CcpA Regulates Arginine Biosynthesis in Staphylococcus aureus through Repression of Proline Catabolism

Abstract

Staphylococcus aureus is a leading cause of community-associated and nosocomial infections. Imperative to the success of S. aureus is the ability to adapt and utilize nutrients that are readily available. Genomic sequencing suggests that S. aureus has the genes required for synthesis of all twenty amino acids. However, in vitro experimentation demonstrates that staphylococci have multiple amino acid auxotrophies, including arginine. Although S. aureus possesses the highly conserved anabolic pathway that synthesizes arginine via glutamate, we demonstrate here that inactivation of ccpA facilitates the synthesis of arginine via the urea cycle utilizing proline as a substrate. Mutations within putA, rocD, arcB1, argG and argH abolished the ability of S. aureus JE2 ccpA::tetL to grow in the absence of arginine, whereas an interruption in argJBCF, arcB2, or proC had no effect. Furthermore, nuclear magnetic resonance demonstrated that JE2 ccpA::ermB produced ¹³C ₅ labeled arginine when grown with ¹³C ₅ proline. Taken together, these data support the conclusion that S. aureus synthesizes arginine from proline during growth on secondary carbon sources. Furthermore, although highly conserved in all sequenced S. aureus genomes, the arginine anabolic pathway (ArgJBCDFGH) is not functional under in vitro growth conditions. Finally, a mutation in argH attenuated virulence in a mouse kidney abscess model in comparison to wild type JE2 demonstrating the importance of arginine biosynthesis in vivo via the urea cycle. However, mutations in argB, argF, and putA did not attenuate virulence suggesting both the glutamate and proline pathways are active and they, or their pathway intermediates, can complement each other in vivo.

Author Summary

Although Staphylococcus aureus encodes the highly conserved arginine biosynthesis pathway via glutamate, arginine is an essential amino acid. We found that a mutation in ccpA, a gene encoding a protein facilitating carbon catabolite repression, mediates arginine biosynthesis under in vitro growth conditions. However, both genetic and biochemical evidence suggested that a S. aureus ccpA mutant synthesizes arginine via proline and the urea cycle, a pathway not demonstrated in bacteria before. Furthermore, an animal model of S. aureus infection demonstrated the importance of arginine biosynthesis in vivo. This new pathway sheds light on important host-pathogen interactions and suggests S. aureus has evolved to address arginine depletion in the host by synthesizing arginine from a readily available substrate such as proline.