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

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. The end products of the menaquinone (MK) biosynthesis pathway are essential to drive both aerobic and anaerobic cellular respirations. 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 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. Furthermore, 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 its role in the extracellular electron transport 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.

Keywords: 1,4-dihydroxy-2-naphthoate; Listeria monocytogenes; Ndh2; extracellular electron transfer; menaquinone; redox homeostasis.

Fig 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 brain-heart infusion (BHI) culture during experimental setup. (B) High-performance liquid chromatography quantification of fermentation products (acetate and lactate) produced and secreted by indicated L. monocytogenes strains ± NOX plasmid complementation grown in BHI media aerobically at 37°C to stationary phase. Means of percentage lactate production from strains were compared to wild-type percentage lactate. (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 independent experiments. (A and B) Data represent the means ± standard deviation of the means from three independent biological replicates. * indicates statistical significance compared to wild-type or indicated comparisons determined by one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparisons test. ns, not significant (**, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001).

Fig 2

Restoration of redox homeostasis rescues virulence defects associated with DHNA deficiency. (A) Indicated L. monocytogenes strains (multiplicity of Infection [MOI] of 10) ± NOX plasmid complementation were tested for cytosolic survival in immortalized IFNAR^−/− bone marrow-derived macrophages (BMDMs) over a 6-hour 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 three independent experiments. Error bars represent the standard deviation of the means of technical triplicates within the representative experiment. (A and B) * indicates statistical significance by one-way ANOVA with Bonferroni’s multiple comparisons test in which means were compared to wild type (A) or ΔmenB at the 8-hour time point (B). (C) Bacterial burdens from the spleen and liver were enumerated at 48-hour 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 represent the median of the group. * indicates statistical significance between wild-type or indicated comparisons determined by Mann-Whitney test. ns, not significant (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001).

Fig 3

DHNA production or supplementation promotes similar effects to NOX complementation in L. monocytogenes. (A) NAD⁺/NADH ratios of indicated L. monocytogenes strains ± 5 µM exogenous DHNA supplementation grown aerobically at 37°C in defined medium to mid-logarithmic phase. Again, ΔmenB was 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, and means were compared to wild type. (B) HPLC quantification of fermentation products (acetate and lactate) produced and secreted by indicated L. monocytogenes strains ± exogenous DHNA supplementation grown in BHI media aerobically at 37°C to stationary phase. Data are an average of three independent biological replicates, and error bars represent the standard deviation of the mean. Means of percentage lactate production of strains were compared to wild-type percentage lactate. (C) Indicated L. monocytogenes strains (MOI of 10) ± DHNA supplementation were tested for cytosolic survival in primary IFNAR^−/− BMDMs over a 6-hour 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. Data represent the means ± standard deviation of the means of three independent biological replicates. In each panel, * indicates statistical significance compared to wild-type or indicated comparisons determined by one-way ANOVA with Bonferroni’s multiple comparisons test. Statistical analysis was performed only at the 8-hour time point for (D). ns, not significant (*, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001).

Fig 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) 5 µM DHNA supplementation aerobically at 37°C and monitored for OD₆₀₀ over 20 hours. (C) ΔmenB/ndh2::Tn L. monocytogenes was grown aerobically in defined medium with either 5 µM DHNA or 5 µM MK and monitored for growth (OD₆₀₀) over 20 hours. All data represent one representative out of three independent biological replicates.

Fig 5

Ndh2 is necessary for DHNA utilization in macrophages ex vivo. Intracellular growth of indicated strains of L. monocytogenes was assayed in both untreated and DHNA-treated BMDMs at an MOI of 0.2. BMDMs were treated with media containing 5 µM DHNA for 30 minutes prior to infection. Growth curves are representative of two independent experiments. Error bars represent the standard deviation of the means of technical triplicates within the representative experiment. * indicates statistical significance relative to ΔmenB at the 8-hour time point determined by one-way ANOVA with Bonferroni’s multiple comparison test. (****, P ≤ 0.0001).