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During aerobic growth, S. aureus relies on acetate overflow metabolism, a process where glucose is incompletely oxidized to acetate, for its bioenergetic needs. Acetate is not immediately captured as a carbon source and is excreted as waste by cells. The underlying factors governing acetate overflow in S. aureus have not been identified. Here, we show that acetate overflow is favored due to a thermodynamic bottleneck in the TCA cycle specifically involving the oxidation of succinate to fumarate by succinate dehydrogenase. This bottleneck reduces flux through the TCA cycle, making it more efficient for S. aureus to generate ATP via acetate overflow metabolism. Additionally, the protein allocation cost of maintaining ATP flux through the restricted TCA cycle is greater than that of acetate overflow metabolism. Finally, we show that the TCA cycle bottleneck provides S. aureus the flexibility to redirect carbon toward maintaining redox balance through lactate overflow when oxygen becomes limiting, albeit at the expense of ATP production through acetate overflow. Overall, our findings suggest that overflow metabolism offers S. aureus distinct bioenergetic advantages over a thermodynamically constrained TCA cycle, potentially supporting its commensal-pathogenic lifestyle., Competing Interests: The authors declare no conflict of interest.

Staphylococcus aureus is a medically important pathogen with high metabolic versatility allowing it to infect various niches within a host. S. aureus utilizes two major transcriptional regulators, namely, CodY and CcpA, to remodel metabolic and virulence gene expression in response to changing environmental conditions. Previous studies revealed that inactivation of either codY or ccpA has a pronounced impact on different aspects of staphylococcal physiology and pathogenesis. To determine the contribution and interplay of these two regulators in modulating central metabolism, virulence, and biofilm development, we constructed and characterized the codY ccpA double mutant in S. aureus UAMS-1. In line with previous studies, we found that CcpA and CodY control the cellular metabolic status by altering carbon flux through the central and overflow metabolic pathways. Our results demonstrate that ccpA inactivation impairs biofilm formation and decreases incorporation of extracellular DNA (eDNA) into the biofilm matrix, whereas disrupting codY resulted in a robust structured biofilm tethered together with eDNA and polysaccharide intercellular adhesin (PIA). Interestingly, inactivation of both codY and ccpA decreases biofilm biomass and reduces eDNA release in the double mutant. Compared with the inactivation of codY , the codY ccpA mutant did not overexpress toxins but maintained overexpression of amino acid metabolism pathways. Furthermore, the codY ccpA mutant produced large amounts of PIA, in contrast to the wild-type strain and ccpA mutant. Combined, the results of this study suggest that the coordinated action of CodY and CcpA modulate central metabolism, virulence gene expression, and biofilm-associated genes to optimize growth on preferred carbon sources until starvation sets in. IMPORTANCE Staphylococcus aureus is a leading cause of biofilm-associated infections, including infective endocarditis, worldwide. A greater understanding of metabolic forces driving biofilm formation in S. aureus is essential for the identification of novel therapeutic targets and for the development of new strategies to combat this medically important pathogen. This study characterizes the interplay and regulation of central metabolism and biofilm development by two global transcriptional regulators, CodY and CcpA. We found that the lack of CcpA and/or CodY have different impacts on intracellular metabolic status leading to a formation of morphologically altered biofilms. Overall, the results of this study provide new insights into our understanding of metabolism-mediated regulation of biofilm development in S. aureus.

Previous studies have found that arginine biosynthesis in Staphylococcus aureus is repressed via carbon catabolite repression (CcpA), and proline is used as a precursor. Unexpectedly, however, robust growth of S. aureus is not observed in complete defined medium lacking both glucose and arginine (CDM-R). Mutants able to grow on agar-containing defined medium lacking arginine (CDM-R) were selected and found to contain mutations within ahrC , encoding the canonical arginine biosynthesis pathway repressor (AhrC), or single nucleotide polymorphisms (SNPs) upstream of the native arginine deiminase (ADI) operon arcA1B1D1C1 . Reverse transcription-PCR (RT-PCR) studies found that mutations within ccpA or ahrC or SNPs identified upstream of arcA1B1D1C1 increased the transcription of both arcB1 and argGH , encoding ornithine carbamoyltransferase and argininosuccinate synthase/lyase, respectively, facilitating arginine biosynthesis. Furthermore, mutations within the AhrC homologue argR2 facilitated robust growth within CDM-R. Complementation with arcB1 or arcA1B1D1C1 , but not argGH , rescued growth in CDM-R. Finally, supplementation of CDM-R with ornithine stimulated growth, as did mutations in genes ( proC and rocA ) that presumably increased the pyrroline-5-carboxylate and ornithine pools. Collectively, these data suggest that the transcriptional regulation of ornithine carbamoyltransferase and, in addition, the availability of intracellular ornithine pools regulate arginine biosynthesis in S. aureus in the absence of glucose. Surprisingly, ~50% of clinical S. aureus isolates were able to grow in CDM-R. These data suggest that S. aureus is selected to repress arginine biosynthesis in environments with or without glucose; however, mutants may be readily selected that facilitate arginine biosynthesis and growth in specific environments lacking arginine. IMPORTANCE Staphylococcus aureus can cause infection in virtually any niche of the human host, suggesting that it has significant metabolic versatility. Indeed, bioinformatic analysis suggests that it has the biosynthetic capability to synthesize all 20 amino acids. Paradoxically, however, it is conditionally auxotrophic for several amino acids, including arginine. Studies in our laboratory are designed to assess the biological function of amino acid auxotrophy in this significant pathogen. This study reveals that the metabolic block repressing arginine biosynthesis in media lacking glucose is the transcriptional repression of ornithine carbamoyltransferase encoded by arcB1 within the native arginine deiminase operon in addition to limited intracellular pools of ornithine. Surprisingly, approximately 50% of S. aureus clinical isolates can grow in media lacking arginine, suggesting that mutations are selected in S. aureus that allow growth in particular niches of the human host.

Staphylococcus aureus (SA) is a major cause of hospital- and community-associated bacterial infections in the U.S. and around the world. These infections have become increasingly difficult to treat due to the propensity to develop antibiotic resistance and form biofilm. To date, no antibiofilm agents are available for clinical use. To add to the repertoire of antibiotics for clinical use and to provide novel agents for combating both SA and biofilm infections, we previously reported marinopyrroles as potent anti-SA agents. In this study, we used fragment-based and bioisostere approaches to design and synthesize a series of novel fluorinated pyrrolomycins for the first time, performed analyses of their physicochemical and drug-like properties, and investigated structure activity relationships and pharmacokinetics. These promising fluorinated pyrrolomycins demonstrate potent antibacterial activity against SA with favorable drug-like properties and pharmacokinetic profiles. Importantly, these compounds kill staphylococcal biofilm-associated cells with a lack of mammalian cell cytotoxicity and no occurrence of bacterial resistance. Our novel fluorinated pyrrolomycin 4 has a clogP value of 4.1, an MIC of 73 ng/mL, MBC of 4 μg/mL, kill staphylococcal-associated biofilm at 8 μg/mL, bioavailability of 35%, and the elimination half-life of 6.04 h and 6.75 h by intravenous and oral administration, respectively. This is the first report of comprehensive drug discovery studies on pyrrolomycin-based antibiotics., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)