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 (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.

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

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.

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.

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.