Brucella Omp25c Modulates Host NAD+/NADH Homeostasis via Interaction with the Mitochondrial Complex I Assembly Factor Ndufaf2

Brucellosis, acting as a typical chronic zoonotic disease, is caused by the invasion of Brucella into the human body. Outer membrane protein 25 (Omp25), specifically localized on the Brucella membrane, is the main virulence factor of Brucella and participates in multiple links of the damage process. Omp25c, a porin protein of Brucella, is a paralog of Omp25 with high sequence identity. NADH dehydrogenase [ubiquinone] complex I assembly factor 2 (Ndufaf2) has a key function in cell energy metabolism, particularly in the formation and activity of the mitochondrial respiratory chain. Loss of Ndufaf2 results in oxidative stress and mitochondrial DNA (mtDNA) deletion. However, the functional relationship between Omp25c and Ndufaf2, the underlying mechanism of the proteins, remains unclear. In this work, we purified the Omp25c and Ndufaf2proteins. Our data revealed that Omp25c directly interacts with Ndufaf2, as determined using Biacore analysis. In addition, assays revealed that Ompa2c reshapes the host cell’s redox environment by decreasing the oxidized nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide (NAD+/NADH) ratioand adenosine triphosphate (ATP) production, whereas Ndufaf2 exerts an opposing regulatory effect; Co-expression results further revealed an antagonistic relationship between the two during metabolic processes. These findings provide a new perspective for elucidating the mechanisms of mitochondrial functional regulation in Brucella–host interactions and lay the theoretical and experimental foundation for drug development targeting metabolic interventions to eliminate intracellular pathogens.