Key driving forces in the biosynthesis of autoinducing peptides required for staphylococcal virulence

Abstract

Staphylococci produce autoinducing peptides (AIPs) as quorumsensing 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.Staphylococcus aureus | quorum sensing | thiolactone | protein homeostasis | thermodynamics of proteolysis

Regulation of Virulence in Staphylococcus aureus : Molecular Mechanisms and Remaining Puzzles

“…This ‘proteolytic cyclization’ process is unusual from a thermodynamic perspective in that it results in the net conversion of a stable peptide bond into a high-energy thioester bond. Reconstitution of this reaction, employing highly purified AgrD peptides and liposome-incorporated AgrB, has confirmed that the thiolactone N-fragment is indeed a kinetically favorable product (Wang et al, 2015). Intriguingly, the proteolytic cyclization exhibited a dynamic equilibrium behavior in the in vitro system.…”

“…Intriguingly, the proteolytic cyclization exhibited a dynamic equilibrium behavior in the in vitro system. The equilibrium constant determined therein revealed that, in order to maintain a sufficient intracellular pool of the thiolactone intermediate to support the rapid production of AIP, S. aureus cells have to efficiently degrade the C-terminal cleavage fragment of AgrD, limiting its half-life to the order of 10 seconds (Wang et al, 2015) (Figure 3C). In other words, the bacterium follows Le Châtelier's principle, harnessing the favorable free energy from the hydrolytic degradation of one cleavage fragment to power the installation of a high-energy thiolactone motif in the other.…”

“…Consequently, AgrB had been surmised to also export and/or cleave the thiolactone intermediate (Zhang et al, 2002). Biochemical studies have since ruled out a role for AgrB in the second proteolysis step (Qiu et al, 2005; Wang et al, 2015) and, while an involvement in AgrD translocation has not been formally ruled out, the absence of an ATP-binding cassette within the enzyme would make such a translocation activity quite extraordinary.…”

Surface-attached molecules control Staphylococcus aureus quorum sensing and biofilm development

“…AIP is processed from the precursor peptide AgrD by AgrB and other proteases, and the AIP is secreted 26,27 . Extracellular AIP is detected by a cognate transmembrane-bound receptor histidine kinase, AgrC, that upon AIP binding, autophosphorylates and subsequently funnels a phosphoryl group to the partner response regulator, AgrA 28,29 (Fig.…”

Structural basis for precursor protein–directed ribosomal peptide macrocyclization

“…The cyclic structures are less susceptible to in vivo degradation and proteolysis and present rigid conformations entropically-favored to bind targets. Several enzymatic strategies are known to catalyze the cyclization of peptides including serine protease-type mechanisms in nonribosomal 23 and ribosomal peptide 24,25 pathways along with ATP-dependent activation paths 10,26 . These approaches often exhibit high degrees of specificity and efficiency that can rival synthetic methods.…”