Conformational Switch to a β-Turn in a Staphylococcal Quorum Sensing Signal Peptide Causes a Dramatic Increase in Potency

Abstract

We report the solution-phase structures of native signal peptides and related analogs capable of either strongly agonizing or antagonizing the AgrC quorum sensing (QS) receptor in the emerging pathogen Staphylococcus epidermidis. Chronic S. epidermidis infections are often recalcitrant to traditional therapies due to antibiotic resistance and formation of robust biofilms. The accessory gene regulator (agr) QS system plays an important role in biofilm formation in this opportunistic pathogen, and the binding of an autoinducing peptide (AIP) signal to its cognate transmembrane receptor (AgrC) is responsible for controlling agr. Small molecules or peptides capable of modulating this binding event are of significant interest as probes to investigate both the agr system and QS as a potential antivirulence target. We used NMR spectroscopy to characterize the structures of the three native S. epidermidis AIP signals and five non-native analogs with distinct activity profiles in the AgrC-I receptor from S. epidermidis. These studies revealed a suite of structural motifs critical for ligand activity. Interestingly, a unique β-turn was present in the macrocycles of the two most potent AgrC-I modulators-in both an agonist and an antagonistthat was distinct from the macrocycle conformation in the less-potent AgrC-I modulators and in the native AIP-I itself. This previously unknown β-turn provides a structural rationale for these ligands' respective biological activity profiles. Development of analogs to reinforce the β-turn resulted in our first antagonist with subnanomolar potency in AgrC-I, while analogs designed to contain a disrupted β-turn were dramatically less potent relative to their parent compounds. Collectively, these studies provide new insights into the AIP:AgrC interactions crucial for QS activation in S. epidermidis and advance the understanding of QS at the molecular level.

Non-Native Peptides Capable of Pan-Activating the agr Quorum Sensing System across Multiple Specificity Groups of Staphylococcus epidermidis

“…In our prior study, we determined key SARs for group-I agr agonism by AIP-I through synthesizing a set of analogues and assaying their agonism and antagonism profiles in a group-I S. epidermidis reporter strain (assumed to act via competitively binding AgrC-I due to their very close structural similarity to AIP-I). , The experimental methods and AIP-I features most pertinent to the current study are highlighted here (shown in Figure A; select assay data in Table ). Briefly, we performed systematic alanine and d -amino acid scans of the AIP-I structure to ascertain the importance of each residue to agonistic activity (Cys4 was not mutated to Ala as it is required for macrocycle formation).…”

“…Our laboratory performed the first study of the structure–activity relationships (SARs) for agr agonism by S. epidermidis AIP-I as well as a NMR structural study of key agonists and antagonists that we identified side-by-side with the native AIPs-I–III . These past studies suggested that a multigroup agonist could be designed based on two observations.…”

“…These past studies suggested that a multigroup agonist could be designed based on two observations. First, agr activation in group-I S. epidermidis was found to be largely dependent on a single exocyclic residue in AIP-I that likely could be incorporated into new scaffolds . Second, AIP-II and AIP-III do not exhibit cross-group inhibition .…”

Diversity in Sensing and Signaling of Bacterial Sensor Histidine Kinases

“…Structural analysis data of the AIP-AgrC complex are not available at present. However, 3-D solution-phase structures of S. epidermidis AIPs and AIP analogs from NMR spectroscopy have been reported, providing new insights into AIP-AgrC interactions [100].…”

Deciphering Single-Bacterium Adhesion Behavior Modulated by Extracellular Electron Transfer

“…), − biosensors, − environmental pollution treatment, , and the biogeochemical cycle . On the other hand, biofilm formation is harmful to hygiene and health. − For instance, infections caused by pathogenic biofilms (e.g., Pseudomonas aeruginosa ) on the surfaces of medical instruments used in surgery are a serious threat to human health and even to life. − Biofilm formation on substrate surfaces is a complex process involving multiple steps (i.e., initial attachment, growth, maturation, detachment, and redevelopment). − Initial bacterial adhesion to substrate surfaces is the first and most critical step in biofilm formation. ,− Therefore, it is desirable to modulate such adhesion in terms of some common characteristics of EAB, which is challenging since the nature of bacteria-surface interactions, especially at the single-bacterium level, is poorly understood.…”