Contribution of NADH Dehydrogenase to ATP Synthase I-Mediated Energy Production in Vibrio vulnificus

Vibrio vulnificus, a halophilic estuarine bacterium, poses a significant threat as an opportunistic pathogen upon ingestion of contaminated seafood or through wound infections. Our previous studies have highlighted a notable metabolic reprogramming in V. vulnificus following infection, with up-regulation of NADH dehydrogenase (complex I) observed in vivo, indicating potential disparities in ATP generation mechanisms compared to in vitro conditions. Proton motive force (PMF) is known to energize ATP synthase I for intracellular ATP production. Leveraging transcriptomic analyses, we hypothesized NADH dehydrogenase's involvement in PMF generation. To investigate this hypothesis, we first assessed the transcriptional expression levels of structural genes linked to complexes I to IV using quantitative real-time polymerase chain reaction (qRT-PCR) in a rat peritoneal infection model. Our findings revealed significant up-regulation in the expression of complex I gene, while genes associated with complexes II to IV exhibited down-regulation. Then, we generated a deletion mutant of complex I (Vv1_2074) and found no significant differences in growth or intracellular ATP levels compared to the wild type under in vitro conditions. However, in a rat peritoneal infection model, the mutant exhibited pronounced in vivo growth retardation, reversible upon gene reversion. Intriguingly, the mutant strain displayed significantly reduced intracellular ATP levels in vivo, fully restored upon reversion. These findings suggest the role of NADH dehydrogenase in PMF generation and subsequent ATP synthesis specifically during established V. vulnificus infections in vivo. Moreover, our findings provide additional experimental evidence supporting the phenomenon of metabolic reprogramming post-infection, thereby reinforcing the validity of our systems biology analyses.