Molecular Mechanisms of Virulence Regulation in Staphylococcus aureus: a Journey into Reconstitutive Biochemistry

Conspectus:

Methodological development in the fields of genetics, chemical biology, and biochemistry over the last several decades have provided researchers with a diverse set of powerful tools to investigate biological processes. Leveraging these innovations in concert, scientists can now characterize biological pathways at a level of complexity ranging from systems biology down to molecular and atomic detail.

Throughout the Account, we illustrate how discoveries made using these tools build on each other to develop a comprehensive understanding of biological pathways. Advancements in genetic sequencing facilitates association of genotypes and phenotypes, independent of biochemical mechanism. Through the biochemical reconstitution of the interactions between biological macromolecules—including the small molecules (ligands and metabolites) and proteins—that participate in these biological pathways, scientists can characterize the specific molecular features that link genotype and phenotype. This facilitates identification of targets within these pathways that can be manipulated to achieve a greater understanding of the biological process or to develop interventions to improve human health outcomes.

Specifically, we describe how this toolbox was leveraged to discover and characterize the molecular biochemistry underlying control of pathogenicity in the Gram-positive bacterium Staphylococcus aureus. Concurrent with advancements in the investigative tools available to the scientific community, we and others reported on the genetic, molecular, and biochemical/biophysical components of this regulatory system. Virulence control in S. aureus is achieved through a chemical system of bacterial cell-to-cell communication indexed to local population density, referred to as quorum sensing (QS). We and our collaborators identified that this QS system is encoded in the accessory gene regulator (agr) operon, and functions via the biosynthesis, secretion, and accumulation of a short peptide signaling molecule—the autoinducing peptide (AIP)—in the local environment correlated with the growth of S. aureus in the same biological niche. Above a threshold concentration, these AIPs bind and activate a cell-surface receptor to stimulate an intracellular response resulting in altered gene expression and bacterial group behaviors. We discovered that chemical modification of these AIPs often generates molecules that exhibit potent inhibition of agr QS, with demonstrated therapeutic potential to treat S. aureus infections. We went on to characterize the biochemical mechanism of signaling molecule biosynthesis and receptor activation in controlled systems through in vitro reconstitution of the constituent enzymes and substrates. Biochemical reconstitution enabled quantitative assessment of biophysical parameters. These efforts culminated in the comprehensive characterization and functional in vitro reconstitution of agr QS in a synthetic system in a minimal model at the interface of genotype, mechanism, and phenotype.

1. Introduction

It is becoming increasingly apparent that complex systems of communication are, in some form, ubiquitous among living organisms.⁴ However, it was not until the 1960s that communication systems were discovered in microorganisms.^{5, 6} Studies by Tomasz and Nealson identified signaling systems in Streptococcus pneumoniae and Vibrio fischeri wherein bacterial behaviors—genetic competence and bioluminescence, respectively—were regulated by secreted biomolecules.^{5, 6} The concentration of these biomolecules, referred to as autoinducers, was found correlate with cell density.^{5, 6} Nealson and colleagues went on to identify the autoinducer in Vibrio fischeri as an acyl-homoserine lactone (AHL).⁷ Over the following decades, a growing body of evidence suggested that similar systems of cellular communication and gene regulation, based on cell-density and autoinducer concentration in a system known as quorum sensing (QS), are widespread among Gram-negative bacteria and are capable of both intraspecies and interspecies information transfer.⁸

In Gram-positive bacteria, the agr circuit in Staphylococcus aureus serves as the model QS system. Gram-negative and Gram-positive QS systems share many key components: a secreted small molecule autoinducer correlated with local population density, a mechanism to identify this signal, and the translation of this stimulus into altered gene expression (Figure 1A).⁸ The key difference lies in the chemical identity of the autoinducer; in Gram-positive bacteria, a small autoinducing peptide (AIP) serves as the signaling molecule for QS.⁸ In S. aureus, upon reaching a threshold concentration of AIP in the local environment, a receptor histidine kinase (RHK) AgrC binds AIP.⁹ This results in a conformational change that promotes AgrC autophosphorylation and phosphoryltransfer to an intracellular response regulator protein, AgrA. Phosphorylated AgrA (P-AgrA) induces the transcription of a number of genes, leading to the regulation of S. aureus cell-adhesion and virulence behaviors. P-AgrA also induces AIP biosynthesis, resulting in a positive feedback loop. AIPs are ribosomally synthesized and post-translationally modified peptides (RiPPs) generated in two steps from a precursor peptide, AgrD. An integral membrane protease, AgrB, cleaves the C-terminal 18 amino acids from AgrD and cyclizes the biosynthetic intermediate by forging a thiolactone between the C-terminus and an internal cysteine in AgrD. This intermediate is then cleaved by a second membrane peptidase, which in some cases has been shown to be MroQ, to yield the mature AIP, which is secreted into the extracellular space (Figure 1B).^{3, 9}

Figure 1.

Overview of Staphylococcus aureus agr quorum sensing. (a) Conceptional framework of bacterial quorum sensing. Through the continuous secretion and recognition of an extracellular signaling molecule, bacteria can coordinate group behaviors relative to local population density. (b) Overview of the S. aureus agr quorum sensing system.

This Account summarizes our progress in identifying the key players in agr quorum sensing in S. aureus, characterizing the molecular details of autoinduction and inhibition in this system, and understanding the biochemical mechanism of signal biosynthesis, signal transduction, behavioral control, and signal propagation. In particular, we highlight how work in our laboratory integrates and builds on staphylococcus microbial genetics with synthetic chemistry and enzymology to achieve a molecular understanding of this system. Furthermore, tools developed in support of these studies have had broad impacts on the field of chemical biology and furthered our ability to study biological phenomena and develop drugs and materials in support of human health.

2. Sequencing and mutagenesis as catalysts for discovery in bacterial quorum sensing

2.1. Annotation and functional characterization of the agr locus

Comprehensive genetic screens performed in the early 1980s by several groups employing phenotypically distinct strains and transposon mutants, led to the identification of many of the disease relevant genes in S. aureus.^10–18 One mutant deficient in hemolytic enterotoxin activity was found to be distinct; the mutation did not occur in the coding region for the toxin.¹⁹ Richard Novick and colleagues sequenced this strain and discovered a locus that encoded a global regulatory system for S. aureus virulence.¹⁹ This system was observed to function through a secreted signaling molecule; spent media produced a replicable phenotype of protein expression and secretion in naïve cultures.¹⁹

Novick, his colleagues, and his collaborators spent the next four decades characterizing this complex genetic locus, termed the accessory gene regulator (agr). Sequencing of this region identified three promoters (P1-P3) controlling the transcription of at least two transcripts. One of these promoters (P2) was demonstrated to control the expression of a polycistronic mRNA (RNAII) encoding a protease (agrB), a short peptide of unknown function (later determined to be agrD), and a canonical two component sensory system (TCS) comprised of a receptor histidine kinase (agrC) and a response regulator (agrA).²⁰ Each of these genes was found to be indispensable for agr signaling.²⁰ Novick and coworkers identified that one of the other promoters, designated P3, regulates the production of RNAIIl—a 514 nucleotide multifunctional transcript that both encodes δ-hemolysin and acts as a regulatory RNA.²¹ Specifically, RNAIII was demonstrated to selectively up- or downregulate gene products through direct interactions with target gene transcripts or potential intermediary protein factors.²¹ Therefore, RNAIII was determined to be the effector of agr gene regulation and control of pathogenicity, establishing both a mechanism and a model for the regulatory activity of this complex polycistronic locus.²¹ The third agr promoter, P1, is suggested to regulate the expression of AgrA.¹⁹ However, this promoter is poorly characterized relative to agr P2 and P3.^{19, 22}

2.2. Discovery of staphyloccocal agr variants and cross-inhibition

In subsequent studies, the Novick group set out to identify the gene product of the uncharacterized open reading frame in RNAII. This gene, designated agrD, was determined to encode the precursor to a peptide pheromone, encoded as a RiPP. Processing of agrD was shown to yield the AIP for S. aureus agr QS.^{1, 23} Efforts to sequence and characterize behaviorally and functionally distinct S. aureus strains led to the discovery of hypervariability in the agr locus, specifically within RNAII. This phenomenon gives rise to four distinct sequences of agrC, agrB, and agrD, each of which encodes a unique AIP-AgrC pair (Figure 2A).^{24, 25} Numbered -I to -IV, these “specificity groups” were found to be cross-inhibitory; for example, while a group-II AIP acts as an agonist for the group-II AgrC, it is a potent inhibitor of the group-I, -III, and -IV receptors (Figure 2A).^{24, 26} This inhibition prevents co-colonization of S. aureus strains with different agr specificity groups in the same niche of an infected host.²⁷ Similar agr-type QS systems, often displaying hypervariability and intra-species cross inhibition, were subsequently identified by sequence homology in many Gram-positive bacteria including L. monocytogenes and E. faecalis (Figure 2B and Table 1).^28–33 Inter-species cross inhibition has also been identified among Staphylococci; in a recent study, 82% of tested Staphylococci produced AIPs inhibit S. aureus agr signaling.^{34, 35} This manifests itself in infection and colonization behaviors, as agr cross-inhibition is implicated as the mechanism by which S. epidermidis is predominantly found over S. aureus on skin.³⁵ Beyond staphylococcal agr systems, unrelated bacteria in the same biological niche have been found to produce small molecule signals that interfere with agr signaling. For example, in Pseudomonas aeruginosa and S. aureus co-colonization of lungs in cystic fibrosis patients, an N-acylhomoserine lactone produced by P. aeruginosa inhibits S. aureus growth via agr interference.³⁶

Figure 2.

Diversity and conservation of agr signaling among Staphylococcus aureus subspecies and other Gram-positive bacteria. (a) Hypervariability in the agr locus spanning the region encoding the C-terminus of AgrB, through AgrD, and the N-terminus of AgrC, results in four subtypes of agr signaling, referred to as specificity groups. Each specificity group encodes one specific AIP, AgrC, and AgrB. Interactions between AIPs and AgrCs from the same specificity group result in agr activation. However, AIPs act as inverse agonists when bound to AgrCs from different specificity groups. (b) Genetic neighborhood network identifying homologues of the agr locus are found in many bacilli. These agr operons exhibit significant sequence similarity, encoding the same biosynthetic pathways (agrD & agrB) and signaling systems (agrC & agrA). Assembled using EFI-GNT.³⁷

Table 1.

agr and agr homologues encoding quorum sensing systems in Gram-positive bacteria

Species	agr locus	Sequence of AIP
Staphylococcus aureus	agr (agrBDCA)	YST(CDFIM)cyc
		GVNA(CSSLF)cyc
		IN(CDFLL)cyc
		YST(CYFIM)cyc
S. epidermidis	agr	DSV(CASYF)cyc
		NASKYNP(CNSYL)cyc
		NAAKYNP(CASYL)cyc
S. lugdunensis	agr	DI(CNAYF)cyc
		DMN(CNGYF)cyc
S. saprophyticus	agr	TINP(CFGYT)cyc
S. pseudintermedius	agr	RIPT(STGFF)cyc
		RIPI(STGFF)cyc
		KIPT(STGFF)cyc
		KYPT(STGFF)cyc
Clostridium botulinum	agrD-I, agrD-II	ACYW(CVYQP)cyc
		ADSA(CHLGI)cyc
C. difficile	agr, agrD-II	ANST(CPWII)cyc
		NSA(SSWVA)cyc
C. perfringens	agrBD	ATSA(CIWFT)cyc
Enterococcus faecalis	fsr (fsrBDCA)	QN(SPNIFGQWM)cyc
Listeria monocytogenes	agr	A(CFMFV)cyc
		(CFMFV)cyc†
Lactobacillus planatarum	lam (lamBDCA)	(CVGIW)cyc

(X₁aaaX₂)_cyc designates the presence of a peptide macrocycle. The side chain of X₁ forms a thiolactone or lactone with the C-terminal carboxylate of X₂

Red amnio acids in the macrocyclic domain indicate conserved hydrophobic motifs

^†The L. monocytogenes AIP, CFMFV, is cyclized through a thiolactone with the N-terminal cysteine side chain.

To characterize agr specificity-group cross-inhibition and quantify agonist and inhibitor strength, biological reporter systems were developed. For each specificity group, agrC and agrA under control of the agr-P2 promoter, and a reporter gene (e.g. blaZ) under control of the agr-P3 promoter was inserted into an agr-null background strain. In the absence of AgrD and AgrB, these strains were incapable of producing their own AIP and would express β-lactamase at a level proportional to the concentration of AIP added to the growth media. These strains have been widely used in diagnostic medicine, the discovery of novel specificity groups, investigations of S. aureus virulence, screens for novel AgrC agonists and inhibitors, and the analysis of the potency of these compounds.^{1–3, 25–27, 34, 38–45}

3. Chemical synthesis of AIPs and derivatives drive understanding of AIP mediated AgrC inhibition and activation

3.1. Confirmation of the molecular identity of S. aureus AIPs

The previous section describes in vivo studies and bacterial genetics that successfully identified many of the key components of the agr virulence-regulation system and established a basic model for its function. However, these studies did not confirm the molecular identity of the signaling molecule responsible for agr signaling in the context of every other exoprotein and secreted product. To identify and characterize the composition and structure of this AIP and other related molecules requires synthesis of the authentic product.

Studies in the Novick group had suggested AIPs contain a macrocycle linked via a thiolactone involving an internal cysteine thiol to the C-terminus.^{23, 24} Since our group was well versed in the manufacture of thioester-containing peptides through our work on native chemical ligation and expressed protein ligation^{46, 47}, the synthesis of these putative AIP structures was of great interest to us, particularly since access to native and non-native agr associated molecules might facilitate investigation of the biochemical processes that underly agr activation and virulence regulation, and perhaps even inform therapeutic development. Thus, was initiated a highly productive and enjoyable collaboration between the Muir and Novick groups, one that has continued for over 25 years and as described below, evolved far beyond the initial focus on AIP synthesis.

A robust synthetic route to the putative AIP-I molecule (i.e., from S. aureus specificity group I) was established, involving an intramolecular transthioesterification step, and the resulting purified peptide found to replicate the phenotype of agr activation produced by spent media, indicating that this molecule, with its unusual thiolactone macrocycle, was necessary and sufficient to activate agr signaling.¹ A similar synthetic strategy was also used to generate the predicted AIP-II, -III, and -IV molecules, confirming that the macrocycle is a conserved feature of the S. aureus autoinducers (Figure 2A).^{1, 25}

Competitive inhibition among the different specificity groups of S. aureus suggests a potential therapeutic modality; given that native activators of one specificity group serve as inhibitors of agr QS in other specificity groups, these molecules could be used to modulate S. aureus virulence. To this end, it is worth noting that in our initial publication on the formal characterization of AIP structures through synthesis (dating from 1999), we showed that a synthetic AIP-II antagonist could be used to attenuate abscess formation in a murine model of group-I S. aureus skin infection (Figure 3A).¹ This represented the first demonstration that pharmacological manipulation of a bacterial quorum sensing pathway could have therapeutic value.

Figure 3.

AgrC inhibition and therapeutic potential of modified AIP homologues. (a) AIP-II was shown to attenuate abscess formation in a murine model of S. aureus skin infection by an agr group-I strain. (b) Overview of AIP SAR model. (c) Truncation of the tail region of AIP-II (tr-AIP-II) generates a global inhibitor model of AgrC. (d) Selected examples of engineered AIP homologues demonstrating AgrC inhibition.^{48, 49} (e) Random nonstandard Peptides Integrated Discovery (RaPID) selection of AgrC inhibitors.⁴² (f) QQ-3, one of several potent multi-group AgrC inhibitors identified by RaPID screening.

3.2. Structure activity relationship studies on the AIP scaffold

The synthetic accessibility and demonstrated therapeutic potential of AIP molecules has led to significant efforts to further investigate the molecular details of agr activation and develop potent and broadly active agr inhibitors. Modifying the native scaffold of AIPs, we were able to identify key residues and motifs that contribute to binding affinity, agonism, and antagonism through the synthesis and investigation of numerous peptides. Alanine substitution of the AIP tail region residues was shown to result in differential activity but relatively minor changes to cross-inhibition.¹ Conversely, an alanine scan of the AIP macrocyclic domain identified that most of the residues in this portion of the molecule significantly influenced receptor binding affinity, with the key motif being a conserved hydrophobic patch consisting of aliphatic and/or aromatic amino acids.^{1, 39} This region was found to be required for AgrC activation and inhibition, and is conserved in most staphylococcal AIPs (Table 1).^{1, 39}

Expansion of the hydrophobic domain size, through the incorporation of additional atoms or additional amino acid residues in the macrocycle, also had significant effects on AIP/AgrC interaction.⁴¹ Constricting the macrocycle by deletion of amino acids or the substitution of a proline completely abolished AgrC binding affinity. Similarly, expansion of the macrocycle domain, even by modifications as small as the insertion of a methylene group, significantly decreased AgrC binding by these AIP analogues.⁴¹ AgrC activation by cognate AIPs was also found to be highly sensitive to the identity of the macrocyclic linker; substituting the thiolactone for a lactone or lactam abolishes AIP agonism while retaining potent cross-strain inhibition.¹ Taken together, these SAR studies give rise to a model of AgrC activation wherein the identity of the tail region amino acids affect agonism and the macrocyclic region affects AgrC binding, establishing a model for the design of molecules that activate or inhibit agr signaling (Figure 3B).

3.3. Therapeutic development of agr modulators based on SAR studies

Consistent with the role of the macrocyclic domain in receptor binding, truncating the tail domain of AIPs generates potent AgrC inhibitors that display no agonism.² In fact, efforts by our group demonstrated that truncation of the tail region of AIP-II generates a potent global inhibitor of S. aureus agr, meaning that this molecule successfully inhibits all four agr specificity groups (Figure 3C).² Diversification of this scaffold yielded a small library of derivatives, all of which yielded low-nanomolar IC₅₀ inhibitors of AgrC from multiple specificity groups.⁵⁰ Building on these efforts, several groups have further elaborated the AIP scaffold in S. aureus and beyond, leading to the generation of an ever expanding set of specific and highly potent agr inhibitors (Figure 3D).^{49, 51–56}

Modern advances in assay development have led to the rise of unbiased library screens to identify novel inhibitors to specific targets. Among the various approaches now available, mRNA display has emerged a powerful platform technology for the discovery of potent binders to receptors of interest.⁵⁷ In collaboration with the group of Hiro Suga, we adapted the Random nonstandard Peptides Integrated Discovery (RaPID) system towards the identification of AgrC-binding compounds, several of which demonstrated potent multi-group inhibition (Figure 3E&F).⁴² Furthermore, this study represents the first selection against a membrane protein target using RaPID, expanding the utility of this technology. High throughput screening approaches leveraging small molecule compound libraries have also been applied to agr inhibitor discovery. Blackwell, Horswill, and others have identified several novel potent agr inhibitors, expanding the chemical space of scaffolds for inhibitor development.^{43, 58} Of these inhibitors, the best characterized is savirin; a small molecule that binds to the DNA binding domain of AgrA and interferes with promoter binding to inhibit agr signaling.⁵⁸ Each of these scaffolds enables the development of chemical biology probes targeting specific components of the agr circuit and provide alternative strategies for therapeutic development against this system.⁵⁹

AIPs have also proven amenable to surface conjugation to develop materials resistant to bacterial colonization. In collaboration with the Bassler group, we demonstrated that surfaces treated with AIP-PEG conjugates resisted S. aureus colonization by stimulating agr-mediated biofilm dispersal and downregulation of cell-adhesion proteins.⁶⁰ Given that hospital acquired bacterial infections are often driven by the development of colony biofilms on implanted medical devices and high-touch surfaces, we anticipate that effective biofilm-resistant materials could significantly reduce medical burden and improve patient outcomes.⁶¹

3.4. Investigations of agr leads to the development of pHis antibodies

A longstanding issue in the biological investigation of phosphohistidine (pHis) as a post translational modification (PTM) is the lack of robust detection methods.⁶² This PTM is best known for its role in bacterial TCS signaling, essential to the mechanism by which AgrC and other RHKs perform transmembrane signal transduction.^63–65 The isolation of natural pHis-modified proteins or the generation of synthetic pHis peptides is complicated by two factors; (1) hydrolysis, as the modification is readily hydrolyzed under acidic conditions, and (2) regio-isomerism, as pHis exists in two isomeric forms (1-pHis and 3-pHis) in vivo, but isomerizes from 1-pHis to 3-pHis during synthesis.^{62, 66} Our group developed synthetic strategies to produce stable phosphohistidine analogues, enabling the development of the first effective pHis antibodies and access to stable modified pHis targets.^66–69 These antibodies were used to study AgrC autophosphorylation and pHis as a functional PTM on several other signaling and metabolic proteins.^67–70 The availability of these antibodies has allowed the identification of many thousands of pHis sites in eukaryotes and prokaryotes, most of which remain functionally uncharacterized.⁷¹

4. Biochemical reconstitution of agr signaling events informs a molecular understanding of this system.

While knockout models are sufficient to connect a phenotype to a genotype, they do not provide causal evidence or provide details about function. Much like synthesis of authentic products confirms the molecular identity of biomolecules, the expression and investigation of specific proteins empowers researchers to characterize their function, enzymology, and structure. In the spirit of Richard Feynman’s axiom “what I cannot create, I do not understand” we set out to create models of agr protein function.

4.1. Engineering, biochemical reconstitution, and structural analysis of AgrC reveals the mechanism of transmembrane signal transduction in agr QS

In parallel with efforts to investigate the role of specific chemical features of AIPs in AgrC binding and agonism (described in 3.2), we embarked on studies to elucidate the biochemical mechanism of AgrC transmembrane signal transduction. RHKs canonically function as dimers.⁷² Activation in model systems derived from E. coli demonstrated that RHK autophosphorylation typically occurs in trans, meaning that the intracellular kinase domain of one monomer acts on the histidine phospho-acceptor of the partner monomer.^{73, 74} In initial studies carried out in collaboration with the Novick group, we confirmed that AgrC functions as a homodimer.⁷⁵ Through the engineering and investigation of obligate mutant AgrC heterodimers in vivo, wherein one AgrC monomer complements the partial activity of the other, we determined that activation of AgrC dimers by AIP occurred in trans (Figure 4A).⁷⁵ These studies also led to the identification of key residues in the AgrC extracellular sensor domain that function to identify cognate AIPs. Using the Group-I and Group-IV AIP/AgrC interactions as a test case, our group and others found that swapping key residues in the central extracellular loop from the sequence of AgrC-I to AgrC-IV switches the receptor from recognizing AIP-I to AIP-IV for activation in vivo.^{76, 77} These studies provide key structural and mechanistic insights into the binding interactions at the AIP/AgrC interface and the position and function of the CA in the biochemical mechanism of AgrC dimer autophosphorylation.

Figure 4.

Structural biology and enzymology inform a model for the biochemical mechanism of AgrC activation. (a) AgrC dimers are activated in trans. (b) A “molecular latch” sequesters the AgrC kinase domain from its phospho-acceptor histidine in the basal state. (c) Biochemical reconstitution of AgrC activity through solubilization in membrane scaffold protein (MSP) lipid nanodiscs. (d) Model for the biochemical mechanism of AgrC activation; AIP agonist binding is predicted to induce structural changes in AgrC that rotate the Dimerization and Histidine phosphotransfer (DHp) helices counterclockwise, releasing the kinase domain from the “molecular latch” and exposing the catalytic histidine.

While no cryo-EM or crystal structures exist of a full-length class 10 HPK, the AgrC sensor domain has been structurally characterized by topology mapping through enzyme accessibility assays and computational prediction. These efforts suggest that AgrC has six to seven transmembrane segments (TMSs).^{76, 78, 79} We solved the crystal structure of the AgrC cytosolic domain, which provided a number of insights into receptor activation. In particular, this structure revealed that, in the basal state, a key arginine residue (R238) within the Dimerization and Histidine phosphotransfer (DHp) domain of the kinase engages the catalytic domain to prevent autophosphorylation of the histidine phospho-acceptor residue through a sequestration mechanism (Figure 4B).⁸⁰ This is consistent with in vivo mutagenesis assays; mutating R238 to remove this “molecular latch” renders AgrC constitutively active.^{80, 81}

To further study AgrC in its active and native state in vitro required the development of a system to functionally reconstitute the full length RHK. While traditional methods to reconstitute integral membrane proteins leverage model membranes such as detergent micelles or liposomes, these strategies often fail to faithfully reconstitute the lipid environment, which can impact the structure and function of proteins. Instead, we leveraged phospholipid bilayer nanodiscs to reconstitute AgrC.^{70, 82} Key to this system is solvent accessibility to both sides of the membrane, allowing us to deliver ligands (AIP) to the sensor domain and substrates (ATP) to the kinase domain simultaneously in the same system (Figure 4C). AgrC was found to be hypersensitive to membrane environment, and was only able to transduce AIP binding to the sensor domain into kinase activation when reconstituted in lipids mimicking the anionic composition of the staphylococcal membrane.^{70, 83} In these reconstituted systems, we were able to directly observe AgrC autophosphorylation and phosphoryltransfer to AgrA, leveraging autoradiography and phosphohistidine antibodies as readouts.^{70, 83}

Together with structural and biochemical insights, we proposed a mechanism for AgrC activation upon AIP binding. Rotation of the central DHp helices of AgrC would disrupt the interaction of the “molecular latch” with the kinase domain. By engineering disulfides into the dimerization helices of AgrC at differential relative positions, varying degrees of rotational torque could be applied to the helical bundle.^{70, 80, 83} Activity assays demonstrated that autophosphorylation of AgrC increases with counterclockwise helical rotation, whereas clockwise helical rotation was inactivating.^{70, 80} Therefore, we postulate that AIP agonist binding induces counterclockwise helical rotation, and an inverse agonist binding induces clockwise helical rotation (Figure 4D).^{70, 80, 83}

4.2. Targeted mutagenesis and biochemical reconstitution of AgrB activity on AgrD facilitates kinetic characterization and investigation of its biochemical mechanism

Early mutagenesis studies in agr established a link between the activity of AgrB and the production of AIP.^{20, 84} Therefore, it was postulated that AgrB cleaves AgrD as part of the AIP biosynthetic pathway.⁸⁵ The model for this interaction predicts that cleavage occurs at the inner leaflet of the cell membrane.⁸⁴ AgrD is highly lipophilic, integrating with the inner leaflet of the cell membrane through its N-terminal amphipathic helix.⁸⁴ Furthermore, AgrB is predicted by sequence homology to be a canonical integral membrane cysteine protease, with a catalytic dyad of histidine (H77) and cysteine (C84) confirmed by mutagenesis.⁸⁵ These key catalytic residues have been shown to be positioned at the intracellular face of the cell membrane as determined by topology mapping studies and computational prediction, suggesting that cleavage occurs inside the cell (Figure 5A & B).^{85, 86}

Figure 5.

Biochemical mechanism of AgrB activity and AIP biosynthesis. (a) Predicted topology, orientation, binding, and activity of AgrB. (b) Binding model of the AgrB dimer and AgrD. At left, ribbon models depict this binding interaction. In these models, one AgrB monomer is shown in grey while the second monomer is shown in blue. AgrD-I shown in orange. In the inset, the cleavage site residues are depicted in black. Only one AgrD is shown for clarity. A hydrophobicity space-filling model is depicted at right. Models generated using AlphaFold Multimer.⁸⁸ (c) Biochemical mechanism and driving forces of AgrD cleavage by AgrB during AIP biosynthesis.

Inspired by the success of the in vitro studies on AgrC, our group undertook the biochemical reconstitution of AgrB, allowing the proteolytic activity of the enzyme on AgrD to be characterized.⁸⁷ These studies identified several key aspects of AgrB enzymology, including its dimeric state and reversible mechanism.⁸⁷ Interestingly, we found that the equilibrium of the cleavage reaction heavily favors the full-length AgrD substrate, as the reaction is thermodynamically unfavorable owing to the generation of the high energy thiolactone in the N-terminal cleavage product.⁸⁷ Biochemical and genetic studies subsequently revealed that the driving force in the conversion of AgrD to AIP biosynthetic intermediate is derived from the preferential degradation of the byproduct—the AgrD C-terminal domain (Figure 5C).⁸⁷ Furthermore, we identified that the newly generated biosynthetic intermediate is stabilized by membrane interaction and protected from recombination with the negatively charged C-terminal domain by sequestration in the membrane.⁸⁷ Notably, this reaction does not produce AIP, instead producing the biosynthetic intermediate AgrD (1–32) thiolactone.

5. Functional Reconstitution of the agr QS pathway

In this final section, we describe functional reconstitution of the agr signaling pathway from purified components, the culmination of our decades long investigations into the molecular mechanisms of this system.

5.1. Identification of the second protease in AIP biosynthesis

The early genetic studies on agr did not identify a second protease in the RNAII agr operon (agrBDCA), or in any of the ORFs controlled by the three agr promoters. Moreover, our in vitro biochemical studies on purified AgrB ruled out that it can perform the second proteolytic cleavage step needed to generate the mature AIP from AgrD.⁸⁷ Thus, there must be a “missing” protease in the AIP biosynthetic pathway. In many other Gram-positive QS systems, AIP biosynthesis and secretion occurs through the cleavage of a N-terminal leader peptide by signal peptidases.^{89, 90} Therefore, Horswill and coworkers investigated the activity of the S. aureus signal peptidase I (SpsB) on short linear peptide analogues of the AIP biosynthetic intermediate.⁹¹ These model peptides were successfully cleaved by truncated analogues of SpsB in vitro, but this activity could not be confirmed in vivo because spsB is an essential gene.⁹¹ Our group further investigated the association of SpsB and AIP biosynthesis, leveraging strains with compensatory mutations for SpsB activity to enable spsB knockout, and determined that ΔspsB had no significant effect on AIP biosynthesis.³

Important clues to the identity of the missing protease in the AIP biosynthetic pathway came from studies employing an S. aureus transposon mutagenesis library which identified a predicted membrane metalloprotease, designated mroQ, whose disruption led to decreased AIP biosynthesis and agr signaling.^{92, 93} We postulated that this protease catalyzed the second and final proteolytic cleavage event in AIP biosynthesis.

The investigation of MroQ echoes the methodologies pioneered by the Novick and Muir groups throughout the last three decades of investigation into S. aureus agr signaling. Mutagenesis studies revealed that mroQ was essential for AIP-I and -II, but not AIP-III biosynthesis.³ Expression and reconstitution of MroQ activity in vitro demonstrated that the protein was necessary and sufficient to cleave the AIP biosynthetic intermediate in groups -I and -II to produce mature AIP (Figure 6A). Through the synthesis of designer chimeric peptide analogues of AgrD (1–32) thiolactone, we also identified the substrate recognition and cleavage site specificity determinants of MroQ and established a model for specificity-group specific recognition and cleavage (Figure 6B).³

Figure 6.

The role of MroQ in AIP biosynthesis. (a) MroQ efficiently and specifically cleaves the AIP biosynthetic intermediate, AgrD (1–32) thiolactone, in vitro. (b) MroQ recognizes sequence features of the AgrD (1–32) thiolactone substrate to direct specificity group-specific cleavage.

5.2. One-pot reconstitution of the entire agr signaling circuit in vitro

With the successful characterization of MroQ, the stage was set to attempt the reconstitution of agr signaling in vitro, an undertaking that we viewed as perhaps the ultimate test of our understanding of this critical QS circuit.³ With this in mind, a one pot model of the agr circuit—featuring AIP biosynthesis, AIP recognition by its cognate receptor, and transmembrane signal transduction—was developed (Figure 7). It is worth emphasizing the technical challenges associated with this undertaking given that all of the components were either integral membrane or membrane associated proteins. Coincubation of purified recombinant AgrD and reconstituted recombinant AgrB, MroQ, and AgrC resulted in AgrC autophosphorylation that was dependent upon AIP agonism, indicative of proper agr signaling (Figure 7). This success of this experiment highlights the degree to which a molecular understanding of the entire signaling pathway, from the intracellular biochemistry (AIP biosynthesis) to extracellular signal recognition and transduction of this signal across the membrane (AgrC activation), had been achieved. It also represented the first synthetic reconstitution of a quorum sensing system in vitro.³

Figure 7.

Biochemical reconstitution of the biosynthetic and receptor sensing arms of the agr circuit. Observed activation of AgrC, as evidenced by autophosphorylation, indicates that we identified and successfully reproduced the necessary and sufficient conditions for agr signaling. Adapted with permission from Zhao et al. 2022.³ Copyright 2022, PNAS.

5.3. Future biochemical investigation of S. aureus agr quorum sensing; remaining puzzles

While the efforts of our group and others have greatly improved our understanding of S. aureus agr QS, there are many avenues for further discovery and innovation in this field. Chief among these outstanding questions is the identity of the second protease in AIP biosynthesis in agr group-III strains. MroQ only functions in AIP-I, AIP-II, and by extension AIP-IV biosynthesis.^{3, 94} Identifying the protease responsible for AIP-III production would greatly improve our understanding of S. aureus evolutionary divergence and putative speciation. Similarly, mroQ homologues have been identified in many species with agr-like QS systems. It would be interesting to explore if MroQ is a universal component of agr QS. If so, inhibition of MroQ and MroQ analogues could potentially be an effective therapeutic for the control of Gram-positive bacterial infections. Medicinal chemistry on the synthetic scaffold of MroQ substrates or small-molecule/mRNA screening assays would be an effective strategy for the development of MroQ inhibitors.

Additionally, we also do not yet understand how AIP is secreted from S. aureus. MroQ may have additional functions as a transporter, or another as-yet-unidentified protein or proteins may facilitate AIP secretion. Alternatively, AIP secretion may occur spontaneously as part of the biosynthesis pathway or freely diffuse across the membrane.⁹⁵ Lastly, despite exhaustive efforts to understand AgrC activation by AIP, we have not yet solved the structure of the AgrC sensor domain in complex with AIPs. A detailed understanding of this binding pocket and the differential binding modes of agonists and inverse agonists would greatly inform the rational design of improved AgrC inhibitors, and potentially bring AgrC inhibition into the clinic as an effective control measure against S. aureus infections.

Advances in microbial genetics, synthetic chemistry, and enzymology are greatly enabling for the investigation of uncharacterized proteins and pathways. We hope that the reconstitutive biochemistry approach laid out in our long research journey into the agr QS, supported by genomics and synthetic chemistry, serves as a model for the future discovery of exciting targets and their functional characterization in S. aureus agr signaling and beyond.

Biographies

Steven P. Bodine was born in Washington, DC, and grew up in Chevy Chase, MD. He attended Middlebury College, performing research under Prof. Roger Sandwick and graduating with a B.A. in Biochemistry in 2016. He then joined the group of Dr. William A. Gahl as a postbaccalaureate research fellow at the National Institutes of Health. From 2018 to 2024, Steven pursued his Ph.D. in Chemistry working with Prof. Tom W. Muir. Dr. Bodine is currently a postdoctoral scholar advised by Prof. Carolyn R. Bertozzi at Stanford University.

Tom W. Muir was born in Stranraer, in the extreme southwest of Scotland. He received his B.Sc. and Ph.D. in Chemistry from the University of Edinburgh in 1989 and 1993 respectively, advised by Prof. Robert Ramage, FRS. He pursued his postdoctoral studies with Prof. Stephen B. H. Kent at The Scripps Research Institute. In 1996, he began his independent career at The Rockefeller University, where he rose through the ranks eventually being appointed the Richard E. Salomon Family Professor and Director of the Pels Center of Chemistry, Biochemistry and Structural Biology. Prof. Muir joined the faculty of the Princeton University Chemistry Department in 2011 as the Van Zandt Williams Jr. Class of ’65 Professor of Chemistry, serving as Chair from 2015–2020.