Regulation of airway fumarate by host and pathogen promotes Staphylococcus aureus pneumonia

Chen, Ying-Tsun; Liu, Zihua; Fucich, Dario; Giulieri, Stefano G.; Liu, Zhe; Wadhwa, Ridhima; Rios, Gustavo; Henschel, Henning; Tyagi, Nupur; Olivier, Françios A. B.; Monk, Ian R.; Shah, Shivang S.; Sridhar, Shwetha H.; Drikic, Marija; Bianco, Colleen; Lohia, Gaurav K.; Beg, Ayesha Z.; Planet, Paul J.; Lewis, Ian A.; Sebra, Robert; Traven, Ana; Hachani, Abderrahman; Stinear, Timothy P.; Howden, Benjamin P.; Boyd, Jeffrey M.; Riquelme, Sebastian A.; Wang, Chu; Prince, Alice; Wong Fok Lung, Tania

doi:10.1038/s41467-025-62453-y

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Published: 01 August 2025

Regulation of airway fumarate by host and pathogen promotes Staphylococcus aureus pneumonia

Nature Communications volume 16, Article number: 7050 (2025) Cite this article

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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.

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Introduction

Staphylococcus aureus frequently causes healthcare-associated pneumonia¹. Antibiotic susceptible as well as resistant strains pose a significant risk for prolonged airway infection and associated morbidity². This risk is heightened in patient populations with underlying pulmonary pathology, such as chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), and following viral infection^3,4. S. aureus is an especially formidable pathogen due to the expression of many surface proteins that thwart innate immune clearance⁵. While toxin-producing epidemic isolates can cause a fulminant necrotizing pneumonia⁶, typically the bacteria adapt to the airway milieu to cause a more indolent but intractable infection. These less immunostimulatory strains are better tolerated by the host as they fail to induce as robust an immune response that is detrimental to the host and pathogen alike. Of note, vaccine strategies that target classic virulence factors have failed⁷, indicating the need for alternative approaches to control staphylococcal infection.

Among the common respiratory pathogens, S. aureus is notable for its substantial metabolic flexibility⁸. In contrast to more fastidious pathogens, S. aureus readily adapts to local carbon sources to optimize its metabolism. The infected airway provides a unique microenvironment with a distinctive set of metabolites and molecules produced by both the host and pathogen including reactive oxygen species, and DNA from lysed or damaged immune and stromal cells. By studying patterns of gene expression in clinical isolates of S. aureus from patients with persistent pneumonia, we observed staphylococcal adaptation to airway metabolites that accumulate during ongoing infection⁹. In a survey of methicillin resistant S. aureus (MRSA) strains isolated from chronic pneumonia, we noted over 100- to 1000-fold increases in the expression of fumC, which encodes the bacterial fumarate hydratase (FH) that converts fumarate to malate¹⁰. Fumarate, a potent proinflammatory metabolite that induces the production of TNFα and IFNβ in stimulated macrophages, is regulated by the host FH to limit immunopathology¹¹. While the immunoregulatory function of host FH is well documented particularly in diseases associated with excess inflammation, such as systemic lupus erythematosus and renal cell carcinomas^11,12,13, less is known about the importance of S. aureus FumC in pneumonia and how it may contribute to the inflammatory response that determines host pathology induced by S. aureus infection.

We hypothesize that the regulation of fumarate is an important component of S. aureus metabolic adaptation to the infected airway. In this report, we illustrate the unexpectedly central role of FumC and fumarate metabolism in orchestrating numerous metabolic processes in S. aureus relevant to its role as a pulmonary pathogen. Our findings raise the possibility of selectively targeting staphylococcal FumC to prevent chronic airway infection.

Results

Conservation of fumC across a global collection of S. aureus isolates

We postulated that the importance of fumC in S. aureus adaptation to the lung is reflected in its conservation across clinical isolates. Specifically, we anticipate fewer non-synonymous substitutions and protein-truncating mutations in fumC compared to other genes. We analyzed a global dataset comprising 820 serially collected isolates from 181 patients with cystic fibrosis (CF) (Fig. 1A, Supplementary Data 1, 2). Using an established statistical genomics model of within-host evolution, we observed a relative mutation rate, λ, less than 1 in fumC from CF isolates (Fig. 1B, Supplementary Data 3, 4). This indicates fewer mutations in fumC from pulmonary isolates than the mean number of mutations across the genome. Similarly, mqo, encoding the malate-quinone oxidoreductase enzyme involved in downstream malate metabolism to oxaloacetate¹³, displayed high conservation, without any mutations in pulmonary isolates (Fig. 1B, C). Conversely, lqo, which encodes a lactate-quinone oxidoreductase for lactate utilization¹⁴, exhibited a substantially higher relative mutation rate (λ = 5.49, p = 1 × 10⁻⁵, Supplementary Data 4).

Fig. 1: S. aureus adapts to the metabolic airway milieu. — **Fig. 1: *S. aureus* adapts to the metabolic airway milieu.**

We next evaluated how the only acquired single nucleotide polymorphism (SNP), E250G, identified in fumC from pulmonary isolates (Supplementary Fig 1A) may affect the enzymatic function. Our prediction of the tetrameric structure of FumC through homology modeling and molecular dynamics (MD) simulation indicated that the glutamate residue 250 (E250) is positioned away from the active site (Supplementary Fig 1B), and that the SNP E250G is unlikely to inhibit its enzymatic activity. The highly negative surface charge (−78) of S. aureus FumC compared to the neutral human FH homolog (Supplementary Fig 1C) suggests differences in substrate affinity and catalytic efficiency. This observation, along with the conserved nature of fumC with minimal mutations in CF isolates, supports the pivotal role of FumC in chronic lung infections and its potential for selective inhibition.

Exogenous fumarate re-directs S. aureus metabolic activity

We explored how high fumarate levels impact S. aureus. We confirmed the presence of substantially elevated levels of fumarate in the infected airways of CF patients as compared with healthy controls (Fig. 1D). S. aureus preferentially utilizes glucose as a carbon source to generate energy (ATP) through glycolysis¹⁵. In the infected lung, where the predominant recruited neutrophils compete with the bacteria for the already limited airway glucose¹⁶, we postulate that S. aureus adapts to fumarate using FumC. We assessed the impact of exogenous fumarate on the transcriptional response of S. aureus USA300 LAC by bulk RNA-seq analysis and observed a major impact on metabolic genes (Fig. 2A, B). The lacA/B/C/D/E/F genes were significantly downregulated by fumarate (Fig. 2A, B), indicating decreased lactose catabolism to galactose and glucose, two main carbon sources utilized by S. aureus for glycolysis. This was consistent with decreased glycolytic rates, which we confirmed by extracellular acidification rate (ECAR), in WT LAC and especially in the ΔfumC mutant that cannot catabolize fumarate (Fig. 2C). We also noted that exogenous fumarate inhibited bacterial oxidative phosphorylation (OXPHOS) represented by the oxygen consumption rate (OCR) (Fig. 2D). The purl/M/N/H genes were downregulated (Fig. 2A), suggesting decreased purine synthesis. In contrast, we observed increased expression of the argG/H genes, which are involved in the urea cycle and promote biofilm formation¹⁷.

Fig. 2: Fumarate imposes metabolic stress on S. aureus. — Fig. 2: Fumarate imposes metabolic stress on *S. aureus.*

The expression of genes associated with pathogenesis is often linked with bacterial metabolism¹⁸. We observed that expression of several such genes were altered in the presence of fumarate (Fig. 2A). The cap5 genes associated with capsule production¹⁹ were upregulated (Fig. 2A). The genes lukS and esxA, encoding a PVL toxin subunit and a Type Seven Secretion System (T7SS) effector respectively, were markedly upregulated upon fumarate exposure (Fig. 2A), as corroborated by qRT-PCR even at lower fumarate concentration (Fig. 2E). We also observed increased expression of perR and katA, genes important in orchestrating antioxidative defenses (Fig. 2E).

The metabolic impact of fumarate in redirecting metabolic activity can be mediated by direct or indirect mechanisms. Fumarate can post-translationally modify both bacterial and mammalian proteins, through covalent binding or succination^12,20,21,22. We performed global profiling of succinated proteins in live S. aureus using a biorthogonal fumarate analogue (FA) probe²³ (Fig. 2F). Among the proteins that were identified, many that were modified at specific cysteines are involved in antioxidative stress responses such as PerR (Fig. 2G, H, Supplementary Data 5). We were intrigued by targets involved in the biogenesis/repair of iron-sulfur (Fe-S) clusters, including Nfu and ScdA (Fig. 2G). We hypothesized that fumarate impairs Fe-S cluster assembly in S. aureus through succination, mirroring its effect in host cells¹², and inhibits downstream Fe-dependent bacterial processes. A number of proteins require Fe-S prosthetic groups for function²⁴, such as the TCA cycle enzyme, aconitase (AcnA/CitB). In S. aureus, defects in Fe-S synthesis result in decreased TCA cycle function and reduced survival within neutrophils²⁵.

We tested the inhibitory effect of fumarate on S. aureus AcnA using the ΔfumC mutant as a genetic model for fumarate accumulation. We observed a significant reduction in AcnA activity in the ΔfumC mutant compared with the parental and complemented strains (Fig. 2I), suggesting that fumarate accumulation may inhibit staphylococcal Fe-S assembly. Metabolite phenotype microarrays (Biolog) also highlighted the inability of the ΔfumC mutant to utilize numerous carbon sources, including TCA cycle intermediates and sugars (Fig. 2J). Thus, the accumulation of excess fumarate has multiple potentially detrimental effects on the bacteria, disrupting the generation of ATP through the TCA cycle, OXPHOS, as well as glycolysis. While these studies indicate the importance of the Fe-independent enzyme FumC²⁶ in regulating the accumulation of fumarate, they also imply that it is involved in many bacterial biosynthetic pathways.

Deletion of fumC impairs major physiological processes in S. aureus

We next addressed the importance of fumC in S. aureus physiology, particularly as relevant to pneumonia. We noted that under conditions that mimic the infected airway, the ΔfumC mutant had a significant growth defect. In artificial sputum media (ASM), which contains fumarate (Supplementary Fig 1D), mucin, DNA, and amino acids²⁷, there was a significant growth attenuation of the ΔfumC mutant compared to the WT and complemented strains (Fig. 3A). This was also observed in Luria broth (LB) rich in peptides but lacking in glucose (Fig. 3B left panel). While the WT strain and the ΔfumC mutant could both grow equally well in the presence of glucose in chemically defined media (CDM) (Fig. 3C left panel), the ΔfumC mutant displayed significant growth impairment in the absence of glucose (Fig. 3C right panel). This growth defect was exacerbated by the addition of fumarate and restored upon supplementation with malate in either LB, ASM or CDM (Fig. 3B middle and right panels, Supplementary Fig 1E, F).

**Fig. 3: FumC promotes bacterial metabolic fitness, antioxidative defense and pathogenesis.**

In addition to effects on growth, the absence of fumC rendered the bacteria more susceptible to oxidative stress (Fig. 3D), consistent with the downregulation of the antioxidative genes perR, trx, aphF, and msrA1/B, but not katA and sod that encode catalase and superoxide dismutase respectively (Fig. 3E). Similarly, the expression of biofilm-associated genes and those encoding α-hemolysin (Hla), LukS (PVL), and the T7SS EsxA effector were significantly downregulated in the ΔfumC mutant (Fig. 3E). As expected, the phenotypes associated with the ΔfumC mutant contrasted with those observed when S. aureus can degrade excess fumarate (Fig. 2E). Of note, the expression of genes encoding the T7SS machinery itself (essA, esaA, and essC) was induced by exogenous fumarate in a fumC-dependent manner (Fig. 3F). Our findings indicate that S. aureus, when exposed to fumarate, depends on fumC to express genes likely important in pathogenesis and in maintaining biosynthetic pathways crucial to preserve its physiology.

FumC orchestrates carbon flux to diverse pathways

We elucidated the precise role of S. aureus FumC in the biosynthetic pathways critical for bacterial growth and fitness (Fig. 4A), properties essential for pathogens. We employed ¹³C₄-labeled fumarate and stable isotope tracing in WT LAC and the ΔfumC mutant to track the metabolic fate of fumarate. Of the total fumarate, 49.5% and 59.4% were ¹³C-labeled in WT LAC and the ΔfumC mutant respectively, particularly as the carbon 4 (C₄) isotopologue (Fig. 4B). This translated into the incorporation of ¹³C into malate as C₁-C₄ isotopologues in WT LAC but not in the ΔfumC mutant (Fig. 4B). The accumulated ¹³C was instead incorporated back into succinate in the ΔfumC strain as C₃-C₄ isotopologues (Fig. 4B). We observed the assimilation of ¹³C derived from labeled fumarate into the TCA cycle metabolites citrate, cis-aconitate and α-ketoglutarate (α-KG) in WT LAC but not the ΔfumC mutant, as predicted (Fig. 4B).

**Fig. 4: FumC serves as a metabolic hub and directs fumarate to major metabolic pathways.**

We detected the substantial integration of labeled carbon from fumarate into argininosuccinate, particularly as the C₄ isotopologue (Fig. 4C). This may serve to rapidly replenish malate and oxaloacetate for gluconeogenesis, hexosamine synthesis and the pentose phosphate pathway (PPP) in the WT strain (Fig. 4A). Accordingly, there was increased integration of ¹³C into the gluconeogenic intermediates, phosphoenol pyruvate (PEP) and fructose 6-phosphate (F6P), in WT LAC but not in the ΔfumC mutant (Fig. 4D). We also detected significant incorporation of ¹³C into N-acetyl-glucosamine-1-phosphate (GlcNAc-1-P) and UDP-N-acetyl-glucosamine (UDP-GlcNAc) (Fig. 4E), which contribute to biofilm formation^9,28 in the WT strain. We confirmed that fumarate promoted biofilm formation in a dose-dependent manner in WT LAC (Fig. 4F), resulting in increased biofilm depth (Fig. 4G).

In addition, we noted the incorporation of labeled carbon into the PPP intermediate, ribose 5-phosphate (R5P) in WT LAC (Fig. 4H). This was not observed in the ΔfumC mutant. Assimilation of ¹³C into R5P correlated with the increased labeling of intermediates in the de novo purine nucleotide metabolism, such as AICAR, AMP, ADP, ATP, GMP, and GDP (Supplementary Fig 1G). In parallel, there was increased labeling of dihydroorate, orotate, and intermediates of the de novo pyrimidine nucleotide synthesis, including UMP, UDP, UTP and CTP, in WT LAC only (Supplementary Fig 1H). Consistently, the incorporation of ¹³C into NAD and the reduced form NADH, which is crucial for energy production, occurs in the WT strain only (Supplementary Fig 1I). Overall, the stable isotope tracing experiment emphasized an unexpectedly central role of FumC as a metabolic hub, directing fumarate utilization into multiple biosynthetic pathways critical for S. aureus survival and proliferation (Fig. 4I).

FumC is critical in pneumonia

We expected S. aureus FumC to be crucial in chronic lung infection. We used a mouse pneumonia model with elevated fumarate levels to more adequately mimic the airway milieu in persistent infections (Fig. 1D). Ptenl^−/− mice, which were previously used to model the airway properties in CF^29,30,31, exhibited a baseline accumulation of the pro-inflammatory metabolite fumarate in the lung (~5 times higher than in BL/6 mice) but not succinate (Fig. 5A). As predicted, in settings with high fumarate levels (Ptenl^−/−), we observed that the ΔfumC mutant was significantly attenuated compared to the WT strain (Fig. 5B). This attenuation was more modest in the bronchoalveolar lavage fluid (BALF) and lung of BL/6 mice (3-fold and 5-fold respectively) (Fig. 5B). Deletion of sucD, encoding the TCA cycle enzyme succinyl-CoA synthetase, had no effect on airway S. aureus load, while the ∆sucA mutant, lacking α-KG dehydrogenase, was attenuated in the lung but not in the BALF (Supplementary Fig 1J). This suggests that not all TCA cycle disruptions negatively impact staphylococcal airway survival and highlights the importance of fumarate metabolism in pulmonary pathogenesis.

Fig. 5: Host fumarate hydratase and S. aureus FumC both regulate fumarate and pathogenesis. — **Fig. 5: Host fumarate hydratase and *S. aureus* FumC both regulate fumarate and pathogenesis.**

To determine if differences in the immune response due to the loss of Ptenl might account for the enhanced ΔfumC clearance, we compared the immune cells (neutrophils, monocytes, alveolar macrophages) and cytokines during infection of both BL/6 and Ptenl^−/− mice. Overall, similar numbers of immune cells were observed in the infected BL/6 and Ptenl^−/− airway (Fig. 5C, D). In the Ptenl^−/− mice, where the difference in infection levels between WT LAC and the ΔfumC mutant was more pronounced (Fig. 5B), we observed a slight reduction in monocyte numbers in the BALF but not in the lung (Fig. 5C, D). While these mice also had lower levels of IL-1β and MIP2, there were no statistical differences in other proinflammatory cytokines such as IL6, KC and TNFα (Fig. 5E). In the BL/6 mice, similar numbers of immune cells and cytokine levels were also detected in response to infection with either the WT or ΔfumC strains (Fig. 5C–E). We confirmed that there were no differences in the uptake of WT LAC and the ΔfumC mutant by phagocytes (Supplementary Fig 1K). Thus, our results indicate that the attenuation of the ΔfumC mutant is not due to a lack of immunostimulation or to differences in the immune response between BL/6 and Ptenl^−/− backgrounds. Instead, our findings underscore the critical role of S. aureus fumarate metabolism during pneumonia.

S. aureus exploits host metabolism

The apparent disparity observed between the profound in vitro effects of fumC on S. aureus metabolism and the modest attenuation of the ΔfumC mutant in BL/6 mice led us to speculate that the bacteria may exploit the host FH to mitigate the absence of fumC in vivo. We observed comparable Fh1 transcript levels in both uninfected and infected murine lungs (Fig. 5F). This indicates that S. aureus sustains host Fh1 expression, in contrast to Gram-negative pathogens and LPS, which typically downregulate it^11,32. The expression of Ass1, which encodes argininosuccinate synthase 1, was also unchanged, unlike Hk2 that was significantly upregulated upon infection with the ΔfumC mutant (Fig. 5F). We next measured the level of malate, the product of either S. aureus FumC or host FH, in the BALF harvested from BL/6 mice infected with WT LAC or the ΔfumC mutant. Similar levels of airway malate were detected during infection with either strain (Fig. 5G, H) despite the inability of the ΔfumC mutant to catabolize fumarate to malate. This suggests that host FH activity may protect S. aureus from the detrimental effects of fumarate in vivo, thereby limiting the impact of fumC deletion through the generation of malate. Our results emphasize that while S. aureus can exploit the host FH to degrade fumarate, it depends significantly on its own FumC enzyme in environments with accumulated fumarate levels typical of chronic lung infections.

Itaconate, another dicarboxylate, accumulates in the chronically infected airway, along with fumarate (Fig. 1D). These two metabolites share structural similarities (Fig. 5I) and could potentially work in a concerted fashion. We wondered if the presence of itaconate might further enhance FumC activity. We monitored the conversion of fumarate to malate by purified FumC³³ in the presence or absence of itaconate (Fig. 5H, I). The significant depletion of added fumarate in the presence of itaconate strongly suggests that itaconate potentiates FumC activity. Our data demonstrate that the metabolism of airway fumarate by S. aureus promotes pneumonia and that this effect is even more biologically significant in the context of other accumulated metabolites such as itaconate.

Discussion

S. aureus is a well-studied pathogen which nonetheless remains among the top three causes of healthcare-associated infection, especially pneumonia¹. Its extensive metabolic flexibility has long been recognized but not effectively targeted in either antibiotic or vaccine development. We uncovered a previously unappreciated role for fumarate metabolism in the overall physiology and pathogenesis of S. aureus, especially in microenvironments with limited glucose such as the airway. Our focus on FumC was initiated by studying clinical isolates that had successfully undergone long term adaptation to the lung. The increased expression¹⁰ and low mutation rate of fumC in S. aureus pneumonia isolates indicate its global epidemiological significance for pulmonary adaptation. This is particularly relevant in the chronically infected airway replete with the host immunometabolites, fumarate and itaconate.

We observed a fascinating dynamic regulation of airway fumarate, an important pro-inflammatory mediator¹¹, by both host and S. aureus fumarate hydratase. For the host, the production of the dicarboxylates fumarate and itaconate offers homeostatic protection against the detrimental effects of excessive inflammation. Fumarate, a product of activated macrophages, induces type I interferon and TNFα production in response to mitochondrial damage¹¹. In contrast, itaconate exhibits immunomodulatory properties; it stimulates major antioxidative mediators such as Nrf2 and ATF3³⁴ and inhibits NLRP3 inflammasome activation³⁵. Just as excess fumarate is detrimental to the host, we show that it is toxic to the bacteria, inhibiting primary metabolic pathways such as glycolysis, the TCA cycle and OXPHOS. The presence of fumC in S. aureus isolates not only enables fumarate degradation, circumventing its negative effects, but also promotes its utilization in anabolic pathways such as gluconeogenesis, and hexosamine production, which are essential for biofilm formation and defense against oxidative stress. We also found that several genes expected to function more directly in pathogenesis were upregulated by fumarate such as those involved in toxin and capsule production and the T7SS.

Having documented the pervasive effects of fumarate on S. aureus biology and the critical role of FumC in numerous metabolic pathways, we also demonstrate its role in vivo, particularly in environments abundant in fumarate, such as the Ptenl^−/− airway. Consistently, we show that the ΔfumC mutant had a more modest limitation during acute pulmonary infection in BL/6 mice. No substantive differences in the immune response to the WT or ΔfumC strains were identified. These observations can be explained by compensatory host FH activity, as confirmed by quantifying equivalent levels of malate in the airway of BL/6 mice. Thus, the host can regulate local fumarate levels to a certain extent, even in the absence of S. aureus FumC, enabling the microorganisms to proliferate. However, in the Ptenl^−/− background with excessive airway fumarate, the lack of fumC resulted in an appreciable loss of bacterial fitness in vivo. This further supports the critical role of FumC in chronic pulmonary infections.

In addition, we observed that the host metabolite itaconate enhances the activity of S. aureus FumC and further promotes the degradation of fumarate and its utilization as a carbon source. Thus, in vivo, ambient fumarate amounts are regulated by both the host FH and S. aureus FumC whose activity seems likely to be boosted by itaconate. It remains to be determined exactly how itaconate accelerates FumC and whether this is unique to S. aureus. Given the overall structural similarities between the host and S. aureus FH, FumC may initially appear to be an unsuitable target for anti-staphylococcal therapy. However, some of the peculiarities of S. aureus FumC structure and charge characteristics may be sufficiently distinct to generate selective bacterial toxicity. These distinctive features could be harnessed for the development of adjuvant therapy to prevent chronic pneumonia.

We highlight two limitations to provide appropriate context for interpreting our findings. Although fumarate is a central focus of this work, its physiological concentrations in the human airway have not yet been determined either in healthy individuals or patients with chronic pulmonary infection. We tested a wide range of fumarate concentrations across multiple media types, including LB, glucose-depleted basal medium, and artificial sputum medium to reflect the likely range of in vivo conditions and to account for the influence of staphylococcal carbon catabolite repression by preferred substrates. Importantly, S. aureus demonstrated FumC-dependent adaptation across this broad range of fumarate concentrations, including levels likely exceeding physiological norms; this adaptation was impaired in the absence of fumC, where fumarate instead exerted toxic effects. Another challenge consists of the variability in expectorated sputum from individuals with CF and induced sputum from healthy controls, an issue inherent to the many studies using clinical materials. These factors may alter sample composition, in part due to dilution effects. We include these limitations here to ensure transparency and support the interpretation of our results within the experimental conditions used.

Methods

All experimental procedures were conducted in accordance with relevant ethical regulations and approved by the appropriate institutional review boards or ethics committees.

Human subjects

All human samples (healthy individuals and CF patients) were obtained from adults (22–44 years of age) in the CF program at Yale University under the Yale IRB Protocol 0102012268 and Columbia Protocols AAAR1395. Males and females are represented in an ~50–50% ratio. Sputum samples from healthy adult individuals were collected after nebulization with 3% hypertonic saline for five minutes on three cycles. To reduce squamous cell contamination, subjects were asked to rinse their mouth with water and clear their throat. CF subjects expectorated sputum spontaneously for our studies. Expectorated sputum samples were collected into specimen cups and placed on ice. Of the 7 CF patients that were chronically infected with Staphylococcus aureus (either MSSA or MRSA), 5 patients were co-infected with other agents, such as Pseudomonas (3 patients), Achromobacter (1 patient) or human influenza (1 patient). No CF patients were infected with Aspergillus, a known itaconate producer. None of the CF patients studied were under CFTR modulator therapy (e.g., Kaleydeco, Orkambi). An informed consent was signed by all subjects.

Mouse experiments

All animal experiments were performed following institutional guidelines at Columbia University and Rutgers New Jersey Medical School (NJMS). Animals were housed and maintained at Columbia University Irving Medical Center (CUIMC) and Rutgers Medical Science Building (MSB) under regular rodent light/dark cycles at 18–23 °C and fed with irradiated regular chow diet (Purina Cat#5053, distributed by Fisher). Animal health was routinely checked by an institutional veterinary. The animal work protocols (AC-AABD5602 and PROTO202400003) were approved by the Institutional Animal Care and Use Committee (IACUC). Animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the NIH, the Animal Welfare Act, and US federal law. C57BL/6 mice, purchased from the Jackson Laboratory (stock number 000664), were acclimated in our facilities. Ptenl^−/− mice²⁹ were bred in our facilities. The animals were infected at 8 to 10 weeks of age. In vivo experiments were performed using roughly 50% female and 50% male animals. During studies, animals were randomly assigned to cages.

In silico fumC conservation

We assembled a collection of serially sequenced S. aureus strains from seven published within-host evolution studies of S. aureus during cystic fibrosis (CF)^{36,37,38,39,40,41,42}. The studies were added if: they included at least two S. aureus strains per patient; sequencing reads were publicly available; minimal metadata were reported: patient identifier, specimen source, date of collection. We supplemented the published studies with unpublished data from a CF study at the Children’s Hospital of Philadelphia. Raw reads and metadata were downloaded from NCBI using the Bioproject identifier provided in the publications (Supplementary Data 2). Metadata were additionally retrieved from the publications. Quality control for reads data was performed as described in ref. ⁴³. Reads were excluded if the coverage was below 20, reads classification using Kraken2, v2.1.1⁴⁴ did not yield S. aureus or <50% reads were classified as S. aureus, or if the assembly size exceeded 3.5 million bp. Reads were assembled using Shovill, v.1.1.0, which implements the Spades assembler⁴⁵. Multi-locus sequence type and resistance genes were inferred from the assemblies using Mlst, v2.19.0 and Abricate, v1.0.1, both available at https://github.com/tseemann.

Within-host evolution analysis was carried out as described in ref. ⁴⁶: for each patient, we used Snippy, v4.6.0 (https://github.com/tseemann/snippy) to map isolate reads to the draft assembly of the earliest strain (if multiple strains were available the reference was selected randomly). The output was filtered by removing variants from reference reads and variants at positions for which the reads coverage of the reference was below 10. The amino-acid sequences of the mutated genes across all episodes were clustered using CD-HIT, v4.8.1⁴⁷ and for each cluster representative we searched for a S. aureus FPR3757⁴⁸ homolog (NCBI accession CP000255) using Blastp, v2.10.1. This generated a list of FPR3757 genes with counts of independent episodes with at least one mutation arising within the host. We then excluded synonymous substitutions and limited the list to genes involved in the tricarboxylic acid cycle (TCA) and surrounding pathways (glycolysis, gluconeogenesis, urea cycle) and calculated the relative mutation rate λ for each gene i as (mutations in gene i/length of gene i)/(mutations in all genes/length of all genes). We inferred the significance of the mutations enrichment by fitting two Poisson models: model 0 as the null hypothesis (neutral evolution), where the number of variants per gene was the product of the mean mutation rate across the genome and the gene length; model 1 estimated the number of variants per gene base on a gene-specific mutation rate while variants in the remaining genes are the product of a mean mutation rate. Model 0 and model 1 were compared using a likelihood ratio test. Scripts for the within-host evolution analysis are available at https://github.com/stefanogg.

Bacterial strains

The bacterial strains used in this study are shown in Supplementary Data 6. WT LAC and its derivative strains, ∆fumC and ∆fumC::fumC were constructed as previously described⁴⁹. The transposon insertions sucA::Tn(ermB) and sucD::Tn(ermB) were sourced from the NARSA transposon mutant library⁵⁰. The alleles were transduced into USA300_LAC using bacteriophage 80α⁵¹. The mutant strains were confirmed via PCR using sucAB and sucCD primers. All strains were grown at 37 °C on plates of Luria-Bertani lacking glucose (LB, Becton Dickinson (BD), 244610) supplemented with 1% agar (w/v, Acros Organics, 400400010). Overnight cultures and subcultures (1/100) were grown in LB broth at 37 °C with shaking. Bacterial inocula were estimated based on the optical density at 600 nm, OD_600nm, and verified by retrospective plating on LB agar plates to determine colony forming units (CFUs). S. aureus recovered from the mouse airways were selected on LB agar plates and BBL CHROMagar Staph aureus plates (BD).

Primers

The primers used in this study are listed in Supplementary Data 7. Full-length fumC was amplified from the genome of WT USA300 FPR3757 using KOD One PCR Master Mix -Blue- (TOYOBO, KMM-201) and cloned into the pET-21b(+) vector by Gibson assembly.

Growth curves

A U-bottomed, clear 96-well plate (Greiner Bio-One #650161) was prepared with LB, chemically defined media (CDM)⁵² or artificial sputum media²⁷. For the experiments that used media supplemented with itaconic acid (Sigma, I29204), L-malic acid (Sigma, M1000), sodium fumarate (Sigma, F1506), or hydrogen peroxide, H₂O₂, (Sigma, H1009), the pH of the media was corrected to 7.0 prior to filter sterilization using 0.20 μm filters (Thermo Fisher Scientific,725-2520). Each well was inoculated with 1.5 × 10⁵ bacteria. Absorbance at 600 nm was read every 10 min for 36 h on the Varioskan™ LUX multimode microplate (Thermo Fisher Scientific, VL0000D0), as the plate was maintained at 37 °C with shaking.

Measurement of biofilms

Log phase cultures were diluted to OD_600nm of 0.1 in LB broth supplemented with or without fumarate (Sigma, F1506) prior to incubation in either 96-well plates (Greiner Bio-One, 650161, 100 µl/well) or 4-well chamber slides (Sarstedt, D-51588, 750 µl/well) for 24 h and 12 h respectively. The plates were processed as follows:

1. Crystal violet staining in 96 well-plates: The OD_600nm was measured prior to crystal violet staining. The plates were fixed with 100% methanol, stained with 1% crystal violet (w/v, Sigma, C6158), washed and dried. Subsequently, 33% acetic acid (v/v, Acros Organics, 222140010) was added to solubilize the dye. The OD_540nm was measured and normalized to the bacterial growth at OD_600nm.

2. Wheat germ agglutinin-Alexa Fluor 555 staining and confocal microscopy: Wheat germ agglutinin (WGA) coupled to Alexa Fluor 555 (5 µg/ml, Thermofisher, W32464) was added into each well and the chamber slides were covered with foil. Each chamber slide was placed on a shaker (horizontal orbit, 60 rpm) in a 37 °C room for 5 min, before being kept stationary in a 37 °C room for 12 h. Confocal imaging of viable biofilms was performed using a Zeiss LSM 980 Airyscan with a 37 °C cage incubator. 16-bit images of WGA-positive cells (1.5x crop area, 1024 × 1024 px frame size) were captured using a 40X oil-immersion objective with the 488 nm laser (670 V gain) set at 1% intensity. Z-stack slices were collected at 1 µm intervals. The z-depth of high-density biofilm layers were calculated as the number of slices (1 µm apart) where extracellular spaces occupied no more than 20% of the image. ImageJ 1.54 f. (National Institutes of Health, Bethesda, MD, USA) was used to calculate the % of extracellular space in an image using thresholding.

Isolation of RNA from bacterial cultures

Bacterial cultures grown in LB with and without 10 mM fumarate were standardized to an OD_600nm of 1.0 prior to centrifugation. Bacterial pellets were incubated in lysis buffer (50 μM Tris-EDTA pH 7.5, 8 U ml⁻¹ mutanolysin (Sigma-Aldrich), 0.018 mg ml⁻¹ lysostaphin (Sigma-Aldrich), 0.05 mg ml⁻¹ lysozyme (Sigma-Aldrich)) at 37 °C for 45 min and TRK lysis buffer (Omega Bio-tek) was added. After 10 min at room temperature, 70% ethanol was added, and samples were transferred to E.Z.N.A. RNA isolation columns (Omega Bio-tek). RNA was isolated following the manufacturer’s instructions and treated with DNase using the DNA-free DNA removal kit (Invitrogen).

Complementary DNA synthesis and qRT-PCR

Multiscribe reverse transcriptase (Applied Biosystems) was used to generate cDNA for qRT–PCRs with Power SYBR Green PCR Mastermix (Applied Biosystems). qRT-PCR was performed using primers for hla, lukS, esxA, per, katA, sod, trx, aphF, msrA1, msrB, capA, icaB and 16S (Supplementary Data 7) and a StepOne Plus thermal cycler (Applied Biosystems). The data were analyzed using the ΔΔC_T method.

RNA-sequencing

WT LAC was grown in LB with or without 100 mM fumarate to late exponential phase. Bacterial pellets were incubated in a cell wall lysis mixture (described above) at 37 °C for 45 min. TRK lysis buffer (Omega Bio-tek, R6834-02) and 70% ethanol were added, and samples were transferred to E.Z.N.A. RNA isolation columns (Omega Bio-tek, R6834-02). RNA was isolated following the manufacturer’s instructions and treated with DNase using the DNAfree DNA removal kit (Fisher Scientific, AM1906). The RNA was precipitated with 0.1 volume 3 M sodium acetate (Thermo Fisher, S209) and 3 volumes of 100% ethanol, recovered by centrifugation, and washed with ice cold 70% ethanol. A ribosomal RNA-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 calls were converted to fatsq files using Bcl2fastqs. Filtered reads were aligned to the LAC_FPR3757 reference genome using STAR-Aligner v2.7.3a. The mapped reads were annotated for read groups and marked for duplicates using the Picard tools v2.22.3. The raw counts were quantified using Subreads:FeatureCounts v1.6.3 and processed for differential gene expression using DEseq2 in R v3.5.3.

In silico protein structure simulation and charge prediction

The predicted structure of S. aureus FumC was constructed using the homology modeling tool from the Schrödinger software package (Schrödinger Release 2022-4: Prime; Schrödinger, Inc: New York, NY, 2022)⁵³. We used the human fumarate hydratase (PDB: 5UPP) with 57% identity as template. The structure of E. coli FumC, which shares a higher identity (60%), could have been utilized, but would have necessitated the de novo construction of longer loops; one loop consisting of 3 amino acids and another loop consisting of 8 amino acids instead of two loops of 3 amino acids and one loop of 1 amino acid. The obtained structure was replicated into four copies, which were superimposed on the four chains of the template tetramer structure and merged. Protonation states of the predicted tetramer were assigned using PROPKA and the structure subjected to a constrained minimization using the OPLS4 force field (Schrödinger Release 2022-4: Protein Preparation Wizard, Schrödinger, LLC, New York, NY, 2022)⁵⁴.

Synthesis of FA-alkyne probe

Monomethyl fumarate (1 g, 1 eq, 7.69 mmol) was dissolved in acetonitrile and cooled to 0 °C. EDCl·HCl (1.6 g, 1.1 eq, 8.46 mmol), HOBt (1.1 g, 1.1 eq, 8.46 mmol) and Et₃N (1.39 mL, 1.3 eq, 2.29 mmol) were added and then stirred for 40 min at 0 °C. Subsequently, propargylamine (0.6 g, 1.3 eq, 10 mmol) was added. The solution was allowed to warm up to room temperature and stirred overnight. The solvent was removed in vacuo, the residue was redissolved in ethylacetate and washed with 1 M HCl, saturated sodium bicarbonate solution and brine. The organic layer was dried over anhydrous sodium sulfate, the resulting crude was dried, and the solvent was removed in vacuo. The resulting crude was purified by flash column chromatography (SiO₂, DCM:MeOH=40:1) to afford the pure compound 1 (820.8 mg, 63.9%) as white solid (Supplementary Fig. 2A). Lithium hydroxide (330.0 mg, 7.86 mmol) was added to a stirring solution of compound 1 (820.8 mg, 4.91 mmol) in a 1:1 mixture of water and tetrahydrofuran (Supplementary Fig. 2B). The reaction mixture was stirred at room temperature and monitored by TLC. After 1 h, the reaction mixture was acidified with 1 M HCl and the solvent was removed in vacuo. The crude product was redissolved in ethylacetate and filtered. The solution was evaporated in vacuo and the crude product was purified by flash column chromatography (SiO₂, DCM:MeOH=20:1 to 10:1) to obtain the pure FAyne (FA-alkyne, 622.1 mg, 82.8%) as white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.96 (t, J = 5.5 Hz, 1H), 6.94 (d, J = 15.5 Hz, 1H), 6.58 (d, J = 15.5 Hz, 1H), 4.01 (dd, J = 5.5, 2.6 Hz, 2H), 3.21 (t, J = 2.5 Hz, 1H) (Supplementary Fig. 2C). ¹³C NMR (101 MHz, DMSO-d₆) δ 166.28, 163.35, 135.86, 131.37, 81.77, 72.78, 28.64 (Supplementary Fig. 2D).

Competitive ABPP workflow for S-succinated cysteines in S. aureus

S. aureus USA300 FPR3757 was cultured overnight in tryptic soy broth (TSB), washed with PBS and concentrated to OD_600nm of 40. For the competition group, 800 μl of bacterial suspension was incubated with 80 μl of fumarate (100 mM stock was dissolved in NaOH and pH was adjusted to neutral) on the ThermoMixer (950 rpm, 30 min, 37 °C). For the control group, fumarate was replaced with the same volume of PBS. After competition, both groups were incubated with 8 μl of 1 M FA-alkyne probe on the ThermoMixer (950 rpm, 37 °C, 1 h). The bacterial suspension was then centrifuged (12,000 rpm, 5 min, 4 °C), the supernatant was decanted, and the pellets were washed once with 1 ml of pre-chilled PBS. Washed pellets were resuspended with 200 μl of 0.1% (v/v) Triton X-100 (Amresco, 0694-1 L) in PBS (hereafter, 0.1% PBST). Bacterial suspension was incubated on the ThermoMixer (1200 rpm, 15 min, 37 °C) after the addition of 3 μl of lysostaphin (5 U/μl, Coolaber, CL6941-1mg). Then, the lysate was supplied with 6 μl of 10% SDS (Thermo Fisher Scientific, AM9822) and sonicated (35% intensity). The lysate was centrifuged (20,000 g, 10 min, RT) to remove the debris and the supernatant was transferred into a new 1.5 ml tube (30 μl of supernatant was also aliquoted for the gel-based ABPP assay). Click reaction was carried out with 106 μl of Click reagent mix (60 μl of 50 mM TBTA ligand, 20 μl of 50 mM CuSO₄, 20 μl of freshly prepared 50 mM TCEP and 6 μl of 20 mM acid-cleavable azide-biotin tag (Confluore, BBBD-19-25 mg) and incubated on the ThermoMixer (1200 rpm, 1 h, 29 °C). The samples were then subjected to streptavidin enrichment and dual enzyme digestion (trypsin and Glu-C) steps before mass spectrometry analysis as previously described⁵⁵.

After the dual enzyme digestion, the peptides were further labeled with a dimethylation reagent (“light” for the control group (i.e., FA probe + vehicle) and “heavy” for the fumarate competition group (i.e., FA probe + fumarate))⁵⁶. Streptavidin beads were washed three times with MS-grade water to thoroughly remove dimethylation reagents. The labeled peptides were released from streptavidin beads by incubating with 200 μl of 2% formic acid on the ThermoMixer (1200 rpm, 25 °C, 90 min). Peptides from the control group and fumarate competition group were combined and dried in a SpeedVac (30 °C). They were then analyzed by HPLC-MS/MS and subjected to quantitative proteomic analysis. A competitive light-to-heavy ratio for each target was calculated. A ratio greater than 1 (or greater than 0 after log2 transformation) suggests that the cysteine site (or protein) undergoes succination by fumarate. The corresponding competition ratios for each cysteine site (or protein) were averaged across three biological replicates, and the q-value (false discovery rate/FDR) was calculated.

Protein purification

Plasmid pET21b-FumC-His₆ was transformed into BL21(DE3). Protein expression was performed overnight at 16 °C in the presence of 0.2 mM IPTG (VWR, 0487-100 G). Bacterial pellets were resuspended in suspension buffer (50 mM Tris-HCl, pH 8.0 (Thermo Fisher Scientific, 15568-025), 150 mM NaCl) and disrupted with sonication. After the lysates were clarified via centrifugation (12,000 rpm, 30 min, 4 °C), the supernatant was loaded onto 5 ml HisSep Ni-NTA 6FF column (YEASEN, 20504ES25) and washed with W1 (50 mM Tris, pH 8.0, 150 mM NaCl, 20 mM imidazole, pH 7.4) and W2 (50 mM Tris, pH 8.0, 150 mM NaCl, 40 mM imidazole, pH 7.4). Protein was eluted with elution buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 300 mM imidazole, pH 7.4) and the eluate was centrifuged (4000 g, 4 °C, swing-out) using Amicon® Ultra-15 Centrifugal Filter Unit to remove imidazole and concentrate protein. The concentrated protein was mixed with 20% glycerol and subjected to BCA assay to measure concentration (Thermo Fisher Scientific, 23225). Finally, the protein was supplied with 1 mM DTT, aliquoted, flash frozen in liquid nitrogen, and stored at −80 °C.

FumC activity

Recombinant FumC was diluted with Tris-HCl (20 mM, pH 8.0) to 1 mg/ml, and incubated with either 1 mM itaconate (pH was adjusted with NaOH to 7.4) and 1 mM DTT or PBS and 1 mM DTT as the negative control (2 h, 37 °C). For the enzymatic assay, the reaction was conducted in a 1.5 ml polystyrene cuvette (BIOFIL, CUV010015). The reaction was initiated by mixing 100 μg of post-incubated FumC and the reaction mixture containing 20 mM Tris-HCl (pH 8.0) and 15 mM fumarate (Sigma-Aldrich, 47910) in a total volume of 1 ml at 25 °C for 30 min. The maximal absorbance of fumarate was determined using a spectrophotometer (IMPLEN, P330) in a 1.5 ml cuvette. Absorbance was read at 289 nm. This enzymatic assay was performed on three biological replicates.

AcnA/CitB activity

AcnA assays were conducted as previously described with slight modifications^57,58. Strains were cultured overnight in TSB before diluting them in fresh TSB to an OD_600nm of 0.1. The cultures were diluted in 0.5 ml or 4 ml of TSB in 10 ml culture tubes. The cells were cultured for 8 h, before they were harvested by centrifugation, and the cell pellets were stored at −80 °C. The cells were thawed anaerobically, resuspended with 200 μl of AcnA buffer (50 mM Tris, 150 mM NaCl, pH 7.4), and lysed by the addition of 4 μg lysostaphin and 8 μg DNase. The cells were incubated at 37 °C until full lysis was observed (~1 h). The cell debris was removed by centrifugation, and AcnA activity was assessed by monitoring the conversion of isocitrate to cis-aconitate spectrophotometrically.

Metabolite phenotype microarray (Biolog)

For Carbon Source Phenotype Microarray™ (Biolog) assays, a stock solution of 2 × 10⁷ bacteria/ml was prepared in 1X IF-Oa buffer (Biolog, 72268) supplemented with 1X Redox Dye Mix A (Biolog, 74221). 100 μl of this stock solution (delivering 2 × 10⁶ bacteria) was added to each well of a PM1 Microplate™ (Biolog, 12111) and the plate was incubated at 37 °C overnight with shaking. Absorbance was read at 590 nm on the SpectraMax M2 plate reader (Molecular Devices).

Bacterial extracellular flux analyses

The XFe24 sensor cartridge (Agilent #102340-100) was calibrated as per the manufacturer’s instructions overnight at 37 °C without CO₂. 500 μl of XF base medium (Agilent #102353-100) was added to each well of a Seahorse XF24 well plate (Agilent #102340-100) and inoculated with 3 × 10⁷ bacteria for a 3 h incubation at 37 °C. The oxygen consumption rate (OCR, indicative of oxidative phosphorylation) and extracellular acidification rate (ECAR, indicative of glycolysis) were again measured on a Seahorse XFe24 Analyzer (Agilent Technologies) using Seahorse Wave Desktop v2.6.0. Glucose (Sigma #G7021) was added at a final concentration of 10 mM followed by the addition of fumarate (Sigma, F1506) to a final concentration of 10 mM.

¹³C₄-fumaric acid labeling and stable isotope tracing

WT LAC and the ΔfumC mutant were grown overnight in LB, then inoculated (1/100) into fresh LB supplemented with ¹³C₄-fumaric acid (Sigma, 606014, pH 7.0), grown at 37 °C to late exponential phase and standardized to an OD_600nm of 14. For metabolite extraction, each culture was diluted with 3 volumes of PBS and centrifuged at 2000 × g for 10 min at 1 °C. The supernatant was discarded, and the pellet was washed with PBS. The pellet (30 μl in volume) was 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 Fisher Scientific) coupled to a Vanquish UPLC system (Thermo Fisher 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. The data in Fig. 4 are presented with correction for natural isotope abundance.

Isolation of murine BMDMs and infection

Murine BMDMs were differentiated as described previously⁹. The femurs and tibias were surgically removed from mice. The bone exterior was sterilized with 70% ethanol and the bone marrow was recovered by flushing with phosphate-buffered saline (PBS, Corning, 20-031). The cell suspension was centrifuged for 6 min at 500 × g and resuspended in ACK lysis buffer (Thermo Fisher Scientific, A1049201) to remove the red blood cells. The lysis solution was quenched with PBS, and the cells were centrifuged again and resuspended in DMEM medium (Corning, 10-013-CV) containing 10% heat-inactivated fetal bovine serum (FBS v/v, Sigma, F4135) and 1% penicillin/streptomycin (P/S, v/v, Corning, 30-002-CI)) supplemented with 20 ng/ml rM-CSF (PeproTech #315-02). The rM-CSF-supplemented media was replenished 3 days after isolation, and the BMDMs were mechanically detached on day 7, centrifuged, resuspended in DMEM medium containing 10% FBS (Sigma), and counted using trypan blue stain (Invitrogen, T10282). The cells were seeded at 1 × 10⁶ cells/ml in a 24 well plate (Agilent, 102340-100) and incubated at 37 °C with 5% CO₂ overnight. The cells were infected at a multiplicity of infection (MOI) of 10 for 30 min and 10 μg ml⁻¹ lysostaphin (Sigma) was then added until the desired time point. The cells were washed, detached using TrypLE Express (Life Technologies), serially diluted, and plated on LB agar. Cell viability was determined by counting live cells using trypan blue (Life Technologies).

Mouse pneumonia model

Mice were infected intranasally with 1 × 10⁷ CFUs of S. aureus in 50 μl PBS. PBS alone was used as a control. The mice were sacrificed at 18 h post infection and the BALF and lung were collected. The lung was homogenized through 40 μm cell strainers (Falcon, 352340). Aliquots of the BALF and lung homogenates were serially diluted and plated on LB agar plates and BBL CHROMagar Staph aureus plates (BD) 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.

Flow cytometry of mouse BALF and lung cells

For the identification of immune cell populations, mouse BALF and lung cells were stained in the presence of counting beads (15.45 μm DragonGreen, Bangs Laboratories Inc., FS07F) with LIVE/DEAD stain (Invitrogen, L23105A) and an antibody mixture of anti-CD45-AF700 (BioLegend, 103127, clone 30-F11), anti-CD11b-AF594 (BioLegend, 101254, clone M1/70), anti-CD11c-Bv605 (BioLegend, 117334, clone N418), anti-SiglecF-AF647 (BD, 562680, clone E50-2440 RUO), anti-Ly6C-Bv421 (BioLegend, 128032, clone HK1.4), and anti-Ly6G-PerCP-Cy5.5 (BioLegend, 127616, clone 1A8), each at a concentration of 1:200 in PBS, for 1 h at 4 °C. After washing, the cells were stored in 2% paraformaldehyde (PFA, Electron Microscopy Sciences, 15714-S) until analysis on the BD LSRII (BD Biosciences) using FACSDiva v9. Flow cytometry was analyzed with FlowJo v10. Mouse BAL and lung cells were identified as follows:

alveolar macrophages: CD45⁺CD11b^+/−SiglecF⁺CD11c⁺;

monocytes: CD45⁺CD11b⁺SiglecF⁻MHCII⁻CD11c⁻Ly6G⁻Ly6C^+/−;

neutrophils: CD45⁺CD11b⁺SiglecF⁻ MHCII⁻CD11c⁻Ly6G⁺Ly6C^+/−.

Cytokine analysis

Cytokine concentrations in mouse BALF supernatants were quantified by Eve Technologies (Calgary, Canada) using a bead-based multiplexing technology.

Semi-targeted metabolomics

Metabolites in the BALF were identified and quantified by high-resolution liquid chromatography mass spectrometry (LC-MS) at the Calgary Metabolomics Research Facility (Calgary, Canada). The metabolites were extracted in a 50% methanol (Alpha Aesar #22909):water (v/v) solution. Sample runs were performed 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 achieved 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 consists of 20 mM ammonium formate pH 3 in mass spectrometry grade water and solvent B consists of mass spectrometry grade acetonitrile with 0.1% formic acid (v/v). The following gradient was used: 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 sample injection volume of 2 μl was used. The mass spectrometer was run 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 signals (±10 ppm) and chromatographic retention times to those observed from commercial metabolite standards (Sigma-Aldrich). The data were analyzed using E-Maven and are listed in Supplementary Data 8-9.

Isolation of RNA from mouse lungs

Total RNA was isolated from excised mouse lungs using TRIzol reagent (Thermo Fisher Scientific, 15596026) according to the manufacturer’s instructions. Residual DNA was then degraded using the TURBO DNA-free Kit (Thermo Fisher Scientific, AM1907) prior to reverse transcription, which was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4374966). Real-time qRT-PCR was performed on the cDNA generated in the previous step using the primers listed in Supplementary Data 7. The data were analyzed using the ΔΔC_T method.

Quantification and statistical analysis

We modeled the number of independent experiments required to reach significance between groups using the computer program JMP. This simulation was based on experimental design, preliminary data and past experience. Analyses were performed assuming a 20% standard deviation and equivalent variances within groups. Significance <0.05 with power 0.8 was used. Experiments in this study were not performed in a blinded fashion. All analyses and graphs were performed using the GraphPad Prism 7a and 8 software. Values in graphs are shown as average ±SEM and data were assumed to follow a normal distribution. For comparison between average values for more than two groups, we performed one-way ANOVA with a multiple posteriori comparison. When studying two or more groups along time, data was analyzed using two-way ANOVA with a multiple posteriori comparison. Differences between two groups were analyzed using a student’s t test for normally distributed data or Mann–Whitney or Kruskal–Wallis test otherwise. Differences were considered significant when a P value under 0.05 (p < 0.05) was obtained. Statistical details of experiments are indicated in each figure legend. No data points were excluded.

Data availability

All data discussed in this study are presented in the published article, including the supplementary files and source data. RNA-Seq data are deposited under accession number GSE276380. Source data are provided with this paper.

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Acknowledgements

We thank Drs. Xiaoyuan Yang, Mengmeng Zhu, and Guoan Zhang from the Proteomics and Metabolomics Core Facility at Weill Cornell Medicine for the staple isotope tracing analysis. We also thank Dr. Min Li (Shanghai Jiao Tong University School of Medicine, China) for sharing S. aureus strain FPR3757, Dr. Clemente J. Britto (Yale University) for providing the sputum samples, which were initially used in our previous study⁹, Drs. Paul Fey and Itidal Reslane (University of Nebraska) and Dr. Barbara Kahl (University of Münster, Germany) for insightful discussions. This study is supported by NIH grant K99/R00 HL157550 to T.W.F.L., NIH grant R01 HL170129 to A.P. and NIAID 1R01AI139100-01, NSF 1750624 and USDA MRF project NE − 1028 awards to JMB. This study is also supported by NIH grant S10RR027050 to the Columbia Center for Translational Immunology (CCTI) Flow Cytometry Core. B.P.H., T.P.S., and A.H. are supported by NHMRC Investigator grants (Australia) APP1196103, GNT1194325, and APP2018880. C.W. is supported by the National Key R&D Program of China (2022YFA1304700). I.A.L. is supported by an Alberta Innovates Translational Health Chair. The Calgary Metabolomics Research Facility (CMRF) is supported by the International Microbiome Centre and the Canada Foundation for Innovation (CFI-JELF 34986). Figure 1a includes the lung icon (“Lungs” by Hycan) and human icon (“Person” by Teewara Soontorn), both from the Noun Project. Royalty-free commercial licenses have been provided. Figure 2f was created in BioRender. Wong, T. (2025) https://BioRender.com/rkq0y88.

Author information

These authors contributed equally: Ying-Tsun Chen, Zihua Liu, Dario Fucich.

Authors and Affiliations

Department of Pediatrics, Columbia University, New York, NY, USA
Ying-Tsun Chen, Dario Fucich, Shivang S. Shah, Gaurav K. Lohia, Ayesha Z. Beg, Sebastian A. Riquelme, Alice Prince & Tania Wong Fok Lung
Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China
Zihua Liu & Chu Wang
Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
Stefano G. Giulieri, Ian R. Monk, Abderrahman Hachani, Timothy P. Stinear & Benjamin P. Howden
Centre for Pathogen Genomics, The University of Melbourne, Melbourne, Australia
Stefano G. Giulieri, Ian R. Monk, Abderrahman Hachani, Timothy P. Stinear & Benjamin P. Howden
Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China
Zhe Liu & Chu Wang
Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, Newark, NJ, USA
Ridhima Wadhwa & Tania Wong Fok Lung
Center for Immunity and Inflammation, Rutgers New Jersey Medical School, Newark, NJ, USA
Ridhima Wadhwa & Tania Wong Fok Lung
Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ, USA
Gustavo Rios, Nupur Tyagi & Jeffrey M. Boyd
Department of Medicinal Chemistry, Uppsala University, Uppsala, Sweden
Henning Henschel
Infection Program and the Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia
Françios A. B. Olivier & Ana Traven
Department of Genetics and Genomic Sciences, Mt. Sinai Icahn School of Medicine, New York, NY, USA
Shwetha H. Sridhar & Robert Sebra
Department of Biological Sciences, University of Calgary, Calgary, Canada
Marija Drikic & Ian A. Lewis
Division of Infectious Disease, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA
Colleen Bianco & Paul J. Planet
Department of Pediatrics and Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Paul J. Planet
Panacent Bio, New York, NY, USA
Robert Sebra
Microbiological Diagnostic Unit Public Health Laboratory, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Australia
Benjamin P. Howden

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Ying-Tsun Chen
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Dario Fucich
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Contributions

Y-T.C., Z.L., and D.F. contributed equally, performing experiments, analyzing data, and editing the manuscript. Z.L. synthesized the fumarate analogue probe. Z.L. and C.W. specifically performed and analyzed the chemoproteomic experiments with the fumarate analogue probe. S.G.G. and A.H. performed in silico analyses on the conservation of fumC in S. aureus isolates. H.H. performed the homology modeling. C.B. and P.J.P. provided whole genome sequences of a collection of S. aureus isolates from cystic fibrosis patients. G.R. and J.M.B. measured S. aureus aconitase activity. N.T. constructed the ΔsucA and ΔsucD mutants in the USA300 LAC background, and R.W. performed in vivo experiments and analyzed data. I.R.M. constructed the bacterial ΔfumC mutant and complemented strain. G.L. and A.Z.B. isolated murine bone marrow-derived macrophages. S.S.S. and S.H.S. analyzed the bulk RNA-seq data. M.D. performed the LC-MS, and S.A.R. provided the data on the relative quantification of fumarate and itaconate in the sputum of patients with cystic fibrosis versus healthy controls. F.A.B.O. performed confocal imaging on biofilms. I.A.L., R.S., T.P.S., B.P.H., J.M.B., A.H., I.R.M., A.T., S.A.R., and C.W. contributed to the experimental design and discussions. A.P. co-wrote and edited the manuscript. T.W.F.L. proposed the central hypothesis, performed experiments, analyzed/interpreted the data, and wrote/edited the manuscript.

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Correspondence to Tania Wong Fok Lung.

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Robert Sebra is a consultant and equity holder for GeneDx and a founder and equity holder of Panacent Bio. All other authors declare no competing interests.

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Chen, YT., Liu, Z., Fucich, D. et al. Regulation of airway fumarate by host and pathogen promotes Staphylococcus aureus pneumonia. Nat Commun 16, 7050 (2025). https://doi.org/10.1038/s41467-025-62453-y

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Received: 15 August 2024
Accepted: 21 July 2025
Published: 01 August 2025
Version of record: 01 August 2025
DOI: https://doi.org/10.1038/s41467-025-62453-y