.\n2) Were all bacterial mutants generated from the same WT batch?\n3) Legend to Fig 1: “Expression of rpoN regulates bacterial response to pulmonary pathogenesis and itaconate.” Maybe “pathogenesis” is rather unspecific and not the perfect term here. Do you mean pathology? Injury? Inflammation? Bacterial reaction on host immune response?\nMinor:\n5) Line 180: metabolites\n6) Line 158: known to associated with", "reviewer_4": "In this manuscript, authors show that conserved cysteine residues of RpoN in Pseudomonas aeruginosa (Pa) PAO1 strain undergo S-itaconation in the presence of itaconate. They then generated a PAO1 strain expressing rpoN lacking the cysteine residues (mutated rpoN) and in several in vitro experiments compare the ability of PAO1 expressing WT rpoN or mutated rpoN to grow, swarm and form biofilms in the presence of itaconate. The strain expressing the mutant rpoN showed a lower ability to form biofilms in vitro and also resulted in reduced bacterial lung infection compared to PAO1 expressing WT rpoN. While studying the effect of S-itaconation on the role of Pa proteins during infection is an interesting idea, the rationale for many experiments performed is either unclear or missing and authors also do not discuss their findings which significantly decreases the impact of the study. Specific examples have been presented below:\n1. From the manuscript, it is unclear if other Pa proteins also undergo itaconation and what is the effect of itaconation on the function of these proteins?\n2. Authors do not discuss if deletion of rpoN increases Pa virulence (fig 1a-b), how does increased rpoN exposure upon itaconate exposure (as would be expected in the airways) help Pa during pathogenesis?\n3. In fig 1c- How the abundance of itaconate was estimated from each sample has not been clearly explained- is this area under the curve? Peak intensity? Is the data normalized?\n4. In figures 1g and extended data fig1b, please show the expression of all genes normalized to ∆rpoN without itaconate. From the current representation, it is not clear whether itaconate can also induce trapC, trapB, ict, quorum sensing and biofilm associated genes in the ∆rpoN strain. This would clarify if there are other factors in addition to rpoN that sense and respond to itaconate.\n5. Lines 63-64: “In vitro in the presence of itaconate rpoN expression protects P. aeruginosa proliferation (Fig. 1h).”\nWhat does protect mean? Why did authors choose to test growth in 5,7.5, 10, 20 and 40mM itaconate? It also appears seems like ∆rpoN is growing faster than WT PAO1 at 5mM and 7mM itaconate, yet authors conclude that rpoN expression protects P. aeruginosa proliferation.\n6. In fig 1j, is there a difference in biofilm formation between the ∆rpoN mutant and WT PAO1 when the strains are grown in 5mM or 7.5mM itaconate? Why did authors choose to determine biofilm formation only in the condition with 20mM itaconate?\n7. In figs 1g and extended data fig1b, what was the concentration of itaconate used?\n8. Studies have shown that lysine can also undergo S-itaconation (PMID: 37271942). Have authors looked at lysine residues in rpoN?\n9. In lines 91-92: “We predicted that C218/275A lacking the cysteine residues essential for itaconate binding would exhibit biologically relevant phenotypes specifically in the presence of itaconate”\nPlease state which biologically relevant phenotypes.\n10. In extended data fig 2f, 7.5mM itaconate was used and ∆rpoN grew slower than WT rpoN. This is inconsistent with fig 1b. It is also unclear why authors keep changing the concentration of itaconate in different experiments.\n11. In figs 3d-e, please normalize all data to a single condition.\n12. If itaconation of cysteine residues does not have any role in swarming, growth in the presence of itaconate, OCR (extended data figure 2e-i), what was the rationale for further pursuing transcriptomics and metabolomic profiling of the WTrpoN and C218/275A mutant?\n13. What was the frequency of strains with deletions in rpoN?\n14. While authors have shown that the conserved cysteine residues in PAO1 can undergo S-itaconation in presence of itaconate, have they tested if this also occurs in clinical isolates?\n15. Minor comments:\nA) Line 288- Fig 1 Expression of rpoN regulates bacterial response to pulmonary pathogenesis and itaconate.\nPlease change to “Expression of rpoN regulates bacterial pathogenesis and host itaconate response”\nB) Line 289: C57BL/NJ6 to “C57BL/6NJ”\nC) Lines 85-87 “For simplicity of notation PAO1 ∆rpoN complemented with plasmid encoded WT rpoN is referred to as “WTrpoN” in contrast to PAO1 ∆rpoN expressing plasmid encoded C218/275A mutant rpoN, referred to as “C218/275A”.\nChange encoded to encoding\nD) Line 549: “M9 minimal media supplemented with glucose an/or succinate”\nChange an to and\nE) Line 182: “We found that P. aeruginosa detoxifies itaconate by covalent binding to cysteine residues of RpoN and that itaconated RpoN boosts bacterial metabolism by promoting use of the preferred Entner Doudoroff Pathway to generate ATP.”\nIsn’t itaconate a metabolite that Pa can utilize and not a toxin and itaconation a post translational modification?\nF) Line 62: Please use consistent terminology: either σ54 or rpoN."}, "author_response": "Point by Point response to reviewers:\n\"Sequestration of the phagocyte metabolite itaconate by P. aeruginosa RpoN promotes successful\npulmonary infection\"\nReviewer #1 (Remarks to the Author):\nIn this manuscript, Beg et al. investigate itaconate’s role as a host-specific signal for P. aeruginosa\nadaptation to the lungs, as well as S-itaconated RpoN’s role in the metabolic shift required for such\nadaptation. The authors observed that in vivo RpoN is important for P. aeruginosa in establishing\ninfection in the lung and shifting the bacteria’s metabolic program. The authors also identified two\nconserved cysteine residues in RpoN required for S-itaconation, and these modifications promote the\nmetabolic shift observed in the presence of itaconate.\nThe article presents a well-written look into the role RpoN in regulating gene expression in the\nitaconate-rich lung environment. To improve its readability, I suggest minor revisions:\n- Combine the methods’ references section with the main text’s references.\n- Keep consistency for temperature notation as it should be, for example, 37 ºC.\n- In Extended data Fig. 2. the diagram in b is not necessary, as cloning, mutagenesis, and\ncomplementation with plasmids are standard protocols; it just seems to use space in an already busy\nfigure.\nWe appreciate the reviewer’s comments and have addressed all of the suggested changes which\nare marked in red throughout the manuscript\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nReviewer #3 (Remarks to the Author):\nBeg and colleagues provide an experimental study showing that sequestration of the phagocyte\nmetabolite itaconate by P. aeruginosa RpoN promotes successful pulmonary infection. The data show\nthe importance of rpoN, a bacterial gene not yet considered a virulence factor in the pathogenesis of\npulmonary infection. The expression of rpoN contributes to the exceptional metabolic versatility of P.\naeruginosa supporting pathogen persistence in the oxidant-rich lung. This finding is of particular\nimportance beyond P. aeruginosa infections as other Gram-negative bacteria and members of the\nESKAPE family express rpoN with conserved cysteine residues in a similar configuration as P.\naeruginosa. Thus, strategies to prevent bacterial adaptation to the itaconate dominated milieu of the\ninfected lung by interfering with RpoN might prove more useful than the development of vaccines\ntargeting individual gene products.\nThe study is well conducted, and the manuscript is nicely written. Particularly, the combination of in\nvitro and in vivo models with rpoN sequence analyses in data sets from clinical isolates of P.\naeruginosa is convincing. However, the following issues need to be raised.\n1) P. aeruginosa is frequently the cause of pneumonia in pre-injured lungs (bronchiectasis, mechanical\nventilation, etc.), and rarely in healthy lungs. Are the current results from very young, healthy mice\ntranslatable into the human clinical situation? This reviewer would suggest to add data from murine CF-\nmodel and VAP model and/or discuss thoroughly.\nWe appreciate the difficulty in applying laboratory models to clinical situations – as we and\nothers recently reviewed this topic in detail. It is especially challenging to model bacterial adaptation to\nthe host as occurs during the more chronic infections that the reviewer mentions.\nBest Practices in the Development and Use of Experimental Models of Bacterial Pneumonia: An Official American\nThoracic Society Workshop Report.\nSamarasinghe AE, Randell SH, Kulkarni HS, Weiser JN, Quinton LJ, Dickson RP, Mizgerd JP, Orihuela CJ,\nParker D, Robinson KM, Prince AS, Evans SE, Kolls JK, Lee JS, Jeyaseelan S, Torres A, Miller LA, Hamilton DJ,\nGómez MI, Moore BB, Walker RL, Barkal LJ, Hook JL, Lau GW, Sharma J, Witzenrath M, Dela Cruz CS.Am J\nRespir Cell Mol Biol. 2025 Aug;73(2):178-199. doi: 10.1165/rcmb.2025-0322ST.PMID: 40748059\nOur central conclusion, that the major host immunometabolite itaconate can modify bacterial gene\nexpression to enhance pathogenicity, is not dependent upon the status of the host, whether old or young,\nwith damaged or native airways. There are abundant data to document the production of itaconate by\nphagocytes in patients and controls as well as in murine models1-3. Itaconate is a key stimulus for the\nmodification of P. aeruginosa metabolism that results in host tolerance to persistent infection1,4. We\npredict that this interaction is actually increased in damaged airways as we have documented increased\nitaconate with chronic infection in CF 1,4. As P. aeruginosa in particular achieves high densities in\ndamaged lungs in CF, with neutrophil dominated inflammation and large amounts of itaconate, there\nwould be a greater likelihood for the organisms to be exposed to the phagocyte metabolite and\naccordingly result in RpoN modification.\nResponses to the reviewer:\n2) Were all bacterial mutants generated from the same WT batch?\nYes, the mutants were generated from the same WT batch, and all the experiments are done from the\nsame frozen (-80 ºC) glycerol stocks.\n3) Legend to Fig 1: “Expression of rpoN regulates bacterial response to pulmonary pathogenesis and\nitaconate.” Maybe “pathogenesis” is rather unspecific and not the perfect term here. Do you mean\npathology? Injury? Inflammation? Bacterial reaction on host immune response?\nThank you for pointing this out, as suggested by the other reviewer, we have modified the\nlegend caption to read “Expression of rpoN regulates bacterial pathogenesis and response to\nitaconate.”\nMinor:\n5) Line 180: metabolites\n6) Line 158: known to associated with\nThese minor changes have been made.\nIn this manuscript, authors show that conserved cysteine residues of RpoN in Pseudomonas\naeruginosa (Pa) PAO1 strain undergo S-itaconation in the presence of itaconate. They then generated\na PAO1 strain expressing rpoN lacking the cysteine residues (mutated rpoN) and in several in vitro\nexperiments compare the ability of PAO1 expressing WT rpoN or mutated rpoN to grow, swarm and\nform biofilms in the presence of itaconate. The strain expressing the mutant rpoN showed a lower\nability to form biofilms in vitro and also resulted in reduced bacterial lung infection compared to PAO1\nexpressing WT rpoN. While studying the effect of S-itaconation on the role of Pa proteins during\ninfection is an interesting idea, the rationale for many experiments performed is either unclear or\nmissing and authors also do not discuss their findings which significantly decreases the impact of the\nstudy. Specific examples have been presented below:\n1. From the manuscript, it is unclear if other Pa proteins also undergo itaconation and what is the effect\nof itaconation on the function of these proteins?\nThe itaconated proteins of P. aeruginosa and sites of itaconation have been published Liu, Z.,\nLiu, D., & Wang, C. (2024). Cell Reports, 2024 5, co- authors on this manuscript. To our knowledge,\nour manuscript is the first documentation of the biologically relevant consequences of specific targets of\nitaconation in P. aeruginosa. We have previously demonstrated the association of itaconate and\naldolase expression in S. aureus6 and effects on glycolysis and biofilm formation but did not precisely\nmap the site and consequences of itaconation at a specific site as was done in the current manuscript.\n2. Authors do not discuss if deletion of rpoN increases Pa virulence (Fig 1a-b), how does increased\nrpoN exposure upon itaconate exposure (as would be expected in the airways) help Pa during\npathogenesis?\nWe avoid the use of the term “virulence” as it implies an acute, fulminant infection, whereas\nmany of the ESKAPE pathogens (S. aureus , K. pneumonia, as well as P. aeruginosa) are also\nassociated with more indolent but progressive and eventually lethal infection, especially pneumonias7.\nIn figure 1 a-c we demonstrate increased numbers of bacteria in the lung, but without a commensurate\nincrease in the host production of damaging cytokines, such as IL-1ß that are typically associated with\n“virulence”, i.e., stimulating a damaging host response.\nWe are unclear exactly what the reviewer means by “increased rpoN exposure” – perhaps\ngreater transcription of rpoN, which would increase its availability for binding itaconate, which in turn\nprotects the bacteria. This connection is important because itaconate is toxic to the organism, as has\nbeen previously reported1.\n3. In fig 1c- How the abundance of itaconate was estimated from each sample has not been clearly\nexplained- is this area under the curve? Peak intensity? Is the data normalized?\nThank you for pointing this out; we have now indicated in the figure legend that the values are\nnormalized to the PBS group (Fig 1c).\n4. In figures 1g and extended data fig1b, please show the expression of all genes normalized to ∆rpoN\nwithout itaconate. From the current representation, it is not clear whether itaconate can also induce\ntrapC, trapB, ict, quorum sensing and biofilm associated genes in the ∆rpoN strain. This would clarify if\nthere are other factors in addition to rpoN that sense and respond to itaconate.\ngene trapC, trapB, ict expression. The revised representation highlights a clear and significant rpoN-\ndependent increase in the expression of these genes in PAO1. (Fig 1g and Extended data Fig 1b)\n5. Lines 63-64: “In vitro in the presence of itaconate rpoN expression protects P. aeruginosa\nproliferation (Fig. 1h).”\nWhat does protect mean? Why did authors choose to test growth in 5,7.5, 10, 20 and 40mM itaconate?\nIt also appears seems like ∆rpoN is growing faster than WT PAO1 at 5mM and 7mM itaconate, yet\nauthors conclude that rpoN expression protects P. aeruginosa proliferation.\n“Protection” refers to the ability of rpoN expression to counteract the toxic effects of itaconate on\nbacterial growth (proliferation). The levels of itaconate vary within the host: relatively low in systemic\ncirculation, as recently reported3, and much higher within phagocytes and the phagolysosome2. While\nhigh concentrations (≥20 mM) have been used to mimic physiological bacterial responses 1,7,8, we\ntested a range of itaconate concentrations in vitro to cover this spectrum and assess effects (Fig 1h,i).\nAs per the reviewer’s suggestion, our repeated experiments show that even at lower concentrations (5\nmM or 7.5 mM), PAO1 grows better than the rpoN mutant.\n6. In fig 1j, is there a difference in biofilm formation between the ∆rpoN mutant and WT PAO1 when the\nstrains are grown in 5mM or 7.5mM itaconate? Why did authors choose to determine biofilm formation\nonly in the condition with 20mM itaconate?\nWe did not include lower itaconate concentrations (5 mM or 7.5 mM), as previous studies1,7\nhave demonstrated that in vitro experiments generally require 20 mM or higher concentrations to elicit\nrobust effects on bacterial physiology.\n7. In figs 1g and extended data fig1b, what was the concentration of itaconate used?\nWe used 20mM itaconate in all experiments unless otherwise specified.\n8. Studies have shown that lysine can also undergo S-itaconation (PMID: 37271942). Have authors\nlooked at lysine residues in rpoN?\nThank you for this interesting comment regarding lysine itaconylation in rpoN. In the paper cited\n(PMID: 37271942) the authors showed lysine could undergo N-itaconylation rather than S-itaconation.\nThe former refers to the acylation on the amine group of lysine while the latter is formed through\nMichael addition between the thiol group of cysteine and itaconate. In that study, Liu et al. used a\npromiscuous antibody that was originally designed to recognize lysine succinylation (due to the\nchemical similarity between succinate and itaconate). They unexpectedly found that this antibody could\nalso recognize lysine itaconylation from LPS-stimulated macrophages. However, there are no reports\non lysine itaconylation in bacteria, yet.\nThere are other reasons that we did not focus on lysine itaconylation: (1) itaconate chemically\nprefers to modify cysteine residues to form S-itaconation. Liu et al. identified only 87 high-confidence\nlysine sites (Figure 3a), whereas Qin et al. from the same group identified over 1,131 S-itaconated\ncysteine sites in the same cell line using a bioorthogonal itaconate probe (Figure 4a, doi:\n10.1021/jacs.9b11962); (2) antibody-based strategies depend heavily on the antibody used and are\ncontext-dependent. There is currently no commercially available anti-lysine itaconylation antibody,\nand although anti-lysine succinylation antibodies exist, they are not an appropriate tool to directly address\nthis question. Therefore, we consider cysteine itaconation (S-itaconation) in rpoN to be the main post-\ntranslational modification that contributes more significantly to rpoN function.\n4\n9. In lines 91-92: “We predicted that C218/275A lacking the cysteine residues essential for itaconate\nbinding would exhibit biologically relevant phenotypes specifically in the presence of itaconate”\nPlease state which biologically relevant phenotypes.\nThere is a large literature detailing phenotypes expected to be important in P. aeruginosa\npneumonia1, including flagella and motility, toxins, extracellular polysaccharides-both alginate and\nbiofilm and others that are expected to be important in the pathogenesis of pneumonia9. Many of\nthese are regulated by RpoN and hence we expected to see effects of RpoN itaconylation on some of\nthese10.\n10. In extended data fig 2f, 7.5mM itaconate was used and ∆rpoN grew slower than WT rpoN. This is\ninconsistent with fig 1b. It is also unclear why authors keep changing the concentration of itaconate in\ndifferent experiments.\nWe thank the reviewer for highlighting this inconsistency. To address this, we repeated the\nexperiments presented in Fig. 2f and Fig. 1h–i under identical conditions. The figures and\ncorresponding extended data have been updated accordingly.\n11. In figs 3d-e, please normalize all data to a single condition.\nThank you for the suggestion, we have updated the figure 3d-e as suggested.\n12. If itaconation of cysteine residues does not have any role in swarming, growth in the presence of\nitaconate, OCR (extended data figure 2e-i), what was the rationale for further pursuing transcriptomics\nand metabolomic profiling of the WTrpoN and C218/275A mutant\nThose observations indicated to us that the major “biologically relevant” effect of itaconation\nmust be metabolic, as has been shown for many human gene products that are modified by itaconate.\n13. What was the frequency of strains with deletions in rpoN?\nWe identified a deletion mutation (Leu139_Ser141) in the rpoN gene, occurring at a frequency\nof 0.648% exclusively in the CF isolates. The mutation profile of the rpoN gene is provided in the\nExtended data file 4.\n14. While authors have shown that the conserved cysteine residues in PAO1 can undergo S-\nitaconation in presence of itaconate, have they tested if this also occurs in clinical isolates?\nWe demonstrate the conservation of these same cysteine residues in the same positions in a\nlarge database of clinical isolates. It seems reasonable to expect these to be susceptible to\nitaconation in vivo. Their conservation despite mutations in other sites of rpoN, underscores the\npotential importance of these S-itaconation sites in the pathogen’s adaptive processes.\n15. Minor comments:\nA) Line 288- Fig 1 Expression of rpoN regulates bacterial response to pulmonary pathogenesis and\nitaconate.\nPlease change to “Expression of rpoN regulates bacterial pathogenesis and host itaconate response”\nThank you for pointing this out , we have slightly modified the figure legend to “Expression of\nrpoN regulates bacterial pathogenesis and response to itaconate.”\nC) Lines 85-87 “For simplicity of notation PAO1 ∆rpoN complemented with plasmid encoded WT rpoN\nis referred to as “WTrpoN” in contrast to PAO1 ∆rpoN expressing plasmid encoded C218/275A mutant\nrpoN, referred to as “C218/275A”.\nChange encoded to encoding – all corrected as suggested\nD) Line 549: “M9 minimal media supplemented with glucose an/or succinate”\nChange an to and\nE) Line 182: “We found that P. aeruginosa detoxifies itaconate by covalent binding to cysteine residues\nof RpoN and that itaconated RpoN boosts bacterial metabolism by promoting use of the preferred\nEntner Doudoroff Pathway to generate ATP.”\nIsn’t itaconate a metabolite that Pa can utilize and not a toxin and itaconation a post translational\nmodification?\nYes, itaconate is directly toxic to the bacteria acting as an oxidant as well as by altering\nmetabolism. Here we show that sequestration of itaconate by RpoN through S-itaconation seems to\nlimit itaconate oxidant toxicity while modifying metabolic activity (e.g. ED pathway in this study).\nWe have slightly modified the sentence (line 182)- “We found that P. aeruginosa limits itaconate toxicity\nby covalent binding to cysteine residues of RpoN and that itaconated RpoN boosts bacterial\nmetabolism by promoting use of the preferred Entner Doudoroff Pathway to generate ATP. ”\nF) Line 62: Please use consistent terminology: either σ54 or rpoN.\nWe appreciate the reviewer’s suggestions and all changes have been addressed and\nhighlighted in red throughout the manuscript.\nIts Metabolism to Promote Biofilm Formation. Cell Metab 31, 1091-1106 e1096,\ndoi:10.1016/j.cmet.2020.04.017 (2020).\ncycle activity at IDH2. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease\n1868, 166530 (2022).\nkinetics. Nature Metabolism, 1-10 (2025).\ninfection. Respiratory Research 21, 326 (2020).\nnovo purine biosynthesis in pathogens. Cell Reports 43 (2024).\nthat suppresses the oxidative burst. Cell Rep 42, 112064,\ndoi:10.1016/j.celrep.2023.112064 (2023).\nNature communications 16, 3729 (2025).\npulmonary infection. Cell Metab 35, 1767-1781 e1766, doi:10.1016/j.cmet.2023.09.001\n(2023).\n6\nintegrating reverse vaccinology with immunoinformatics approaches. Scientific Reports 15,\n10425 (2025).\nsecretion system in Pseudomonas aeruginosa PAO1. Journal of bacteriology 200,\n10.1128/jb. 00205-00218 (2018).\n7"}]

Decision letter