Fig 1.
Influence of NAD+ regeneration on Spn physiology.
(A) Scheme of NAD+/NADH-associated metabolism in Spn and the targets tested in this study by mutation: ldh, lactate dehydrogenase; adh and adhE, alcohol dehydrogenase; pdhC, pyruvate dehydrogenase complex; nox, NADH oxidase. (B) Levels of NAD(H) measured in designated Spn and the ratio of NAD+/NADH (n = 3). (C) Intracellular ATP concentration and (D) growth curve of mutant Spn and wild-type (WT) control (n = 4). ATP concentration was measured during exponential phase of growth (OD620 = 0.4). The data underlying Fig 1B, 1C and 1D can be found in S1 Data. Statistical analyses were performed using a one-way ANOVA with Dunnett post hoc test. Asterisks indicate statistical significance: *, P ≤ 0.05, **, P ≤ 0.01; ***, P ≤ 0.001, ****, P ≤ 0.0001.
Fig 2.
Disruption of NAD+ regeneration alters Spn virulence factor production and in vivo fitness.
(A) Representative immuno dot blot for capsular polysaccharide using anti-serotype 4 antibody (done in triplicate) and relative capsule amount on the bacterial surface as determined using an FITC dextran exclusion assay. Pneumococci with greater capsule have a larger mean area per diplococcus. For each strain, pneumococci in 30 objective images were analyzed. (B) Representative immunoblot for pneumolysin and PspA using lab-generated polyclonal antibody. (C) Results of a competitive index assay for WT Spn (W) and designated mutants (M) following 1:1 coinfection of mice in a pneumonia model (n = 5–6). Burden in the lungs at 48-hour infection was used for the comparative analysis. The data underlying Fig 2A and 2C can be found in S1 Data. Statistical analyses were done using a one-way ANOVA with Dunnett post hoc test compared to its WT. Asterisks indicate statistical significance: *, P ≤ 0.05, **, P ≤ 0.01; ****, P ≤ 0.0001.
Fig 3.
Disruption of NAD+ regeneration genes influences antibiotic susceptibility.
Spn wild type and its isogenic mutants were enumerated following treatment with erythromycin (50 μg/ml), gentamicin (5 μg/ml), chloramphenicol (10 μg/ml), penicillin (10 μg/ml), vancomycin (10 μg/ml), and rifampicin (50 μg/ml). The data underlying this figure can be found in S1 Data. Statistics analyses was done using a Mann–Whitney t test with comparison made between wild type antibiotic-treated versus mutant antibiotic-treated samples (n ≥ 3) Asterisks indicate statistical significance: *, P ≤ 0.05, **, P ≤ 0.01; ***, P ≤ 0.001.
Fig 4.
Efflux capability of Spn TIGR4 and mutants.
Efflux pump capability of Spn TIGR4 WT and its isogenic mutants was performed using EtBr fluorescence measurement. The relative fluorescence level of EtBr compared to its initial time point (T0), i.e., the ability to pump out the fluorescent stain, was displayed from each sample, which were monitored every 5 minutes for 30 minutes. The data underlying this figure can be found in S1 Data. (n = 6)
Fig 5.
Fomepizole treatment decreases the NAD(H) pool, impairs ATP production, and enhances ribosomal targeting antibiotic efficacy.
(A) A schematic of the fomepizole mechanism. Fomepizole treatment of Spn for 2 hours resulted in (B) alteration of the NAD(H) pool (n ≥ 4) and (C) reduced intracellular ATP concentrations (n ≥ 4). (D) Heat plots of microdilution checkerboard assay, an assay for bacterial viability, following addition of fomepizole and erythromycin, kanamycin, gentamicin, rifampicin, penicillin, and vancomycin. At least 3 independent assays were performed. (n ≥ 3). The data underlying Fig 5B, 5C and 5D can be found in S1 Data. Statistical analyses were done with the Mann–Whitney t test and one-way ANOVA. Asterisks indicate statistical significance: *, P ≤ 0.05, **, P ≤ 0.01; ***, P ≤ 0.001.
Table 1.
MIC alteration of Spn TIGR4 by the combination of antibiotics and fomepizole.
Fig 6.
Fomepizole enhances erythromycin susceptibility in multidrug-resistant Spn pneumonia and invasive disease.
The potential of fomepizole as an adjuvant was tested in a pneumonia model using multidrug-resistant Spn serotype 35B. (A) Infection was done intratracheally, with erythromycin and fomepizole injected intraperitoneally 18 hours post-infection. At 48 hours post-infection, animals were killed, and the bacterial burden in the (B) lungs, (C) spleen, and (D) heart were determined (n ≥ 8). Statistical analyses were done using a Kruskal–Wallis ANOVA test with Dunnett post hoc test with control group. Asterisks indicate statistical significance: *, P ≤ 0.05, **, P ≤ 0.01; ***, P ≤ 0.001.