Quorum-sensing, intra- and inter-species competition in the staphylococci

Abstract

In Gram-positive bacteria such as Staphylococcus aureus and the coagulase-negative staphylococci (CoNS), the accessory gene regulator (agr) is a highly conserved but polymorphic quorum-sensing system involved in colonization, virulence and biofilm development. Signalling via agr depends on the interaction of an autoinducing peptide (AIP) with AgrC, a transmembrane sensor kinase that, once phosphorylated activates the response regulator AgrA. This in turn autoinduces AIP biosynthesis and drives target gene expression directly via AgrA or via the post-transcriptional regulator, RNAIII. In this review we describe the molecular mechanisms underlying the agr-mediated generation of, and response to, AIPs and the molecular basis of AIP-dependent activation and inhibition of AgrC. How the environment impacts on agr functionality is considered and the consequences of agr dysfunction for infection explored. We also discuss the concept of AIP-driven competitive interference between S. aureus and the CoNS and its anti-infective potential.

Keywords: Staphylococcus aureus; agr; autoinducing peptides; inter-bacterial competition; quorum sensing; staphylococci.

Fig. 1.

The agr QS system in S. aureus negatively (-) regulates the production of capsular polysaccharides and multiple cell-wall proteins involved in host protein interactions including immunoglobulin, fibronectin and fibrinogen binding proteins. agr positively (+) regulates the expression of diverse virulence factor genes including those coding for tissue degrading exoenzymes, haemolysins, enterotoxins, exfoliative toxins leukocidins and phenol soluble modulins.

Fig. 2.

(a) Schematic of the staphylococcal agr quorum-sensing system. The agrBDCA locus is composed of two divergent transcripts, RNAII and RNAIII, driven by the agrP2 and agrP3 promoters, respectively. AgrD, the pro-peptide precursor of the autoinducing peptide (AIP) is processed at the cytoplasmic membrane by AgrB and MroQ such that AIPs are released extracellularly. AIPs bind to and activate the AgrC receptor, a membrane-bound histidine sensor kinase resulting in phosphorylation of the response regulator AgrA and activation of the agrP2 and agrP3 promoters. This drives the autoinduction circuitry to generate more AIP signal molecules and induces expression of virulence genes either indirectly via RNAIII or directly via the target gene promoters. (b) Schematic showing the hypervariable region (in green/cyan) of the agrBDCA locus incorporating agrD and giving rise to the different agr groups. Amino acid residues marking the beginning and ends of the variable regions are numbered (adapted from [142]). (c) Generalized AIP structure and summary table showing the amino acid sequences of the AIPs belonging to each of the four S. aureus agr groups and their cross-group activities. The brackets denote the amino acid residues within the macrocycle. In S. aureus , the AIP N-terminal tails have two, three or four amino acid residues.

Fig. 3.

Schematic showing the processing of AgrD pro-peptides to generate the active cyclic AIP signal molecules. (a) Amino acid sequences of S. aureus AgrD1-D4 showing the AgrB cleavage site and the MroQ sites for AgrD1, D2 and D4. MroQ does not cleave AgrD3. (b) Processing of AgrD by AgrB and MroQ to release N-AgrD and the cyclic AIP. (c) Schematic showing the formation and release of the AIP and N-AgrD at the cytoplasmic membrane. Cleavage of AgrD by AgrB releases a 14 amino acid C-terminal peptide (AgrD-C), which is degraded in the cytoplasm. N-AgrD-AIP is cleaved by MroQ to release N-AgrD and the mature AIP. The mechanism by which the AIP and N-AgrD are exported is not known.

Fig. 4.

Conformation of membrane-embedded ternary complex AgrB₂/AgrD after molecular dynamics simulations. The two AgrB proteins are inequivalent with AgrB-I (cornflower blue) guiding substrate AgrD (fuchsia) to the active site involving catalytic AgrB-II (blue). AgrD residues C28 and M32 are close to each other and to catalytic AgrB-II C84. Key interactions stabilizing the complex include AgrB-I K139-D33 and AgrB-I R70-E34 contacts with AgrD, and AgrB-II H77-F30 via π-interactions. Sidechains are colour-coded aqua (C28, C84 AgrB-II), grey (M32 AgrD), pink (F30 AgrD), purple (H77 AgrB-II), fuchsia (E34 AgrD), green (D33 AgrD), yellow (K139 AgrB-I) and orange (R70 AgrB-I). All four AgrB termini are on the cytosolic side (top) consistent with six transmembrane domain topology of AgrB. The (AgrB)₂/AgrD complex orientation here is shown with cytosol at the top offering a better view of the cytosol-accessible active site (adapted from [51]).

Fig. 5.

Topological model of transmembrane endopeptidase MroQ. The homology model of MroQ (unpublished) was obtained from I-TASSER [143] and annealed in atomistic MD simulations using NAMD [144] within a membrane patch built in CHARMM-GUI [145]. The model reveals an eight TMD topology – lateral view (left); and axial view (right). Residues Glu141 and Glu142 and His180 and His 213 required for MroQ functionality are shown within the membrane region where they have access to the hydrophobic AgrD substrate.

Fig. 6.

Architecture of the AgrC and AgrA two-component sensor and response regulator proteins. Models of the homodimeric sensor kinase AgrC (left) (orange/cornflower blue) and response regulator AgrA (right) (cyan) associated with the agrP2 promoter site. Initial conformations of AgrC and AgrA were obtained from AlphaFold [146]. The AgrC dimer was built using ClusPro [147]. Lipid phosphates are shown to mark the membrane interface (P atoms are shown in cyan). His239, responsible for AgrC autophosphorylation, is shown in purple; Arg238 (yellow) and Gln305 (fuchsia) are important for the molecular ‘latch’ that stabilizes AgrC in the ‘off state’ [67]. The AgrA model was superimposed onto the structure of the AgrA C-terminal DNA-binding domain [PDB:3BS1] [71]. Asp59 (green) phosphorylation by AgrC is important for activation of AgrA. His169 (purple), Asn201 (fuchsia) and Arg233 (orange-red) are responsible for DNA recognition.

Fig. 7.

SAR for S. aureus AIP-1 showing how minor modifications to the peptide sequence including Ala-scanning influence activity. Substitution of the Asp residue (d⁵ ) with Ala to give (Ala⁵)AIP-1 converted AIP-1 from an activator of AgrC1 to a potent cross group inhibitor. IC₅₀ data from [29].

Fig. 8.

(a) Intracellular uptake of S. aureus into non-professional epithelial and endothelial cells is independent of agr expression. Once internalized, agr expression precedes endosomal escape by facilitating endosomal lysis via α-haemolysin or the PSMs. This enables S. aureus to replicate and persist within the cytoplasm protected from host immune defences and antibiotics or to lyse the host cells and establish further rounds of uptake and release leading to tissue destruction. (b) Fluorescence microscopy of S. aureus transformed with an agrP3-gfp reporter invading mammary epithelial cells and stained with DAPI (DNA; blue) and an anti-tubulin-Cy3 conjugate (red; microtubules). After 2 h incubation, intracellular staphylococcal cells are observed (white arrow) with red cell walls as the Cy3 antibody conjugate has bound to protein A showing that agr has not yet been activated. As the infection proceeds to 6 h agr expression has clearly been induced as observed by the high level of gfp expression (green) in the bacterial cells (adapted from [14]).