Staphylococcus aureus encodes four differentially regulated pyruvate transporters

The success of Staphylococcus aureus as a pathogen is attributable, in part, to its ability to exploit the diverse nutrient sources available during infection. Critical to this success are the pathways involving pyruvate that serve as a nexus for energy production, oxidative metabolism, and biosynthetic processes. When available, bacteria acquire pyruvate from the environment to fuel growth. Recently, LrgAB was identified as a pyruvate transporter under microaerobic conditions, leading us to speculate that S. aureus encodes other pyruvate transporters that are active during aerobic growth. In this study, we used the toxic pyruvate analog, 3-fluoropyruvic acid (3-FP), to isolate mutants with impaired pyruvate uptake. Whole-genome sequencing (WGS) of these mutants revealed mutations in two genes, lctP and lldP. Pyruvate uptake was significantly delayed when both lctP and lldP were inactivated. Although LldP and LctP were annotated as L-lactate permeases, ¹⁴C-pyruvate uptake assays confirmed that they function as pyruvate transporters. Despite a reduction in pyruvate uptake, the lctP lldP mutant did not have a growth defect in media with pyruvate, indicating that there may be an additional pyruvate importer. Reassessment of 3-FP susceptibility of the lctP lldP mutant revealed a zone of inhibition, confirming there is another transporter. WGS of 3-FP-resistant lldP lctP mutants identified B7H15_13955, an annotated MFS transporter, as the fourth transporter. Importantly, inactivation of all four genes completely eliminated pyruvate uptake, suggesting we have identified all the pyruvate transporters. These findings reveal that S. aureus employs multiple pyruvate transporters to support pyruvate metabolism under aerobic and anaerobic conditions.IMPORTANCEPyruvate is a key metabolite that supports bacterial energy production in many conditions. While the LrgAB system was previously implicated in pyruvate import under microaerobic conditions, the transporters that enable Staphylococcus aureus pyruvate acquisition during aerobic growth have remained undefined. We identified lctP and lldP, two genes annotated as lactate transporters, and B7H15_13955 as additional pyruvate transporters. Through genetic inactivation, pyruvate consumption, growth, and ¹⁴C-pyruvate uptake assays, we demonstrate that LctP, LldP, and B7H15_13955 are capable of pyruvate import, and with LrgAB, comprise a regulated network for pyruvate acquisition. This discovery fills a critical gap in our understanding of S. aureus metabolic adaptation and reveals that this pathogen is equipped with multiple systems to import pyruvate under diverse environmental conditions.