ABSTRACT Staphylococcus aureus infections are a major cause of morbidity and mortality in the United States and across the globe. This is largely due to the evolution of multidrug-resistance and the ability of the bacterium to adapt its metabolism and bioenergetics to infect nearly every site of the human body. Thus, there is a significant need for the development of effective therapeutics against this organism. There are several critical gaps in our knowledge of how metabolic pathways used by S. aureus in different environments influence this pathogen virulence in vitro and inside the host. Therefore, our long-term goal is to elucidate how S. aureus receives signals from the environment in the host (e.g. oxygen concentration, nutrients), senses its own redox poise and energy status, and triggers metabolic changes, including the production of virulence factors. Our previous studies identified two respiratory enzymes in S. aureus called type 2 NADH dehydrogenases (NDH-2s: NdhC and NdhF) and revealed their importance for animal infection and organ colonization, and the production of virulence factors and biofilm formation in vitro. These results lead to our central hypothesis that the NADH-dependent respiratory chain is primarily responsible for controlling the NADH/NAD+ and MQH2/MQ (menaquinol/menaquinone) pools that are used by the cell to monitor its redox status and which we propose are major elements regulating virulence via specific global regulators. Guided by strong preliminary data, we propose to pursue three Specific Aims: (1) Determine the metabolic pathways utilized both in the presence and absence of each of the two NADH dehydrogenase enzymes, and the molecular mechanisms by which these enzymes modulate the production of α-toxin; (2) Determine why the presence of both NADH dehydrogenase enzymes is important for biofilm formation; (3) Determine if menaquinol is the signaling molecule that directly induces SrrB and SaeS autokinase activity in the SaeRS and SrrAB two-component systems, which are both critical for the regulation of virulence and biofilm formation. To accomplish these Aims, we have assembled a powerful team to employ a multidisciplinary approach that combines metabolomics, proteomics, genetics, microfluidics technology and biochemistry. Collectively, our proposed studies will have a broad impact on the field by uncovering the role of the respiratory chain in connecting environment signals with the intracellular redox poise that regulates S. aureus virulence. In the long term, these studies may reveal novel therapeutic targets to treat S. aureus-related diseases.