Key driving forces in the biosynthesis of autoinducing peptides required for staphylococcal virulence.
Staphylococci produce autoinducing peptides (AIPs) as quorum-sensing signals that regulate virulence. These AIPs feature a thiolactone macrocycle that connects the peptide C terminus to the side chain of an internal cysteine. AIPs are processed from ribosomally synthesized precursors [accessory gene regulator D (AgrD)] through two proteolytic events. Formation of the thiolactone is coupled to the first of these and involves the activity of the integral membrane protease AgrB. This step is expected to be thermodynamically unfavorable, and therefore, it is unclear how AIP-producing bacteria produce sufficient amounts of the thiolactone-containing intermediate to drive quorum sensing. Herein, we present the in vitro reconstitution of the AgrB-dependent proteolysis of an AgrD precursor from Staphylococcus aureus. Our data show that efficient thiolactone production is driven by two unanticipated features of the system: (i) membrane association of the thiolactone-containing intermediate, which stabilizes the macrocycle, and (ii) rapid degradation of the C-terminal proteolysis fragment AgrD(C), which affects the reaction equilibrium position. Cell-based studies confirm the intimate link between AIP production and intracellular AgrD(C) levels. Thus, our studies explain the chemical principles that drive AIP production, including uncovering a hitherto unknown link between quorum sensing and peptide turnover.
Department of Chemistry, Frick Chemistry Laboratory, Princeton University, Princeton, NJ 08544; Graduate Program, The Rockefeller University, New York, NY 10065;
Boyuan
B
Wang
Department of Chemistry, Frick Chemistry Laboratory, Princeton University, Princeton, NJ 08544;
Aishan
A
Zhao
Department of Microbiology, Skirball Institute, New York University Medical Center, New York, NY 10016.
Richard P
RP
Novick
Department of Chemistry, Frick Chemistry Laboratory, Princeton University, Princeton, NJ 08544; muir@princeton.edu.
Tom W
TW
Muir
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D002474:Cell-Free System; D015964:Gene Expression Regulation, Bacterial; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D011510:Proteolipids; D059748:Proteolysis; D053038:Quorum Sensing / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D013816:Thermodynamics; D014774:Virulence / Q000502:physiology*
C414533:AgrB protein, Staphylococcus aureus; C414532:AgrD protein, Staphylococcus; D001426:Bacterial Proteins; D010456:Peptides, Cyclic; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; C020485:proteoliposomes
Staphylococcus aureus; protein homeostasis; quorum sensing; thermodynamics of proteolysis; thiolactone