Regulation of airway fumarate by host and pathogen promotes S. aureus pneumonia

Abstract

Staphylococcus aureus is a leading cause of healthcare-associated pneumonia, contributing significantly to morbidity and mortality worldwide. As a ubiquitous colonizer of the upper respiratory tract, S. aureus must undergo substantial metabolic adaptation to achieve persistent infection in the distinctive microenvironment of the lung. We observed that fumC, which encodes the enzyme that converts fumarate to malate, is highly conserved with low mutation rates in S. aureus isolates from chronic lung infections. Fumarate, a pro-inflammatory metabolite produced by macrophages during infection, is regulated by the host fumarate hydratase (FH) to limit inflammation. Here, we demonstrate that fumarate, which accumulates in the chronically infected lung, is detrimental to S. aureus, blocking primary metabolic pathways such as glycolysis and oxidative phosphorylation (OXPHOS). This creates a metabolic bottleneck that drives staphylococcal FH (FumC) activity for airway adaptation. FumC not only degrades fumarate but also directs its utilization into critical pathways including the tricarboxylic acid (TCA) cycle, gluconeogenesis and hexosamine synthesis to maintain metabolic fitness and form a protective biofilm. Itaconate, another abundant immunometabolite in the infected airway enhances FumC activity, in synergy with fumarate. In a mouse model of pneumonia, a ΔfumC mutant displays significant attenuation compared to its parent and complemented strains, particularly in fumarate- and itaconate-replete conditions. Our findings underscore the pivotal role of immunometabolites in promoting S. aureus pulmonary adaptation.

Extended Data Figure 1.. FumC is essential for bacterial metabolic fitness.

(A) Map of fumC showing the position of de novo mutations in both pulmonary isolates and nasal colonizers. (B) Simulated structure of S. aureus FumC tetramer depicting the position of the SNP E250G (carbon atoms in purple) with respect to the catalytic site. Citrate and malate ligands bound to the active and B sites respectively, are shown with carbon atoms in green. Ligand positions were created by alignment with PDB ID: 6OS7, and 1FUR. (C) Protein surface colored by electrostatic potential, where red symbolizes negative charge, and blue positive charge. The left panel displays human fumarate hydratase (PDB: 5UPP). The right panel illustrates the constructed model of S. aureus FumC. (D) Relative level of fumarate in chemically defined media (CDM), Luria-Bertani broth (LB) and artificial sputum media (ASM), n = 2 per condition. (E) Growth curve of WT LAC, the ΔfumC mutant and complemented strain in LB (left panel), ASM (middle panel) or CDM (right panel) supplemented with or without 10 mM malate; n = 3 biological samples in triplicate (3 independent experiments). (F-H) ¹³C-fumarate labeling of metabolites involved in the (F) de novo purine nucleotide synthesis, (G) de novo pyrimidine nucleotide synthesis, or (H) coenzyme synthesis in WT LAC and the ΔfumC mutant; n = 3 biological replicates (1 independent experiment). (I) Bacterial burden from the BALF (left panel) and lung (right panel) of BL/6 infected with WT LAC and the ΔsucA and ΔsucD mutants (n = 5 per condition). The dotted line indicates the limit of detection. (J) Intracellular bacterial survival at 1.5 h and 4 h post-infection in BMDMs from WT BL/6 mice relative to the number of viable BMDMs and bacterial inocula; n = 2 independent experiments. Data are shown as mean ± SEM. Statistical significance is determined by two-tailed t-student test with FDR correction for (E-I) and Mann-Whitney non-parametric U test for (D), *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Extended Data Figure 2.. Synthesis of the FA-alkyne probe.

(A-B) Chemical reactions for the production of (A) compound 1 and (B) the FA-alkyne probe. (C-D) Validation of the structure of the FA-alkyne probe by (C) proton nuclear magnetic resonance (¹H NMR) and (D) carbon-13 nuclear magnetic resonance (¹³C NMR).

Figure 1.. S. aureus adapts to the metabolic airway milieu.

(A) Phylogenetic tree illustrating within-host evolution of S. aureus during persistent pulmonary infection (cystic fibrosis, blue clade) and nasal carriage (red clade). This model assumes genetic bottlenecks upon transmission and expansion of a single lineage during infection or colonization. Only mutations acquired within the host (i.e. on blue or red branches) are considered in the analysis. (B) Output of the convergence analysis: the size of the dots is proportional to the number of independent (i.e. acquired de novo within the host) protein-altering mutations in genes of the TCA cycle and surrounding pathways. The relative mutation rate is equivalent to a rate ratio in Poisson models and calculated as: (mutations in gene x/length of gene x)/(mutations in all genes/length of all genes). A rate < 1 (grey shading) indicates less mutations than the mean across the genome, suggesting that the gene is conserved during infection or colonization. GLY: glycolysis; GLN: gluconeogenesis; PPP: pentose phosphate pathway; TCA: tricarboxylic acid/Krebs cycle; UREA: urea cycle. (C) Gene maps with position, type, and evolutionary niche of the de novo mutations. (D) Relative levels of fumarate and itaconate in the sputum of healthy subjects (HS) and patients with cystic fibrosis (CF).

Figure 2.. Fumarate imposes metabolic stress on S. aureus.

(A) Volcano plot showing significantly differentially expressed genes in WT LAC in the presence (100 mM) and absence of fumarate above the dotted line; n = 3 biological samples (1 experiment). (B) Pathway enrichment analysis of genes from (A) by Gene Ontology depicting significant differences in metabolic pathways in the presence and absence of fumarate. (C) Glycolysis and (D) oxidative phosphorylation (OXPHOS) of S. aureus, as measured by the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) respectively, using the Seahorse extracellular flux analyzer. Glucose was injected, followed by three sequential additions of either the vehicle (left panel) or itaconate (right panel). Each data point is the mean ± SEM; n = 4 biological samples (4 independent experiments) with at least 3 technical replicates. The asterisks denote statistical differences at each time point between left and right panels by one-way ANOVA. (E) Heatmap illustrating variations in gene expression in WT LAC cultured with (10 mM) and without fumarate, as determined by qRT-PCR. The differences in gene expression are relative to WT LAC without fumarate. Statistical analysis was conducted using the Mann-Whitney non-parametric U test for each gene, with significance denoted as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. (F) Schematic diagram outlining the workflow for the chemoproteomic profiling of succinated S. aureus proteins by the fumarate analogue (FA) probe. (G) Volcano plot of the quantified FA-captured sites that are competed for by fumarate in live S. aureus FPR3757. The threshold for the −log10(q value/FDR) is set at 1.3; n = 3 biological samples (3 independent experiments) (H) Gene ontology analysis for the succinated sites from the in situ profiling. (I) AcnA/CitB activity in WT LAC, the ΔfumC mutant and complemented strain. Each data point is the mean ± SEM.; n = 3 biological samples (3 independent experiments) in triplicate. Statistical analysis was conducted by one-way ANOVA, ***p < 0.001. (J) Carbon source assimilation of WT LAC, the ΔfumC mutant and complemented strain (PM1 Biolog; n = 3 biological replicates from 3 independent experiments). The color intensity in the heatmap corresponds to the absorbance of the bacterial strains (OD_590nm) as a readout of bacterial respiration in the presence of the indicated carbon source, normalized to the absorbance (OD_590nm) of WT LAC in the same carbon source. Asterisks denote statistically significant differences as determined by two-way ANOVA.

Figure 3.. FumC promotes bacterial metabolic fitness, antioxidative defense and pathogenesis.

(A-C) Growth curve of WT LAC, the ΔfumC mutant and complemented strain in (A) artificial sputum media (ASM), (B) Luria Bertani (LB) broth lacking glucose (left panel) and supplemented with fumarate (middle panel) or malate (right panel) at a final concentration of 10 mM or (C) chemically defined media (CDM) supplemented with (left panel) and without (middle panel) glucose. (D) Growth curve of WT LAC, the ΔfumC mutant and complemented strain in LB with and without hydrogen peroxide (H₂O₂) at a final concentration of 0.5 mM. Data are shown as mean ± SEM from n ≥ 3 biological replicates (≥ 3 independent experiments) at least in triplicate. Statistical significance is determined by two-tailed t-student test with FDR correction. (E) Heatmap showing variations in gene expression in WT LAC, the ΔfumC mutant and complemented strain, as determined by qRT-PCR. The differences in gene expression are relative to WT LAC. (F) Expression of genes encoding the T7SS machinery by WT LAC (left panel) and the ΔfumC mutant (right panel) in the presence of increasing fumarate concentrations. The differences in gene expression are relative to WT LAC or the ΔfumC mutant grown in the absence of fumarate. For (D-E), statistical analyses were conducted using the two-tailed t-student test with FDR correction for each gene, with significance denoted as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 4.. FumC serves as a metabolic hub and directs fumarate to major metabolic pathways.

(A) Schematic diagram depicting key metabolic pathways in S. aureus. (B-E, H) ¹³C₄-fumarate labeling of metabolites involved in the (B) TCA cycle or (C) aspartate-argininosccuinate shunt, (D) gluconeogenesis, (E) hexosamine synthesis and (H) PPP in WT LAC and the ΔfumC mutant. (F) Biofilm formation by WT LAC, the ΔfumC mutant, and the complemented strain in LB broth (without glucose) with increasing concentrations of fumarate, as evaluated by crystal violet staining. (G) Biofilm depth of WT LAC in the presence of increasing fumarate concentrations, measured using wheat germ agglutinin-Alexa Fluor 555 staining and confocal microscopy. For (B-H), data are shown as mean ± SEM from n = 3 biological replicates (1 independent experiment), or n = 2 biological replicates for (G). Statistical significance is determined by two-tailed t-student test with FDR correction. (I) Diagram summarizing the anabolic pathways fueled by S. aureus FumC.

Figure 5.. Host fumarate hydratase and S. aureus FumC both regulate fumarate and pathogenesis.

(A) Relative level of fumarate (left panel) and succinate (right panel) in the BALF of uninfected BL/6 and Ptenl^−/− mice. (B) Bacterial burden from the BALF (left panel) and lung (right panel) of BL/6 and Ptenl^−/− mice infected with WT LAC, the ΔfumC mutant and complemented strain; BL/6 infected with WT LAC (n = 11), ΔfumC mutant (n = 13), and ΔfumC::fumC (n = 7), Ptenl^−/− infected with WT LAC (n = 6), ΔfumC mutant (n = 5). The above-mentioned total number of mice per group were from at least 2 independent experiments The dotted line indicates the limit of detection. (C-D) Innate immune cells (monocytes left panel, neutrophils middle panel and alveolar macrophages right panel) from the (C) BALF and (D) lungs of uninfected and infected mice from (A). (E) Cytokine measurements from the BALF of uninfected and infected mice from (A). (F) Expression of murine Fh1, Ass1 and Hk2 from uninfected and infected mouse lungs (n = 5 per condition) by qRT-PCR (left panel) and schematic diagram showing the metabolic reactions catalyzed by the encoded enzymes fumarate hydratase, argininosuccinate synthase and hexokinase (right panel). (G) Absolute level of malate in the BALF of uninfected (PBS) BL/6 mice and mice infected with WT LAC or the ΔfumC mutant. (H) Schematic diagram of reaction catalyzed by FumC. (I) FumC activity assessed by the absorbance of fumarate (left panel). Fumarate was supplied as a substrate for FumC, preincubated with and without 1 mM itaconate (chemical structure, right panel). Data represent mean values ± SEM. Statistical analysis was performed by Mann-Whitney non-parametric U test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.