Revision as of 05:58, 10 October 2017

Detection

Quorum sensing of Staphylococcus aureus

Staphylococcus aureus is an opportunistic and invasive pathogen that utilizes quorum sensing (QS), a cell-to-cell signaling mechanism, to strengthen its ability to cause disease in humans. QS allows S. aureus to monitor their surroundings and population size, and regulate the production of virulence factors. As shown in Fig. 1, QS in S. aureus is regulated by the agr operon which consists of two transcription units agrBDCA and RNAIII. The genes agrBDCA are controlled by an inducible promoter termed P2. When regulatory proteins bind to P2, these four genes start to be transcribed and then translated to give four different proteins which are the transmembrane protein AgrB, the precursor peptide AgrD, the receptor protein AgrC and the regulator protein AgrA. The agrD encodes precursor peptides and will be post-transcriptionally processed by AgrB to generate a functional QS signal molecule auto-inducing peptides (AIPs) (George and Muir, 2007). When AIPs are secreted from the cells into the external environment, it can be detected by AgrC present on the S. aureus cell surface. The binding of AIP to AgrC phosphorylates AgrA which has a higher affinity to interact with P2 than the un-phosphorylated form (Koenig et al., 2004). Upon binding to the P2 promoter, AgrA upregulates the transcription of the agr gene and leads to a higher production of AIP.

**Figure 1 The agr system. The arrows with a filled head show the positive feedback loop.**

Auto-inducing peptides (AIPs)

AIPs secreted by Staphylococcus aureus are seven to nine amino acids in length and have the C-terminal five residues constrained as a thiolactone ring through a cysteine side chain (Fig. 2). There are four different classes in the Agr systems which are referred to as Agr-I, Agr-II, Agr-III, and Agr-IV and each is capable of recognizing a unique AIP structure which is respectively AIP-I, AIP-II, AIP-III and AIP-IV. S. aureus may also be classified into four groups/strains (I to IV) by the class of the AIPs produced. Among the four classes of AIPs, the five-residue thiolactone ring structure is always conserved, while the other ring and tail residues differ. Similarly, the proteins involved in signal biosynthesis (AgrB and AgrD) and surface receptor binding (AgrC) also show variability. Interestingly, different AIP signals cross-inhibit the activity of the others (Mayville et al. 1999). For example, group I S. aureus’ s quorum sensing can be activated by AIP-I but is inhibited by the AIPs produced by group II or III S. aureus strains. Since AIP-I and AIP-IV differ by only one amino acid, they are grouped together and can function interchangeably. Indeed, Lyon et al. (2002) demonstrates that, the three AIP groups (group I/IV, group II and group III) can cross-inhibit each other with binding constants in the low nanomolar range. Therefore, when conducting experiments, the groups of S.aureus strains and the classes of AIPs must match to avoid QS down-regulation. In our project, we chose the group I S. aureus strain and AIP-I to verify our parts efficiency.

**Figure 2 The structure of four AIP signals of S. aureus and their cross-inhibitory interactions.**

The sensing device

Our project tried to incorporate a part of the QS system as the sensing device to detect the existence of S. aureus and upregulate multiple network elements. The detection of AIP-I signal by AgrC results in auto-phosphorylation of AgrC followed by transfer of the phosphate group to AgrA. Phosphorylated AgrA has a higher affinity for binding to P2 and upregulates the target genes.

The reporter protein

In this year’s project, we have checked a range of reporter genes. The luciferase reporter gene and the green fluorescent protein (GFP) reporter gene were considered in the initial plan. Thereafter, in the light of long shipment time of the luciferase assay kit, we turned to utilize sfGFP as the reporter. Most existing variants of GFP often mis-fold when expressed as fusion proteins. Though there exist some better-folded variants of GFPs that can be utilized as protein fusion tags, yet the fused proteins can reduce the folding yield and ﬂuorescence of these GFPs. Hence, we adopted to employ sfGFP (superfolder GFP) developed by Jean-Denis Pedelacq’s team (2006) as our visualizing reporter tool, which is a more robustly folded version of GFP. The merit of sfGFP is that the ﬂuorescence from this protein is independent of mis-folding of the fusion partner and is directly proportional to total expression level regardless of the solubility of the fusion, thus superfolder GFP makes itself a robust reporter of both fusion protein expression and pathway (will show later). Hence, sfGFP was used as a reporter in our project this year for its superb folding stability.