Influence of the AgrC-AgrA Complex on the Response Time of Staphylococcus aureus Quorum Sensing

Abstract

The Staphylococcus aureus agr quorum-sensing system plays a major role in the transition from the persistent to the virulent phenotype. S. aureus agr type I to IV strains are characterized by mutations in the sensor domain of the histidine kinase AgrC and differences in the sequences of the secreted autoinducing peptides (AIP). Here we demonstrate that interactions between the cytosolic domain of AgrC (AgrC Cyto ) and the response regulator domain of AgrA (AgrA RR ) dictate the spontaneity of the cellular response to AIP stimuli. The crystal structure of AgrC Cyto provided a basis for a mechanistic model of AgrC-AgrA interactions. This model enabled an analysis of the biochemical and biophysical parameters of AgrC-AgrA interactions in the context of the conformational features of the AgrC-AgrA complex. This analysis revealed distinct sequence and conformational features that determine the affinity, specificity, and kinetics of the phosphotransfer reaction. This step, which governs the response time for transcriptional reengineering triggered by an AIP stimulus, is independent of the agr type and similar for agonist and antagonist stimuli. These experimental data could serve as a basis on which to validate simulations of the quorum-sensing response and for strategies that employ the agr quorum-sensing system to combat biofilm formation in S. aureus infections. Multiple pore-forming toxins, immune evasion factors, and adhesins contribute to acute and chronic Staphylococcus aureus infections in humans (1). The expression of most of these virulence factors is controlled by the accessory gene regulator (agr), a two-component regulatory system in S. aureus (2). The sequence composition of the autoinducing peptide (AIP) with a five-member thiolactone ring provides a basis on which to distinguish between different S. aureus strains, also referred to as agr types I to IV. The interaction of the AIP with AgrC, a membranebound histidine kinase, regulates the catalytic activity of AgrC. Mutations in AgrC across S. aureus strains of different agr types are confined to the ectodomain. This observation is consistent with experimental data that suggest that the signal recognition mechanism is confined to the extracellular and membrane-associated components (3). The subsequent steps of signal transduction involve phosphorylation of AgrA by AgrC, followed by the interaction of phosphorylated AgrA with cognate promoters to trigger agr-dependent reengineering of the transcriptional profile. This step incorporates signal amplification due to AgrA-induced expression from the P2 promoter and expression of RNAIII, a downstream effector that regulates the expression of virulence factors and other transcriptional regulators (4). Prominent changes in the transcriptional profile induced by AgrA include the expression of the characterized immune response suppressors ␣ and ␤ phenol-soluble modulins (5). The production and secretion of the AIPs involve the action of the permease AgrB on the AgrD propeptide (6).The sensor module of AgrC (resid...

Genotypic and phenotypic diversity ofStaphylococcus aureusfrom cystic fibrosis lung infections and their interactions withPseudomonas aeruginosa

“…The accessory gene regulatory ( agr ) quorum sensing system controls multiple virulence factors in S. aureus and is thought to be vital in infection [36-38]. There are four known types of this system based on mutations and polymorphisms in the histidine kinase and autoinducer peptides [39]. Among our isolates, there were 24 agr type I, 35 type II, and 6 type III (Table 1).…”

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

“…In all HPK10 sequences, an Asn residue substitutes for the conserved “G1-box” Asp, which normally hydrogen-bonds to the N-6 amino group on the adenine base of the nucleotide. A recent crystal structure of the AgrC CA domain indicates that this Asn residue indeed takes the place of the canonical Asp (Srivastava et al, 2014). Therefore, the substitution is likely responsible for the exceptionally weak affinity between full-length AgrC and ATP: the K m is about 2 mM (Wang et al, 2014) (Figure 5B).…”

The Staphylococcus aureus Two-Component System AgrAC Displays Four Distinct Genomic Arrangements That Delineate Genomic Virulence Factor Signatures

“…We further looked at the specific amino acid variations in the AgrC protein that may have contributed to the distinctive variations in the biochemical properties among the four agr types. For this analysis, we compared the protein sequences of AgrC for each agr Type against a type I AgrC (AgrC-I) from S. aureus (strain COL) as a reference sequence (UniProt: A0A0H2WWL2) (Srivastava et al, 2014). This comparison was limited to AgrC proteins forming canonical gene arrangements, as those in noncanonical arrangements were mostly truncated by insertion sequences and hence amino acid variations would not be correctly identified.…”

“…Finally, we inspected variations which occurred together (referred to as co-occurring mutations) in the cytoplasmic domain, of which the CA domain has been crystallized (Srivastava et al, 2014) to understand the stability of the AgrC proteins for each Type. The mutation of P247 to a threonine corresponds to a phosphotransfer specificity residue (A268) found in a previous study of a similar two-component system (Podgornaia et al, 2013) and does not seem to be a type specific mutation itself.…”

“…Another potential consequence would be an increase in the likelihood of non-cognate receptors to bind (Srivastava et al, 2014;Studer et al, 2013).…”