The Na+-Translocating NADH:Quinone Oxidoreductase Enhances Oxidative Stress in the Cytoplasm of Vibrio cholerae
ABSTRACTWe searched for a source of reactive oxygen species (ROS) in the cytoplasm of the human pathogenVibrio choleraeand addressed the mechanism of ROS formation using the dye 2′,7′-dichlorofluorescein diacetate (DCFH-DA) in respiring cells. By comparingV. choleraestrains with or without active Na+-translocating NADH:quinone oxidoreductase (Na+-NQR), this respiratory sodium ion redox pump was identified as a producer of ROSin vivo. The amount of cytoplasmic ROS detected inV. choleraecells producing variants of Na+-NQR correlated well with rates of superoxide formation by the corresponding membrane fractions. Membranes from wild-typeV. choleraeshowed increased superoxide production activity (9.8 ± 0.6 μmol superoxide min−1mg−1membrane protein) compared to membranes from the mutant lacking Na+-NQR (0.18 ± 0.01 μmol min−1mg−1). Overexpression of plasmid-encoded Na+-NQR in thenqrdeletion strain resulted in a drastic increase in the formation of superoxide (42.6 ± 2.8 μmol min−1mg−1). By analyzing a variant of Na+-NQR devoid of quinone reduction activity, we identified the reduced flavin adenine dinucleotide (FAD) cofactor of cytoplasmic NqrF subunit as the site for intracellular superoxide formation inV. cholerae. The impact of superoxide formation by the Na+-NQR on the virulence ofV. choleraeis discussed.IMPORTANCEIn several studies, it was demonstrated that the Na+-NQR inV. choleraeaffects virulence in a yet unknown manner. We identified the reduced FAD cofactor in the NADH-oxidizing NqrF subunit of the Na+-NQR as the site of superoxide formation in the cytoplasm ofV. cholerae. Our study provides the framework to understand how reactive oxygen species formed during respiration could participate in the regulated expression of virulence factors during the transition from aerobic to microaerophilic (intestinal) habitats. This hypothesis may turn out to be right for many other pathogens which, likeV. cholerae, depend on the Na+-NQR as the sole electrogenic NADH dehydrogenase.