The branched-chain amino acids (BCAAs [Ile, Leu, and Val]) represent important nutrients in bacterial physiology, with roles that range from supporting protein synthesis to signaling and fine-tuning the adaptation to amino acid starvation. In some pathogenic bacteria, the adaptation to amino acid starvation includes induction of virulence gene expression: thus, BCAAs support not only proliferation during infection, but also the evasion of host defenses.

Staphylococcus aureus requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for S. aureus physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed S. aureus uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that S. aureus can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a trans-acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a cis-acting leucine-responsive attenuator, uncovering how S. aureus regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in S. aureus, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on S. aureus environmental adaptation and virulence via CodY.

Summary The branched-chain amino acids (BCAAs) are vital to both growth and virulence of the human pathogen Staphylococcus aureus. In addition to supporting protein synthesis, the BCAAs serve as precursors for branched-chain fatty acids (BCFAs), which are predominant membrane fatty acids, and, in association with the global regulatory protein CodY, the BCAAs are key co-regulators of virulence factors. Despite these critical functions, S. aureus represses Leu and Val synthesis, instead preferring to acquire them from the extracellular milieu. We previously identified BrnQ1 as a BCAA transporter, yet a brnQ1 mutant remained capable of BCAA acquisition. Here, we describe BcaP as an additional BCAA transporter, and determine that it plays a secondary role to BrnQ1 during S. aureus growth in a chemically defined medium. Furthermore, membrane fatty acid composition analysis revealed that BrnQ1, and not BcaP, is required for transporting Leu and Val to be used for iso-BCFA synthesis. Despite a predominant role for BrnQ1 in vitro, both BrnQ1 and BcaP are required for S. aureus fitness in vivo in a hematogenous spread infection model and a nasal colonisation model. These data demonstrate the importance of BrnQ1 and BcaP for growth, environmental adaptation and virulence of S. aureus.

Staphylococcus aureus is a significant cause of human infection. Here, we demonstrate that mutations in the transcriptional repressor of purine biosynthesis, purR, enhance the pathogenic potential of S. aureus. Indeed, systemic infection with purR mutants causes accelerated mortality in mice, which is due to aberrant up-regulation of fibronectin binding proteins (FnBPs). Remarkably, purR mutations can arise upon exposure of S. aureus to stress, such as an intact immune system. In humans, naturally occurring anti-FnBP antibodies exist that, while not protective against recurrent S. aureus infection, ostensibly protect against hypervirulent S. aureus infections. Vaccination studies support this notion, where anti-Fnb antibodies in mice protect against purR hypervirulence. These findings provide a novel link between purine metabolism and virulence in S. aureus.

The branched-chain amino acids (BCAAs; Ile, Leu, and Val) not only are important nutrients for the growth of Staphylococcus aureus but also are corepressors for CodY, which regulates virulence gene expression, implicating BCAAs as an important link between the metabolic state of the cell and virulence. BCAAs are either synthesized intracellularly or acquired from the environment. S. aureus encodes three putative BCAA transporters, designated BrnQ1, BrnQ2, and BrnQ3; their functions have not yet been formally tested. In this study, we mutated all three brnQ paralogs so as to characterize their substrate specificities and their roles in growth in vitro and in vivo. We demonstrated that in the community-associated, methicillin-resistant S. aureus (CA-MRSA) strain USA300, BrnQ1 is involved in uptake of all three BCAAs, BrnQ2 transports Ile, and BrnQ3 does not have a significant role in BCAA transport under the conditions tested. Of the three, only BrnQ1 is essential for USA300 to grow in a chemically defined medium that is limited for Leu or Val. Interestingly, we observed that a brnQ2 mutant grew better than USA300 in media limited for Leu and Val, owing to the fact that this mutation leads to overexpression of brnQ1. In a murine infection model, the brnQ1 mutant was attenuated, but in contrast, brnQ2 mutants had significantly increased virulence compared to that of USA300, a phenotype we suggest is at least partially linked to enhanced in vivo scavenging of Leu and Val through BrnQ1. These data uncover a hitherto-undiscovered connection between nutrient acquisition and virulence in CA-MRSA. Staphylococcus aureus is a highly successful human pathogen that succeeds at infecting virtually every body site, causing skin, soft tissue, respiratory, bone, joint, and endovascular infections (1). Maintenance of metabolic homeostasis is important for its infection process, as the majority of genes necessary for infection identified in large-scale signature-tagged mutagenesis (STM) screens fall into the categories of metabolism, transport, and biosynthesis (2, 3). Acquisition of host-derived nutrients, specifically amino acids, appears to be an important mechanism of meeting nutritional needs, as a large proportion of attenuated STM strains contain mutations in amino acid transporters (3).Amino acid transporters are ubiquitous in bacteria and are typically selective for transporting either one amino acid or several amino acids with related structures (4). The branched-chain amino acids (BCAAs; Ile, Leu, and Val) are hydrophobic amino acids typically found in the core in globular proteins or in the transmembrane domain in cell surface proteins (5). The transport mechanisms described for acquisition of the BCAAs in bacteria include secondary transporters belonging to the Leu, Ile, Val:cation symporter (LIVCS) family (e.g., BrnQ, BraB, BraZ, and BcaP) (4, 6-9) and the LIV-I ABC transporter (10-13). With the exception of an ABC transporter in Streptococcus pneumoniae (14), secondary transport, which couples the movement of an ion...