Decision letter: Listeria monocytogenes requires cellular respiration for NAD+ regeneration and pathogenesis

Abstract

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Cellular respiration is essential for multiple bacterial pathogens and a validated antibiotic target. In addition to driving oxidative phosphorylation, bacterial respiration has a variety of ancillary functions that obscure its contribution to pathogenesis. We find here that the intracellular pathogen Listeria monocytogenes encodes two respiratory pathways which are partially functionally redundant and indispensable for pathogenesis. Loss of respiration decreased NAD+ regeneration, but this could be specifically reversed by heterologous expression of a water-forming NADH oxidase (NOX). NOX expression fully rescued intracellular growth defects and increased L. monocytogenes loads >1000-fold in a mouse infection model. Consistent with NAD+ regeneration maintaining L. monocytogenes viability and enabling immune evasion, a respiration-deficient strain exhibited elevated bacteriolysis within the host cytosol and NOX expression rescued this phenotype. These studies show that NAD+ regeneration represents a major role of L. monocytogenes respiration and highlight the nuanced relationship between bacterial metabolism, physiology, and pathogenesis. Editor's evaluation In this study, authors report that a major role of respiration in Listeria monocytogenes pathogenicity, is to control redox balance (NAD+ regeneration) rather than generation of proton motive force. This is a new way of perceiving respiration that should be of interest to the microbiology community and broader readership. https://doi.org/10.7554/eLife.75424.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Cellular respiration is one of the main ways organisms make energy. It works by linking the oxidation of an electron donor (like sugar) to the reduction of an electron acceptor (like oxygen). Electrons pass between the two molecules along what is known as an ‘electron transport chain’. This process generates a force that powers the production of adenosine triphosphate (ATP), a molecule that cells use to store energy. Respiration is a common way for cells to replenish their energy stores, but it is not the only way. A simpler process that does not require a separate electron acceptor or an electron transport chain is called fermentation. Many bacteria have the capacity to perform both respiration and fermentation and do so in a context-dependent manner. Research has shown that respiration can contribute to bacterial diseases, like tuberculosis and listeriosis (a disease caused by the foodborne pathogen Listeria monocytogenes). Indeed, some antibiotics even target bacterial respiration. Despite being often discussed in the context of generating ATP, respiration is also important for many other cellular processes, including maintaining the balance of reduced and oxidized nicotinamide adenine dinucleotide (NAD) cofactors. Because of these multiple functions, the exact role respiration plays in disease is unknown. To find out more, Rivera-Lugo, Deng et al. developed strains of the bacterial pathogen Listeria monocytogenes that lacked some of the genes used in respiration. The resulting bacteria were still able to produce energy, but they became much worse at infecting mammalian cells. The use of a genetic tool that restored the balance of reduced and oxidized NAD cofactors revived the ability of respiration-deficient L. monocytogenes to infect mammalian cells, indicating that this balance is what the bacterium requires to infect. Research into respiration tends to focus on its role in generating ATP. But these results show that for some bacteria, this might not be the most important part of the process. Understanding the other roles of respiration could change the way that researchers develop antibacterial drugs in the future. This in turn could help with the growing problem of antibiotic resistance. Introduction Distinct metabolic strategies allow microbes to extract energy from diverse surroundings and colonize nearly every part of the earth. Microbial energy metabolisms vary greatly but can be generally categorized as possessing fermentative or respiratory properties. Cellular respiration is classically described by a multistep process that initiates with the enzymatic oxidation of organic matter and the accompanying reduction of NAD+ (nicotinamide adenine dinucleotide) to NADH. Respiration of fermentable sugars typically starts with glycolysis, which generates pyruvate and NADH. Pyruvate then enters the tricarboxylic acid (TCA) cycle, where its oxidation to carbon dioxide is coupled to the production of additional NADH. NADH generated by glycolysis and the TCA cycle is then oxidized by NADH dehydrogenase to regenerate NAD+ and the resulting electrons are transferred via an electron transport chain to a terminal electron acceptor. While mammals strictly use oxygen as a respiratory electron acceptor, microbes reside in diverse oxygen-limited environments and have varying and diverse capabilities to use disparate non-oxygen respiratory electron acceptors. Whatever the electron acceptor, electron transfer in the electron transport chain is often coupled to proton pumping across the bacterial inner membrane. This generates a proton gradient or proton motive force, which powers a variety of processes, including ATP production by ATP synthase. Respiratory pathways are important for several aspects of bacterial physiology. Respiration’s role in establishing the proton motive force allows bacteria to generate ATP from non-fermentable energy sources (which are not amenable to ATP production by substrate-level phosphorylation) and increases ATP yields from fermentable energy sources. In addition to these roles in ATP production, respiratory electron transport chains are directly involved in many other aspects of bacterial physiology, including the regulation of cytosolic pH, transmembrane solute transport, ferredoxin-dependent metabolisms, protein secretion, protein folding, disulfide formation, and flagellar motility (Bader et al., 1999; Driessen et al., 2000; Driessen and Nouwen, 2008; Manson et al., 1977; Slonczewski et al., 2009; Driessen et al., 2000; Tremblay et al., 2013; Wilharm et al., 2004). Beyond the proton motive force, respiration functions to regenerate NAD+, which is essential for enabling the continued function of glycolysis and other metabolic processes. By obviating fermentative mechanisms of NAD+ regeneration, respiration increases metabolic flexibility, which, among other metabolic consequences, can enhance ATP production by substrate-level phosphorylation (Hunt et al., 2010). Bacterial pathogens reside within a host where they must employ fermentative or respiratory metabolisms to power growth. Pathogen respiratory processes have been linked to host-pathogen conflict in several contexts. Phagocytic cells target bacteria by producing reactive nitrogen species that inhibit aerobic respiration (Richardson et al., 2008). Aggregatibacter actinomycetemcomitans, Salmonella enterica, Streptococcus agalactiae, and Staphylococcus aureus mutants with impaired aerobic respiration are attenuated in murine models of systemic disease (Craig et al., 2013; Hammer et al., 2013; Jones-Carson et al., 2016; Lencina et al., 2018; Lewin et al., 2019; Rivera-Chávez et al., 2016). Aerobic respiration is vital for Mycobacterium tuberculosis pathogenesis and persister cell survival, making respiratory systems validated anti-tuberculosis drug targets (Cook et al., 2014; Hasenoehrl et al., 2020). Respiratory processes that use oxygen, tetrathionate, and nitrate as electron acceptors are important for the growth of S. enterica and Escherichia coli in the mammalian intestinal lumen (Rivera-Chávez et al., 2016; Winter et al., 2010; Winter et al., 2013). While several studies have linked respiration in bacterial pathogens to the use of specific electron donors (i.e. non-fermentable energy sources) within the intestinal lumen, the particular respiratory functions important for systemic bacterial infections remain largely unexplained (Ali et al., 2014; Faber et al., 2017; Gillis et al., 2018; Spiga et al., 2017; Thiennimitr et al., 2011). Listeria monocytogenes is a human pathogen that, after being ingested on contaminated food, can gain access to the host cell cytosol and use actin-based motility to spread from cell to cell (Freitag et al., 2009). L. monocytogenes has two respiratory-like electron transport chains. One electron transport chain is dedicated to aerobic respiration and uses a menaquinone intermediate and QoxAB (aa3) or CydAB (bd) cytochrome oxidases for terminal electron transfer to O2 (Figure 1A; Corbett et al., 2017). We recently identified a second flavin-based electron transport chain that transfers electrons to extracytosolic acceptors (including ferric iron and fumarate) via a putative demethylmenaquinone intermediate and can promote growth in anaerobic conditions (Figure 1A; Light et al., 2018; Light et al., 2019; Zeng et al., 2021). Final electron transfer steps in this flavin-based electron transport mechanism are catalyzed by PplA and FrdA, which are post-translationally linked to an essential cofactor by the flavin mononucleotide transferase (FmnB) (Light et al., 2018; Méheust et al., 2021). Figure 1 with 1 supplement see all Download asset Open asset Respiration impacts L. monocytogenes growth and fermentative output. (A) Proposed respiratory electron transport chains in L. monocytogenes. Different NADH dehydrogenases likely transfer electrons to distinct but presently unidentified quinones (Qa and Qb). FmnB catalyzes assembly of essential components of the electron transport chain, PplA and FrdA, that can transfer electrons to ferric iron and fumarate, respectively. Other proteins involved in the terminal electron transfer steps are noted. (B) Optical density of L. monocytogenes strains aerobically grown in nutrient-rich media, with the anaerobically grown wildtype strain provided for context. The means and standard deviations from three independent experiments are shown. (C) Fermentation products of L. monocytogenes strains grown to stationary phase in nutrient-rich media under aerobic and anaerobic conditions. Error bars show standard deviations. Results from three independent experiments are shown. (D) Proposed pathways for L. monocytogenes sugar metabolism. The predicted number of NADH generated (+) or consumed (−) in each step is indicated. PplA, peptide pheromone-encoding lipoprotein A; FrdA, fumarate reductase; ΔQC, ΔqoxA/ΔcydAB; ΔQC/fmnB, ΔqoxA/ΔcydAB/fmnB::tn; GLC, glucose; Ack, acetate kinase; Pdh, pyruvate dehydrogenase; Pfl, pyruvate formate-lyase; DMK, demethylmenaquinone. Figure 1—source data 1 Source data for Figure 1B. https://cdn.elifesciences.org/articles/75424/elife-75424-fig1-data1-v2.xlsx Download elife-75424-fig1-data1-v2.xlsx Figure 1—source data 2 Source data for Figure 1C. https://cdn.elifesciences.org/articles/75424/elife-75424-fig1-data2-v2.xlsx Download elife-75424-fig1-data2-v2.xlsx L. monocytogenes resembles fermentative microbes in lacking a functional TCA cycle (Trivett and Meyer, 1971). Despite thus being unable to completely oxidize sugar substrates, previous studies have shown that aerobic respiration is important for the systemic spread of L. monocytogenes (Chen et al., 2017; Corbett et al., 2017; Stritzker et al., 2004). Microbes that similarly contain a respiratory electron transport chain but lack a TCA cycle are considered to employ a respiro-fermentative metabolism (Pedersen et al., 2012). Respiro-fermentative metabolisms tune the cell’s fermentative output and often manifest with the respiratory regeneration of NAD+ enabling a shift from the production of reduced (e.g. lactic acid and ethanol) to oxidized (e.g. acetic acid) fermentation products. In respiro-fermentative lactic acid bacteria closely related to L. monocytogenes, cellular respiration results in a modest growth enhancement, but is generally dispensable (Duwat et al., 2001; Pedersen et al., 2012). The studies presented here sought to address the role of respiration in L. monocytogenes pathogenesis. Our results confirm that L. monocytogenes exhibits a respiro-fermentative metabolism and show that its two respiratory systems are partially functionally redundant under aerobic conditions. We find that the respiration-deficient L. monocytogenes strains exhibit severely attenuated virulence and lyse within the cytosol of infected cells. Finally, we selectively abrogate the effect of diminished NAD+ regeneration in respiration-deficient L. monocytogenes strains by heterologous expression of a water-forming NADH oxidase (NOX) and find that this restores virulence. These results thus elucidate the basis of L. monocytogenes cellular respiration and demonstrate that NAD+ regeneration represents a key function of this activity in L. monocytogenes pathogenesis. Results L. monocytogenes’ electron transport chains have distinct roles in aerobic and anaerobic growth We selected previously characterized ΔqoxA/ΔcydAB (ΔQC) and ΔfmnB L. monocytogenes strains to study the role of aerobic respiration and extracellular electron transfer, respectively (Chen et al., 2017; Light et al., 2018). In addition, we generated a ΔqoxA/ΔcydAB/fmnB::tn (ΔQC/fmnB) L. monocytogenes strain to test for functional redundancies of aerobic respiration and extracellular electron transfer. Initial studies measured the growth of these strains on nutritionally rich brain heart infusion (BHI) media in the presence/absence of electron acceptors. Compared to anaerobic conditions that lacked an electron acceptor, we found that aeration led to a relatively modest increase in growth of wildtype and ΔfmnB strains (Figure 1B and Figure 1—figure supplement 1a). This growth enhancement could be attributed to aerobic respiration, as aerobic growth of the ΔQC strain resembled anaerobically cultured strains (Figure 1B and Figure 1—figure supplement 1a). Similarly, in anaerobic conditions, inclusion of the extracellular electron acceptors, ferric iron and fumarate, resulted in a small growth enhancement of wildtype L. monocytogenes (Figure 1—figure supplement 1b). This phenotype could be attributed to extracellular electron transfer, as ferric iron or fumarate failed to stimulate growth of the ΔfmnB strain (Figure 1—figure supplement 1b). These findings are consistent with aerobic respiration and extracellular electron transfer possessing distinct roles in aerobic and anaerobic environments, respectively. The ΔQC/fmnB strain exhibited the most striking growth pattern. This strain lacked a phenotype under anaerobic conditions but had impaired aerobic growth, even relative to the ΔQC strain (Figure 1B). Notably, ΔQC/fmnB was the sole strain tested with a substantially reduced growth rate in the presence of oxygen (Figure 1B). These observations suggest that aerobic extracellular electron transfer activity can partially compensate for the loss of aerobic respiration and that oxygen inhibits L. monocytogenes growth in the absence of both electron transport chains. Respiration alters L. monocytogenes’ fermentative output Respiration is classically defined by the complete oxidation of an electron donor (e.g. glucose) to carbon dioxide in the TCA cycle. However, L. monocytogenes lacks a TCA cycle and instead converts sugars into multiple fermentation products (Romick et al., 1996). We thus asked how respiration impacts L. monocytogenes’ fermentative output. Under anaerobic conditions that lacked an alternative electron acceptor, L. monocytogenes exhibited a pattern of mixed acid fermentation, with lactic acid being most abundant and ethanol, formic acid, and acetic acid being produced at lower levels (Figure 1C). By contrast, under aerobic conditions L. monocytogenes almost exclusively produced acetic acid (Figure 1C). Consistent with respiration being partially responsible for the distinct aerobic vs. anaerobic responses, ΔQC and ΔQC/fmnB strains failed to undergo drastic shifts in fermentative output when grown in aerobic conditions. The ΔQC strain mainly produced lactic acid in the presence of oxygen, and this trend was even more pronounced in the ΔQC/fmnB strain, which almost exclusively produced lactic acid (Figure 1C). These results show that aerobic respiration induces a shift to acetic acid production and support the conclusion that L. monocytogenes’ two electron transport chains are partially functionally redundant in aerobic conditions. A comparison of fermentative outputs across the experimental conditions also clarifies the basis of central energy metabolism in L. monocytogenes. A classical glycolytic metabolism in L. monocytogenes likely generates ATP and NADH. In the absence of oxygen or an alternative electron acceptor, NAD+ is regenerated by coupling NADH oxidation to the reduction of pyruvate to lactate or ethanol. In the presence of oxygen, NADH oxidation is coupled to the reduction of oxygen, and pyruvate is converted to acetate. Moreover, the pattern of anaerobic formate production is consistent with aerobic acetyl-CoA production through pyruvate dehydrogenase and anaerobic production through pyruvate formate-lyase (Figure 1D). Collectively, these observations suggest that L. monocytogenes prioritizes balancing NAD+/NADH levels in the absence of an electron acceptor and maximizing ATP production in the presence of oxygen. In the absence of oxygen, NAD+/NADH redox homeostasis is achieved by minimizing NADH produced in acetyl-CoA biosynthesis and by consuming NADH in lactate/ethanol fermentation (Figure 1D). In the presence of oxygen, ATP yields are maximized through respiration and increased substrate-level phosphorylation by acetate kinase activity (Figure 1D). Respiratory capabilities are essential for L. monocytogenes pathogenesis We next asked about the role of cellular respiration in intracellular L. monocytogenes growth and pathogenesis. The ΔfmnB mutant deficient for extracellular electron transfer was previously shown to resemble the wildtype L. monocytogenes strain in a murine model of infection (Light et al., 2018). We found that this mutant also did not differ from wildtype L. monocytogenes in growth in bone marrow-derived macrophages (BMMs) and a plaque assay that monitors bacterial growth and cell-to-cell spread (Figure 2A and B). Consistent with previous reports, the ΔQC strain deficient for aerobic respiration was attenuated in the plaque assay and murine model of infection, but resembled wildtype L. monocytogenes in macrophage growth (Figure 2A–C; Chen et al., 2017; Corbett et al., 2017). Combining mutations that resulted in the loss of both extracellular electron transfer and aerobic respiration produced even more pronounced phenotypes. The ΔQC/fmnB strain did not grow intracellularly in macrophages and fell below the limit of detection in the plaque assay and murine infection model (Figure 2A–C). Consistent with this phenotype reflecting a loss of respiratory activity, we observed that a mutant that targeted the two respiratory NADH dehydrogenases resulted in a similar phenotype in the plaque assay (Figure 2A). These results thus demonstrate that respiratory activities are essential for L. monocytogenes virulence, and that the organism’s two respiratory pathways are partially functionally redundant within a mammalian host. Figure 2 Download asset Open asset Respiration is required for L. monocytogenes virulence. (A) Plaque formation by cell-to-cell spread of L. monocytogenes strains in monolayers of mouse L2 fibroblast cells. The mean plaque size of each strain is shown as a percentage relative to the wildtype plaque size. Error bars represent standard deviations of the mean plaque size from two independent experiments. Statistical analysis was performed using one-way ANOVA and Dunnett’s post-test comparing wildtype to all the other strains. ****, p<0.0001; ns, no significant difference (p>0.05). (B) Intracellular growth of L. monocytogenes strains in murine bone marrow-derived macrophages (BMMs). At 1-hour post-infection, infected BMMs were treated with 50 μg/mL of gentamicin to kill extracellular bacteria. Colony-forming units (CFU) were enumerated at the indicated times. Results are representative of two independent experiments. (C) Bacterial burdens in murine spleens and livers 48 hours post-intravenous infection with indicated L. monocytogenes strains. The median values of the CFUs are denoted by black bars. The dashed lines represent the limit of detection. Data were combined from two independent experiments, n = 10 mice per strain. Statistical significance was evaluated using one-way ANOVA and Dunnett’s post-test using wildtype as the control. ****, p<0.0001. ΔQC, ΔqoxA/ΔcydAB; ΔQC/fmnB, ΔqoxA/ΔcydAB/fmnB::tn; Δndh1/ndh2, Δndh1/ndh2::tn. Figure 2—source data 1 Source data for Figure 2A. https://cdn.elifesciences.org/articles/75424/elife-75424-fig2-data1-v2.xlsx Download elife-75424-fig2-data1-v2.xlsx Figure 2—source data 2 Source data for Figure 2B. https://cdn.elifesciences.org/articles/75424/elife-75424-fig2-data2-v2.xlsx Download elife-75424-fig2-data2-v2.xlsx Figure 2—source data 3 Source data for Figure 2C. https://cdn.elifesciences.org/articles/75424/elife-75424-fig2-data3-v2.xlsx Download elife-75424-fig2-data3-v2.xlsx Expression of NOX restores NAD+ levels in L. monocytogenes respiration mutants Cellular respiration both regenerates NAD+ and establishes a proton motive force that is important for various aspects of bacterial physiology. The involvement of respiration in these two distinct processes can confound the analysis of respiration-impaired phenotypes. However, the heterologous expression of water-forming NADH oxidase (NOX) has been used to decouple these functionalities in mammalian cells (Figure 3A; Titov et al., 2016). Because NOX regenerates NAD+ without pumping protons across the membrane, its introduction to a respiration-deficient cell can correct the NAD+/NADH imbalance, thereby isolating the role of the proton motive force in the phenotype (Lopez de Felipe et al., 1998; Titov et al., 2016). Figure 3 with 1 supplement see all Download asset Open asset Water-forming NADH oxidase (NOX) restores redox homeostasis in respiration-deficient L. monocytogenes strains. (A) Reaction catalyzed by the Lactococcus lactis water-forming NOX, which is the same as aerobic respiration without the generation of a proton motive force. (B) NAD+/NADH ratios of parent and NOX-complemented L. monocytogenes strains grown aerobically in nutrient-rich media to mid-logarithmic phase. Results from three independent experiments are presented as means and standard deviations. Statistical significance was calculated using one-way ANOVA and Dunnett’s post-test using the wildtype parent strain as the control. ****, p<0.0001; ***, p<0.001; **, p<0.01; ns, not statistically significant (p>0.05). (C) Fermentation products of L. monocytogenes strains grown in nutrient-rich media under aerobic conditions. Error bars show standard deviations. Results from three independent experiments are shown. ΔQC, ΔqoxA/ΔcydAB; ΔQC/fmnB, ΔqoxA/ΔcydAB/fmnB::tn; + NOX, strains complemented with L. lactis nox. Figure 3—source data 1 Source data for Figure 3B. https://cdn.elifesciences.org/articles/75424/elife-75424-fig3-data1-v2.xlsx Download elife-75424-fig3-data1-v2.xlsx Figure 3—source data 2 Source data for Figure 3C. https://cdn.elifesciences.org/articles/75424/elife-75424-fig3-data2-v2.xlsx Download elife-75424-fig3-data2-v2.xlsx To address which aspect of cellular respiration was important for L. monocytogenes pathogenesis, we introduced the previously characterized Lactococcus lactis water-forming NOX to the genome of respiration-deficient L. monocytogenes strains (Heux et al., 2006; Neves et al., 2002a; Neves et al., 2002b). We confirmed that the ΔQC and ΔQC/fmnB strains exhibited decreased NAD+/NADH levels and that constitutive expression of NOX rescued this phenotype (Figure 3B). Consistent with the altered fermentative output of the ΔQC/fmnB strain resulting from impaired NAD+ regeneration, we observed that NOX expression restored the predominance of acetic acid production to the aerobically grown cells (Figure 3C). To confirm that NOX expression specifically impacts NAD+/NADH-dependent phenotypes, we tested the effect of NOX expression on bacterial motility. Consistent with respiration impacting flagellar function through the proton motive force, we found that ΔQC/fmnB exhibited impaired bacterial motility and that this phenotype was resilient to NOX expression (Manson et al., 1977; Figure 3—figure supplement 1). These experiments thus provide evidence that NOX expression provides a tool to specifically manipulate the NAD+/NADH ratio in L. monocytogenes. Respiration is critical for regenerating NAD+ during L. monocytogenes pathogenesis We next sought to dissect the relative importance of respiration in generating a proton motive force versus maintaining redox homeostasis for L. monocytogenes virulence. We tested NOX-expressing ΔQC and ΔQC/fmnB strains for macrophage growth, plaque formation, and in the murine infection model. Expression of NOX almost fully rescued the plaque and macrophage growth phenotypes of the ΔQC and ΔQC/fmnB strains (Figure 4A and B). NOX expression also partially rescued L. monocytogenes virulence in the murine infection model (Figure 4C). Notably, NOX expression had a greater impact on the L. monocytogenes load in the spleen than the liver, suggesting distinct functions of respiration for L. monocytogenes colonization of these two organs (Figure 4C). These results thus suggest that NAD+ regeneration represents the primary role of respiration in L. monocytogenes pathogenesis to an organ-specific extent. Figure 4 Download asset Open asset NOX expression restores virulence to respiration-deficient L. monocytogenes strains. (A) Plaque formation by cell-to-cell spread of L. monocytogenes strains in monolayers of mouse L2 fibroblast cells. The mean plaque size of each strain is shown as a percentage relative to the wildtype plaque size. Error bars represent standard deviations of the mean plaque size from two independent experiments. Statistical analysis was performed using the unpaired two-tailed t test. ****, p<0.0001; ns, no significant difference (p>0.05). (B) Intracellular growth of L. monocytogenes strains in murine bone marrow-derived macrophages (BMMs). At 1-hour post-infection, infected BMMs were treated with 50 μg/mL of gentamicin to kill extracellular bacteria. Colony-forming units (CFU) were enumerated at the indicated times. Results are representative of three independent experiments. (C) Bacterial burdens in murine spleens and livers 48 hours post-intravenous infection with indicated L. monocytogenes strains. The median values of the CFUs are denoted by black bars. The dashed lines represent the limit of detection. Data were combined from two independent experiments, n = 10 mice per strain, but for the wildtype +NOX strain (n = 9 mice). Statistical significance was evaluated using one-way ANOVA and Dunnett’s post-test using the wildtype control strain to compare with the NOX-complemented strains. Significance between the parental and the NOX-complemented strains was determined using the unpaired two-tailed t test. ****, p<0.0001; **, p<0.01; ns, no significant difference (p>0.05). ΔQC, ΔqoxA/ΔcydAB; ΔQC/fmnB, ΔqoxA/ΔcydAB/fmnB::tn; Δndh1/ndh2, Δndh1/ndh2::tn; + NOX, strains complemented with Lactococcus lactis nox. Figure 4—source data 1 Source data for Figure 4A. https://cdn.elifesciences.org/articles/75424/elife-75424-fig4-data1-v2.xlsx Download elife-75424-fig4-data1-v2.xlsx Figure 4—source data 2 Source data for Figure 4B. https://cdn.elifesciences.org/articles/75424/elife-75424-fig4-data2-v2.xlsx Download elife-75424-fig4-data2-v2.xlsx Figure 4—source data 3 Source data for Figure 4C. https://cdn.elifesciences.org/articles/75424/elife-75424-fig4-data3-v2.xlsx Download elife-75424-fig4-data3-v2.xlsx Impaired redox homeostasis is associated with increased cytosolic L. monocytogenes lysis We next asked why respiration-mediated redox homeostasis was critical for L. monocytogenes pathogenesis. We reasoned that previous descriptions of L. monocytogenes quinone biosynthesis mutants might provide a clue. Quinones are a family of redox-active cofactors that have essential functions in respiratory electron transport chains (Collins and Jones, 1981). Our previous studies suggested that distinct quinones function in flavin-based electron transfer and aerobic respiration (Light et al., 2018). A separate set of studies found that L. monocytogenes quinone biosynthesis mutants exhibited divergent phenotypes. L. monocytogenes strains defective in upstream steps of the quinone biosynthesis pathway (menB, menC, menD, menE, and menF) exhibited increased bacteriolysis in the cytosol of host cells and were severely attenuated for virulence (Figure 5A). By contrast, L. monocytogenes strains defective in downstream steps of the quinone biosynthesis pathway (menA and menG) did not exhibit increased cytosolic bacteriolysis and had less severe virulence phenotypes (Chen et al., 2019, Chen et al., 2017; Smith et al., 2021; Figure 5A). These divergent phenotypic responses resemble the loss of aerobic respiration versus the loss of aerobic respiration plus flavin-based electron transfer observed in our studies. The distinct virulence phenotype of quinone biosynthesi