Abstract by Benjamin Svejdal Bejder

Many Gram-positive bacteria can perform cell-to-cell communication, often referred to as quorum sensing (QS), to orchestrate various biological processes though the secretion and detection of signalling peptides referred to as autoinducing peptides (AIPs). The Staphylococcus aureus accessory gene regulator (agr) system is a well-studied QS system that employs thiolactone-containing AIPs to co-ordinately modulate gene expression. In S. aureus the activation of the agr system is intricately linked to the production a large arsenal of virulence factors that are important for its pathogenesis. All staphylococci have the genetic potential to produce AIPs and the known AIP structures display a large chemical diversity. Nevertheless, the AIPs share certain characteristics making them able to interfere with QS of one another. This QS interference has been proposed to play a role in social interactions between staphylococci in complex microbial communities like on human or animal skin. Many other Gram-positive bacteria contain homologs of the S. aureus agr locus and some have also been linked to virulence, although their physiological function is overall not understood to the same degree. Nevertheless, the conserved nature of these systems imply a certain biological importance and they have been the focus of numerous studies, uncovering important aspects of bacterial physiology and gene regulation.

In this thesis, we focused on elucidating QS mechanisms in Gram-positive bacteria, particularly through the study of AIPs and their role in cell-to-cell communication.

We conducted an extensive systematic study on QS interference using synthetic AIPs, encompassing over 200 potential interactions by AIPs naturally produced by 17 staphylococcal species. Here we report interactions of AIPs with agr systems of S. aureus, Staphylococcus epidermidis and Staphylococcus lugdunensis in a comparable dataset. While previous studies had largely focused on AIPs interfering with agr systems of S. aureus, this work uncovered novel cross-activators and inhibitors, including the most potent inhibitory interaction against S. lugdunensis agr-I observed for an AIP produced by Staphylococcus simulans.

Utilising an improved native chemical ligation (NCL) trapping methodology for AIP identification, we provide evidence for the production of chemically diverse AIPs, as a result of variable N-terminal cleavage from a single AgrD precursor peptide in Staphylococcus chromogenes. We found that the different lengths of AIPs produced by an S. chromogenes isolate affected its own agr system differently, producing two self-activators and a weak self-inhibitor. Preliminary studies hint at the presence of secreted enzymes responsible for variable AIP exotail lengths, presenting a novel phenomenon with potential implications for niche competition.

Finally, our study on AIPs produced by Listeria monocytogenes presented novel insights about their structure, with potential implications for differential agr regulation. We demonstrated that predicted exotail-free thiolactone-containing AIPs from L. monocytogenes can undergo rapid intramolecular rearrangement via S®N acyl shift to form the corresponding homodetic peptide, in a highly pH-dependent manner. We identified only the homodetic peptide from L. monocytogenes culture and subsequently screened both AIP configurations alongside a series of synthetic structural analogues of the short-lived exotail-free thiolactone AIP in luminescence-based reporter strain assays for agr activity of L. monocytogenes. We found evidence to support that in spite of its short-lived nature, the exotail-free thiolactone-containing AIP is highly potent and the rearrangement is potentially a signal turn-off mechanism.