New Results

Listeria monocytogenes requires DHNA-dependent intracellular redox homeostasis facilitated by Ndh2 for survival and virulence

Hans B. Smith, Kijeong Lee, David M. Stevenson, Daniel Amador-Noguez, John-Demian Sauer
doi: https://doi.org/10.1101/2023.01.13.524026

ABSTRACT

Listeria monocytogenes is a remarkably well-adapted facultative intracellular pathogen that can thrive in a wide range of ecological niches. L. monocytogenes maximizes its ability to generate energy from diverse carbon sources using a respiro-fermentative metabolism that can function under both aerobic and anaerobic conditions. Cellular respiration maintains redox homeostasis by regenerating NAD+ while also generating a proton motive force (PMF). The end products of the menaquinone (MK) biosynthesis pathway are essential to drive both aerobic and anaerobic cellular respiration. We previously demonstrated that intermediates in the MK biosynthesis pathway, notably 1,4-dihydroxy-2-naphthoate (DHNA), are required for the survival and virulence of L. monocytogenes independent of their role in respiration. Furthermore, we found that restoration of NAD+/NADH ratio through expression of water-forming NADH oxidase (NOX) could rescue phenotypes associated with DHNA deficiency. Here we extend these findings to demonstrate that endogenous production or direct supplementation of DHNA restored both the cellular redox homeostasis and metabolic output of fermentation in L. monocytogenes. Further, exogenous supplementation of DHNA rescues the in vitro growth and ex vivo virulence of L. monocytogenes DHNA-deficient mutants. Finally, we demonstrate that exogenous DHNA restores redox balance in L. monocytogenes specifically through the recently annotated NADH dehydrogenase Ndh2, independent of the extracellular electron transport (EET) pathway. These data suggest that the production of DHNA may represent an additional layer of metabolic adaptability by L. monocytogenes to drive energy metabolism in the absence of respiration-favorable conditions.

INTRODUCTION

Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen that is exceptionally well-adapted to survive and replicate in the restrictive mammalian host cytosol (1-3). Bacteria that lack the specific adaptations required to survive or replicate in the host niche are effectively cleared (4-7), often by triggering host defense mechanisms comprised of innate immune pathways (8-13). L. monocytogenes utilizes its internalin proteins to facilitate invasion into the host cell where it becomes captured in a phagosome (14, 15). The pore-forming cytolysin listeriolysin O (LLO) then facilitates escape from the phagosome into the cytosol (14, 16), where L. monocytogenes can utilize ActA to mediate actin-based motility by hijacking the host’s actin machinery (17-20). Using this motility, L. monocytogenes moves into adjacent cells where they again invade the cytosol by expressing LLO and two phospholipase Cs, PlcA and PlcB, enabling it to restart its life cycle (14, 21).

L. monocytogenes can also thrive in a diverse range of ecological niches that contain highly variable pools of fermentable and non-fermentable carbon sources (2, 22). L. monocytogenes employs both fermentative and respiratory metabolic mechanisms to maximize its energy output from scavenged nutrients (22, 23). In contrast to canonical respiratory organisms however, L. monocytogenes contains an incomplete tricarboxylic acid (TCA) cycle and is therefore unable to fully oxidize its carbon substrates (24). Accordingly, L. monocytogenes utilizes a respiro-fermentative metabolism characterized by glycolysis-derived pyruvate that is funneled into the fermentative production of acetate, generating ATP through substrate-level phosphorylation (SLP) via the activity of acetate kinase (24, 34). During the respiro-fermentative process, the activity of L. monocytogenes’ respiratory electron transport chain (ETC) enables it to regenerate NAD+, without having to rely upon lactate dehydrogenase, while also producing a functional proton motive force (PMF) (22, 24, 34). Further lending to its diverse metabolic adaptability, L. monocytogenes possesses two distinct respiratory ETCs that allow it to respire both aerobically and anaerobically (25). The aerobic ETC in L. monocytogenes mediates electron transfer from a type II NADH dehydrogenase, Ndh1, to a membrane-bound menaquinone (MK) and subsequently to terminal cytochrome oxidases QoxAB (aa3) or CydAB (bd) for final transfer to O2 (26, 27). In contrast, the recently annotated anaerobic respiratory pathway in L. monocytogenes uses a flavin-based ETC to drive extracellular electron transfer (EET) to extracytosolic acceptors such as fumarate or ferric ion using a novel NADH dehydrogenase (Ndh2) and an alternative demethylmenaquinone (DMK) intermediate (25, 28). Both of the respiratory ETC in L. monocytogenes rely upon the MK biosynthesis pathway to generate their respective quinone electron acceptors, with the biosynthetic intermediate 1,4-dihydroxy-2-naphthoate (DHNA) functioning as a mutual branching point (Fig. S1) (25).

The requirement for L. monocytogenes to perform cellular respiration during infection has been well documented (29-32). However, understanding the specific contributions of maintaining cellular redox homeostasis via NAD+ regeneration versus the production of a functional PMF to achieve virulence has remained elusive. Further complicating our ability to dissect the specific contributions that cellular respiration may have during infection, the MK intermediates DHNA-CoA and DHNA have recently been reported to be required for the survival and virulence of L. monocytogenes independent of MK synthesis and aerobic respiration (29, 31, 32). Importantly, although it was observed that the supplementation of exogenous DHNA could rescue the in vitro growth of a DHNA-deficient L. monocytogenes mutant, this rescue did not coincide with the restoration of its PMF (31). Therefore, although DHNA-deficient strains of L. monocytogenes possess the downstream enzymes to produce MK or DMK, these data suggest that exogenous DHNA is not utilized to promote either aerobic or anaerobic cellular respiration. Recent work from Rivera-Lugo et al. sought to dissect the relative importance of maintaining redox homeostasis versus PMF generation for the pathogenesis of L. monocytogenes using a water-forming NADH oxidase (NOX) that specifically regenerates NAD+ independent of respiration and PMF function (34). Through the heterologous expression of NOX in respiration-deficient strains of L. monocytogenes, it was concluded that the regeneration of NAD+ represents a major role for cellular respiration during pathogenesis.

The studies presented here sought to define the respiration-independent mechanisms of DHNA utilization to promote the survival and virulence of L. monocytogenes. Consistent with observations from Rivera-Lugo et. al, in the absence of respiration, the ex vivo and in vivo virulence defects associated with DHNA-deficiency were a result of impaired redox homeostasis which could be rescued upon ectopic NOX expression. Similarly, exogenous DHNA supplementation rescues the in vitro and ex vivo growth and cytosolic survival of DHNA-deficient mutants. Indeed, DHNA-dependent rescue by direct supplementation resulted in a restored cellular redox homeostasis with a concurrent shift of fermentative flux from lactate production to acetate in L. monocytogenes, independent of respiration. We further go on to show that the recently annotated anaerobic-specific Ndh2 is essential for DHNA-deficient L. monocytogenes mutants to utilize exogenous DHNA for growth in defined medium, independent of its canonical role in EET, suggesting that Ndh2 is the NADH dehydrogenase specifically required for the restoration of redox homeostasis via DHNA. Taken together, these data suggest that the endogenous production of DHNA can be utilized by L. monocytogenes to restore both its intracellular redox homeostasis and fermentative metabolic flux through an undefined mechanism requiring Ndh2.

RESULTS

Redox homeostasis via NOX shifts fermentative output and rescues in vitro growth of DHNA-deficient L. monocytogenes

Two main outcomes of cellular respiration include 1) maintaining intracellular redox homeostasis by regenerating NAD+ from NADH and 2) the generation of a PMF to drive oxidative phosphorylation and various other aspects of bacterial physiology. A recent study employed a water-forming NADH oxidase (NOX) expression system in L. monocytogenes to dissect the relative importance of cellular respiration in maintaining redox homeostasis versus PMF generation (34). We had previously demonstrated that L. monocytogenes mutants lacking the key MK biosynthetic intermediate DHNA were attenuated, in part, independent of loss of respiration (29, 31, 32). We hypothesized that restoration of NAD+ pools might rescue these virulence defects similar to the rescue observed for mutants lacking components of the respiratory chains (34). To test this hypothesis, we assessed NAD+/NADH levels in ΔmenB, ΔmenI and ΔmenA mutants +/-expression of NOX in trans. The inability to generate endogenous DHNA by the ΔmenB mutant results in a severely diminished redox homeostasis as measured by the ratio of oxidized NAD+ to reduced NADH. This imbalance was significantly restored by ectopic expression of NOX to a level similar to the ΔmenA mutant (Fig. 1A). The ΔmenI mutant, which can generate DHNA-CoA, displays an intermediate phenotype between ΔmenB and ΔmenA levels, which is similarly rescued upon NOX expression (Fig. 1A), consistent with possible respiration independent roles for DHNA in NAD+/NADH redox balancing.

Figure 1.
Figure 1.

Redox homeostasis via NOX shifts fermentative output and rescues in vitro growth of DHNA-deficient L. monocytogenes. (A) NAD+/NADH ratios of indicated L. monocytogenes strains +/-NOX plasmid complementation grown aerobically at 37°C in defined medium to mid-logarithmic phase (OD600 0.4-0.6). ΔmenB mutant fails to grow in defined medium, thus these culture samples were spiked with 2 × 108 total CFU from an overnight BHI culture during experimental setup. (B) HPLC quantification of fermentation products (Lactate and Acetate) produced and secreted by indicated L. monocytogenes strains +/-NOX plasmid complementation grown in BHI media aerobically at 37°C to stationary phase. (C) L. monocytogenes strains +/-NOX plasmid complementation were grown in defined medium at 37°C. OD600 was monitored for 20 hours. Data are representative of three (A, C) or two (B) independent experiments. ns, not significant; WT, wild-type

L. monocytogenes employs a respiro-fermentative metabolism due to an incomplete TCA cycle, characterized by the funneling of pyruvate towards the fermentative production of acetate (23, 24). Respiration-deficient mutants of L. monocytogenes are impaired in their ability to maintain cellular redox homeostasis and as a result nearly exclusively produce lactate rather than acetate as a metabolic byproduct (34). To test whether impaired redox homeostasis due to DHNA-deficiency would similarly result in the predominant production of lactate, we analyzed fermentation byproducts in bacterial supernatants using high-performance liquid chromatography (HPLC). As expected, wild-type L. monocytogenes predominantly generated acetate whereas DHNA-deficient ΔmenB had a drastic shift to lactate production (Fig. 1B). Heterologous NOX expression rescued ΔmenB acetate production back to wild-type levels, consistent with restored redox homeostasis driving acetate production to generate ATP (Fig. 1B). Consistent with the results seen in our NAD+/NADH experiments, the ΔmenI mutant displayed an intermediate phenotype by producing similar levels of acetate and lactate, which was also fully restored to wild-type upon NOX expression (Fig. 1B). The ΔmenA mutant produced slightly more lactate and less acetate when compared to wild-type, likely attributed to the difference in redox homeostasis observed previously (Fig. 1A, B).

Finally, we have previously shown that the production of DHNA is critical for L. monocytogenes in vitro growth in chemically defined medium (29, 31, 32). To test whether restoration of redox homeostasis can rescue this growth defect, we’ve assayed for in vitro growth of the above mutants complemented with NOX in defined medium. As expected, ΔmenB showed the largest growth defect followed by ΔmenI, and both mutants showed wild-type level growth upon NOX complementation (Fig. 1C). Together, these data suggest that metabolic defects associated with DHNA deficiency in L. monocytogenes are due to NAD+/NADH redox imbalances and that restoration of this balance can rescue ΔmenB mutant growth and carbon metabolism in L. monocytogenes.

Restoration of redox homeostasis rescues virulence defects associated with DHNA-deficiency

Based on the restoration of in vitro growth of ΔmenB mutants via expression of NOX, we hypothesized that restoration of NAD+ pools would similarly rescue virulence defects of DHNA-deficient mutants. DHNA-deficient mutants are susceptible to cytosolic killing in the macrophage cytosol, therefore we assessed cytosolic survival of ΔmenB, ΔmenI, and ΔmenA with or without expression of NOX in trans (29, 35). As hypothesized, ΔmenB and ΔmenI displayed increased cytosolic killing and NOX expression rescued their survival in the macrophage cytosol (Fig. 2A). Rescue by NOX expression was specific to mutants with disrupted NAD+/NADH redox homeostasis as NOX expression was unable to rescue cytosolic survival of a ΔglmR mutant susceptible to cytosolic killing due to cell wall defects (Fig 2A) (33, 35, 36). Consistent with NAD+ pool restoration supporting cytosolic survival, ΔmenB mutant replication in the macrophage cytosol was also rescued upon expression of NOX in trans (Fig. 2B).

Figure 2.
Figure 2.

Restoration of redox homeostasis rescues virulence defects associated with DHNA-deficiency. (A) Indicated L. monocytogenes strains (MOI of 10) +/-NOX plasmid complementation were tested for cytosolic survival in immortalized IFNAR-/- bone marrow-derived macrophages (BMDM) over a 6 hr infection. Data are normalized to wild-type levels of bacteriolysis and presented as the standard deviation of the means from three independent experiments. (B) Intracellular growth of wild-type, ΔmenB, or ΔmenB-NOX was determined in BMDMs following infection at an MOI of 0.2. Growth curves are representative of at least three independent experiments. Error bars represent the standard deviation of the means of technical triplicates within the representative experiment. (C) Bacterial burdens from the spleen and liver were enumerated at 48 hr post-intravenous infection with 1 × 105 total CFU of indicated L. monocytogenes strains +/-NOX plasmid complementation. Data are representative of results from two independent experiments. Horizontal bars represent the limits of detection and the bars associated with the individual strains represents the mean of the group. ns, not significant.

Finally, we had previously demonstrated that DHNA-deficient mutants are more attenuated in vivo than respiration-deficient mutants, suggesting that DHNA contributes to virulence in a respiration independent manner (29, 31, 32). To determine if the respiration independent function of DHNA during in vivo infection is due to NAD+/NADH homeostasis defects, we assessed virulence of ΔmenB, ΔmenI, and ΔmenA mutant L. monocytogenes with and without expression of NOX in trans. Ectopic NOX expression rescued the in vivo burden of ΔmenB mutants by ∼100-fold in the spleen and liver (Fig. 2C) and a similar rescue for ΔmenI mutants in the liver following NOX expression is also observed (Fig. 2C). Interestingly, there was little to no change in the in vivo virulence of ΔmenA upon the introduction of NOX (Fig 2C). This is in agreeance with our previous results that showed both redox homeostasis and acetate production of the ΔmenA mutant was also not significantly altered upon NOX expression (Fig 1A, B). Taken together, these data suggest that in L. monocytogenes maintaining cellular redox homeostasis in the absence of DHNA is sufficient to promote survival and virulence both ex vivo and in vivo.

DHNA production or supplementation promotes similar effects to NOX complementation in L. monocytogenes

We have previously demonstrated that exogenous addition of either purified DHNA or culture supernatant from DHNA sufficient strains of L. monocytogenes could rescue the in vitro growth of DHNA-deficient L. monocytogenes in defined media (31), suggesting that L. monocytogenes, like other bacteria including Propionibacterium spp. and Lactobacillus spp., may secrete DHNA (43, 45). To test the hypothesis that L. monocytogenes secretes DHNA, we assayed culture supernatants for DHNA via mass spectrometry. As hypothesized, wild-type L. monocytogenes contained abundant levels of DHNA, while ΔmenB mutants contained no detectable extracellular DHNA (Fig. S2). Given that exogenous DHNA could rescue the in vitro growth of DHNA-deficient L. monocytogenes mutants and that DHNA-deficient mutants could similarly be rescued by NAD+ regeneration through NOX expression, we hypothesized that exogenous DHNA could act to restore NAD+ levels in ΔmenB mutants. To test this hypothesis, we measured cellular NAD+/NADH with or without DHNA supplementation. Consistent with the results observed with NOX expression, the exogenous supplementation of DHNA rescued redox homeostasis of ΔmenB mutants to levels similar to those seen with ΔmenA mutants, suggesting that exogenous DHNA might be utilized in a similar fashion to DHNA produced endogenously (Fig. 3A). Consistent with DHNA supplementation of ΔmenB rescuing cellular redox homeostasis, exogenous DHNA also shifted the metabolic flux of ΔmenB back towards acetate production, similar to ΔmenA levels (Fig. 3B). Importantly, we had previously demonstrated that exogenous DHNA does not restore respiration and membrane potential (31). Taken together, these data suggest that DHNA, independent of its role in respiration, restores cellular redox homeostasis, subsequently shifting the fermentative output from lactate back towards acetate that likely drives ATP production through acetate kinase (24, 34).

Figure 3.
Figure 3.

DHNA production or supplementation promotes similar effects to NOX complementation in L. monocytogenes. (A) NAD+/NADH ratios of indicated L. monocytogenes strains +/-exogenous DHNA supplementation grown aerobically at 37°C in defined medium to mid-logarithmic phase. Again, ΔmenB were spiked with 2 × 108 total CFU from an overnight BHI culture during experimental setup. Data are presented as the standard deviation of the means from three independent experiments. (B) HPLC quantification of fermentation products (Lactate and Acetate) produced and secreted by indicated L. monocytogenes strains +/-exogenous DHNA supplementation grown in BHI media aerobically at 37°C to stationary phase. Data are representative of two independent experiments. (C) Indicated L. monocytogenes strains (MOI of 10) +/-DHNA supplementation were tested for cytosolic survival in primary IFNAR -/- BMDMs over a 6 hr infection. Data are normalized to wild-type levels of bacteriolysis and presented as the standard deviation of the means from three independent experiments. (D) Intracellular growth of wild-type, ΔmenB, or ΔmenA was determined in BMDMs following infection at an MOI of 0.2. Growth curves are representative of at least three independent experiments. Error bars represent the standard deviation of the means of technical triplicates within the representative experiment. ns, not significant.

Having previously observed that DHNA can restore NAD+ redox homeostasis and that NOX-dependent NAD+ restoration could restore virulence defects of ΔmenB mutants, we hypothesized that exogenous DHNA supplementation during infection may similarly rescue the cytosolic survival and intracellular growth of DHNA-deficient L. monocytogenes. Indeed, the addition of exogenous DHNA during macrophage infection with ΔmenB or ΔmenI mutants restored their cytosolic survival back to wild-type and ΔmenA levels (Fig. 3C). Importantly, as observed with NOX expression, DHNA supplementation did not rescue the cytosolic survival of ΔglmR mutants whose virulence phenotypes are due to cell wall stress response defects (Fig. 3C) (33, 36), demonstrating that the rescue of cytosolic survival by DHNA is specific to DHNA-deficient L. monocytogenes. Accordingly, supplementing DHNA during macrophage infection also rescued the ability of ΔmenB mutants to replicate intracellularly to levels similar of that during ΔmenA infection (Fig 3D). Taken together, these results demonstrate that exogenously provided DHNA can balance NAD+/NADH redox homeostasis thereby potentiating L. monocytogenes virulence.

ndh2 is conditionally essential for DHNA utilization in vitro

Although DHNA can drive regeneration of NAD+ in L. monocytogenes upon exogenous supplementation, it does not restore membrane potential suggesting that it is not simply imported and used to synthesize MK as described in Streptococci (44, 53). We hypothesized that the two annotated L. monocytogenes’ NADH dehydrogenases encoded by ndh1 (LMRG_02734) and ndh2 (LMRG_02183), respectively, may utilize DHNA independent of the respiratory pathways to facilitate NAD+/NADH homeostasis (23, 25). To test this hypothesis, we generated Δndh1/menB::Tn and ndh2::Tn/ΔmenB mutants and assayed for growth with or without 5μM exogenous DHNA in defined medium. As expected, both double mutants were unable to grow without exogenous DHNA due them being a ΔmenB mutant (Fig. 4A). DHNA supplementation rescued growth of the Δndh1/menB::Tn mutant suggesting that Ndh1 is not required for DHNA-dependent NAD+/NADH redox homeostasis. In contrast, the ndh2::Tn/ΔmenB mutant was unable to grow in the presence of exogenous DHNA (Fig. 4B). ndh2 is required for the function of the recently described EET pathway in L. monocytogenes (25), therefore we hypothesized that EET may be necessary to utilize DHNA for NAD+/NADH redox homeostasis. To test this hypothesis, we transduced pplA::Tn, dmkA::Tn, eetA::Tn, and fmnA::Tn mutations into a ΔmenB background. The growth of all four of these double mutants were rescued upon DHNA supplementation in defined medium (Fig. S3).

Figure 4.
Figure 4.

ndh2 is conditionally essential for DHNA utilization in vitro. Indicated strains of L. monocytogenes were grown in defined medium without (A) or with (B) 5μM DHNA supplementation aerobically at 37°C and monitored for OD600 over 20 hr. (C) ndh2::Tn/ΔmenB L. monocytogenes was grown aerobically in defined medium with either 5μM DHNA or 5μM MK and monitored for growth (OD600) over 20 hr. All data represent one representative out of three biological replicates.

Finally, exogenous MK supplementation can restore not only growth of DHNA deficient mutants but also their membrane potential (31, 32), likely through direct insertion of MK in the membrane and subsequent restoration of the aerobic respiratory chain. To ensure that ndh2::Tn/ΔmenB mutants are not more generally incapable of growing in defined media, we supplemented ndh2::Tn/ΔmenB mutants with either DHNA or MK directly.

Supplementation of MK rescued growth of ndh2::Tn/ΔmenB in defined medium unlike DHNA, showing that this mutant is specifically dysfunctional in the use of DHNA as a redox homeostasis substrate (Fig. 4C). Taken together, these data suggest that Ndh2 facilitates DHNA-dependent NAD+/NADH redox homeostasis in the absence of respiration in L. monocytogenes.

DISCUSSION

Cytosolic pathogens require specific adaptations to survive and replicate within the host. In L. monocytogenes, MK biosynthetic intermediate DHNA is among those factors necessary for cytosolic survival, independent of its known role in MK synthesis and cellular respiration (29, 31). In the present study, we sought to address the respiration-independent mechanism by which DHNA is required for the survival and virulence of L. monocytogenes. Utilizing a heterologous NOX expression system, we demonstrated that virulence defects associated with loss of DHNA could be rescued by restoration of NAD+/NADH homeostasis (Fig. 1, 2). We then found that exogenous DHNA supplementation restores NAD+/NADH balance, cytosolic survival, and intracellular replication of the DHNA-deficient mutant ΔmenB (Fig.3). Balancing of redox homeostasis also coincided with a marked shift in fermentative flux from lactate to acetate upon DHNA production or supplementation (Fig. 3B) to maximize ATP production via SLP through the activity of acetate kinase (34, 37). Lastly, we provide evidence that Ndh2 is the NADH dehydrogenase responsible for restoring redox homeostasis during extracellular DHNA utilization, independent of its role in EET (Fig. 4).

Although we’ve demonstrated that Ndh2 is conditionally essential for DHNA utilization in L. monocytogenes, it is still unclear how Ndh2 utilizes DHNA to maintain intracellular redox homeostasis. One possibility is that DHNA, or one of its derivatives, may be used as an alternative quinone to directly accept electrons from Ndh2, regenerating NAD+ similar to the system recently described in Shewanella oneidensis MR-1. Mevers et al. recently demonstrated that a derivative of DHNA, 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), could serve as a novel electron shuttle that functioned to promote redox balance and energy metabolism (38). The authors went on to show that ACNQ is produced non-enzymatically from extracellular DHNA under oxidizing conditions in the presence of a nitrogen donor (i.e. ammonium or amino acids) (38). We have confirmed that indeed, DHNA is secreted by wild-type L. monocytogenes (Fig. S2) and extracellular DHNA is readily converted to ACNQ in our defined medium based on mass spectrometry analysis (data not shown). Based on this model, it is possible that DHNA produced by L. monocytogenes is secreted outside of the cell to shuttle electrons away where it is then freely oxidized non-enzymatically in the local environment to form ACNQ. Newly formed ACNQ would then be imported back into L. monocytogenes to be reduced again through the activity of Ndh2. The repeated oxidation and reduction of DHNA and/or ACNQ is the hallmark of an “electron shuttle” and is one of the proposed mechanisms of EET in S. oneidensis (38, 39). A strikingly similar model has been described in Pseudomonas aeruginosa in which endogenous production of phenazine is cyclically reduced intracellularly, shuttled outside of the cell, and oxidized by a terminal electron acceptor where it is then imported again by the cell (41). Studies to determine whether DHNA/ACNQ fuels an Ndh2-dependent electron shuttle to maintain intracellular redox homeostasis or whether DHNA works via an alternative mechanism are currently ongoing.

It has been proposed that in addition to serving as an electron shuttle by P. aeruginosa, secreted phenazine may be used as a shared resource by the surrounding microbial community to fuel their own redox shuttling (42). The function of phenazine as a shared metabolite is also similar to what has been previously documented with the secretion of DHNA being used as a shared resource to fuel metabolic processes of other localized microbes (31, 43–45). Furthermore, a recent study by Tejedor-Sanz et al. reported that the homofermentative lactic acid bacteria Lactiplantibacillus plantarum contains the EET gene locus previously annotated in L. monocytogenes, however it is missing the upstream genes necessary for quinone biosynthesis (40). Upon addition of exogenous DHNA, L. plantarum was observed to employ an Ndh2-dependent form of EET that functioned to increase intracellular redox homeostasis by enhancing metabolic flux through fermentative pathways, generating additional lactate, while increasing ATP generation through SLP (40). Importantly, the capacity of DHNA supplementation to induce EET in L. plantarum did not coincide with the generation of a PMF to drive oxidative phosphorylation, similar to the phenotypes observed in L. monocytogenes. Whether there are functions of L. monocytogenes secreted DHNA as a shared metabolite in complex microbial communities such as those found in the intestine during the early stages of infection will require additional future studies.

Overall, we’ve shown that L. monocytogenes can utilize DHNA to maintain redox homeostasis through the anaerobic-specific NADH dehydrogenase Ndh2, independent of other EET proteins. Utilization of extracellular DHNA can aid DHNA-deficient L. monocytogenes mutants to restore their ability to grow and replicate within the cytosol by potentially driving a yet unclear method of energy metabolism. Pathways involved in unique energy metabolism by various pathogens are increasingly viewed as attractive drug targets and as such future studies utilizing the important model pathogen L. monocytogenes to understand the mechanisms of DHNA-dependent redox homeostasis could provide novel insights into the generation of new antimicrobials.

MATERIALS AND METHODS

Bacterial strains, plasmid construction, and growth conditions in vitro

L. monocytogenes strain 10403S is referred to as the wild-type strain, and all other strains used in this study are isogenic derivatives of this parental strain. Vectors were conjugated into L. monocytogenes by Escherichia coli strain S17 or SM10 (47). The integrative vector pIMK2 was used for constitutive expression of L. monocytogenes genes for complementation (48).

L. monocytogenes strains were grown at 37°C or 30°C in brain heart infusion (BHI) medium (237500; VWR) or defined medium supplemented with glucose as the sole carbon source. Defined medium is identical to the formulation described by Smith et al. (220). Escherichia coli strains were grown in Luria-Bertani (LB) broth at 37°C. Antibiotics were used at concentrations of 100 μg/ml carbenicillin (IB02020; IBI Scientific), 10 μg/ml chloramphenicol (190321; MP Biomedicals), 2 μg/ml erythromycin (227330050; Acros Organics), or 30 μg/ml kanamycin (BP906-5; Fisher Scientific) when appropriate. Medium, where indicated, was supplemented with 5 μM 1,4-dihydroxy-2-naphthoate (DHNA) (281255; Sigma) or 5 μM menaquinone (MK) (V9378; Sigma).

Phage Transduction

Phage transductions were performed as previously described (310). Briefly, MACK L. monocytogenes was grown overnight in 3mL LB at 30°C stationary to propagate U153 phage stocks. MACK cultures were pelleted and resuspended in LB + 10mM CaSO4 + 10mM MgCl2 and added into LB + 0.7% agar + 10mM CaSO4 + 10mM MgCl2 at 42°C. This mixture was immediately poured on BHI plates and incubated overnight at 30°C. U153 phage plaques were collected and soaked out with 10mM Tris (pH7.5) + 10mM CaSO4 + 10mM MgCl2. Donor plaque soak-outs were propagated the same way and were filter-sterilized using a 0.2μm syringe filter (09-740-113; Fisher Scientific) and additionally kept sterile by adding 500μL chloroform. Recipient ΔmenB strain was infected with these donor soak-outs for 30 minutes at room temperature and subsequently plated on BHI agar with erythromycin for selection at 37°C.

Intracellular bacteriolysis assay

Standard intracellular bacteriolysis assays were performed as previously described (29). Briefly, primary or immortalized bone marrow-derived IFNAR -/- macrophages (5 × 105 per well of 24-well plates) were grown in a monolayer overnight in 500 μL volume. L. monocytogenes strains carrying the bacteriolysis reporter pBHE573 (35) were grown at 30°C without shaking overnight. Cultures were then diluted to a final concentration of 5 × 108 CFU/mL in PBS and used to infect macrophages at a MOI of 10. At 1 hr postinfection, media were removed and replaced with media containing 50 μg/ml gentamicin. At 6 hr post infection, media from the wells were aspirated and macrophages were lysed using TNT lysis buffer (20 mM Tris, 200 mM NaCl, 1% Triton [pH 8.0]). Cell lysates were transferred to opaque 96-well plates, and luciferin reagent was added and assayed for luciferase activity (Synergy HT, BioTek; Winooski, VT).

Intracellular growth assay

Bone marrow-derived macrophages (BMDMs) were prepared from C57BL/6 mice as previously described (51). BMDMs were plated on coverslips at 5 × 106 cells per 60mm dish and allowed to adhere overnight. BMDMs were then infected at an MOI of 0.2 with their respective strain and infection proceeded for 8 hr. At 30 min postinfection, media were removed and replaced with media containing 50 μg/ml gentamicin. Total CFU were quantified at various time points as previously described (50).

NAD+ and NADH measurements

L. monocytogenes strains were grown in defined medium at 37°C with shaking to mid-logarithmic phase (OD600 0.4-0.6). Cultures were centrifuged and then resuspended in PBS. Resuspended bacteria were then lysed (2 × 108 total CFU) by a 1:1 addition of 1% dodecyltrimethylammonium bromide (DTAB) (AC409310250; Fisher Scientific) for 5 min with agitation. Lysates were then processed to measure NAD+ and NADH levels using the NAD/NADH-Glo assay (Promega, G9071) per the manufacturer’s protocol.

Fermentation byproduct measurements

Cultures of L. monocytogenes were grown in BHI at 37°C with shaking overnight. Bacteria were then centrifuged and 1 mL of the resulting supernatant was filtered through a 0.2μm-pore-size syringe filter (09-740-113; Fisher Scientific). Supernatant samples were next treated with 2μL of H2SO4 to precipitate any components that might be incompatible with the running buffer. The samples were then centrifuged at 16000 × g for 10 min and then 200μL of each sample transferred to an HPLC vial. HPLC analysis was performed using a ThermoFisher (Waltham, MA) Ultimate 3000 UHPLC system equipped with a UV detector (210 nm). Compounds were separated on a 250 × 4.6 mm Rezex© ROA-Organic acid LC column (Phenomenex Torrance, CA) run with a flow rate of 0.2 mL min−1 and at a column temperature of 50 °C. The samples were held at 4 °C prior to injection. Separation was isocratic with a mobile phase of HPLC grade water acidified with 0.015 N H2SO4 (415 μL L−1). At least two standard sets were run along with each sample set. Standards were 100, 20, 4, and 0.8mM concentrations of lactate or acetate. The resultant data was analyzed using the Thermofisher Chromeleon 7 software package.

Acute virulence assay

All techniques were reviewed and approved by the University of Wisconsin — Madison Institutional Animal Care and Use Committee (IACUC) under the protocol M02501. Female C57BL/6 mice (6 to 8 weeks of age; purchased from Charles River) were used for the purposes of this study. L. monocytogenes strains were grown in BHI medium at 30°C without shaking overnight. These cultures were then back-diluted the following day 1:5 into fresh BHI medium and grown at 37°C with shaking until mid-exponential phase (OD600 0.4-0.6). Bacteria were diluted in PBS to a concentration of 5 × 105 CFU/mL and mice were injected intravenously with 1 × 105 total CFU. At 48 hr postinfection, spleens and livers were harvested and homogenized in 0.1% Nonidet P-40 in PBS. Homogenates were then plated on LB plates to enumerate CFU and quantify bacterial burdens.

Statistical analysis

Statistical significance analysis (GraphPad Prism, version 6.0h) was determined by one-way analysis of variance (ANOVA) with a Dunnett’s posttest comparing wild-type to all other indicated strains or by one-way ANOVA with Tukey’s multiple comparisons test unless otherwise stated (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001).

FUNDING INFORMATION

This work was funded by the National Institutes of Health (T32007215 [HBS] and R01AI137070 [J-D S]). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Figure S1.
Figure S1.

Menaquinone biosynthetic pathway in Listeria monocytogenes. Chorismate is generated by the upstream shikimate biosynthesis pathway and is converted to DHNA by the series of listed enzymes (MenFDCHEBI). Red arrows indicate DHNA branching point towards aerobic respiration. Blue arrow indicates DHNA branching point towards anaerobic respiration. Corresponding gene locus numbers for L. monocytogenes strains EGD-e (Lmo) and 10403S (LMRG; parental strain used in this study) are listed underneath reaction arrows. OSB, o-succinylbenzoate; DMK, demethylmenaquinone.

Figure S2.
Figure S2.

Detection of secreted DHNA by mass spectrometry. Detection of DHNA from the cell-free supernatants of overnight aerobic cultures of wildtype or ΔmenB strains by mass spectrometry. Data were analyzed via MAVEN. Error bars represent the standard deviation of the means from two independent experiments.

Figure S3.
Figure S3.

Four separate genes other than ndh2 that are part of the L. monocytogenes EET locus did not display growth defects. Indicated strains were grown in defined medium at 37°C with the addition of 5μM DHNA. OD600 was monitored for 20 hours. Data represents one representative out of three biological replicates.

View this table:
Table S3.1

Strains used in this study.

View this table:
Table S3.2

Plasmids used in this study.

ACKNOWLEDGEMENTS

We would like to thank Dr. Samuel Light for providing the vector pPL2-NOX, expressing the water-forming NADH oxidase for integration into Listeria monocytogenes.

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Posted January 14, 2023.
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Listeria monocytogenes requires DHNA-dependent intracellular redox homeostasis facilitated by Ndh2 for survival and virulence
Hans B. Smith, Kijeong Lee, David M. Stevenson, Daniel Amador-Noguez, John-Demian Sauer
bioRxiv 2023.01.13.524026; doi: https://doi.org/10.1101/2023.01.13.524026
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