Team:XJTLU-CHINA/Detection
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.
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.
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.
Nisin production in Lactococcus lactis
We used the promoter PnisA as the promoter of the circuit because it can be induced by nisin. Nisin is a 34 amino acid anti-microbial peptide produced by the probiotic bacterium Lactococcus lactis. It can lead to cell death by binding to Lipid II, an essential precursor for cell-wall synthesis, and forming small pores in the cytoplasmic membrane which can lead to leakage of small molecules such as ATP. It is widely used as food preservative due to its broad host spectrum. In the food industry, nisin is not chemically synthesized, but it is obtained from the culture of L. lactis in natural substrates, such as milk or dextrose. Biosynthesis of nisin is a complex process. It is encoded by a cluster of 11 genes, of which the first gene, nisA, encodes the precursor of nisin. The nisB, nisC, nisP, and nisT genes are involved in the modification, translocation and processing of nisin. The nisI, nisF, nisE, and nisG genes are for the immunity against nisin. The regulation on the expression of the nisin genes are controlled by nisR and nisK, which are, respectively, the response regulator and the sensor kinase of the bacterial two-component signal transduction systems (TCS). NisK is a histidine–protein kinase that resides in the cytoplasmic membrane and act as a receptor for the mature nisin molecule. Upon binding of nisin to NisK, NisK auto-phosphorylates and subsequently transfers the phosphate group to NisR. The activated NisR induces transcription from two of the three promoters in the nisin gene cluster: PnisA and PnisF.
The strain Lactococcus lactis NZ9000
The strain of bacteria we chose, Lactococcus lactis NZ9000, is created by integrating the nisK and nisR genes for the signal transduction system into the pepN gene (broad range amino peptidase) of L. lactis subsp. cremoris MG1363 (nisin-negative). When a gene of interest is subsequently placed behind the inducible promoter PnisA in the plasmid pNZ8148, the expression of that gene can be induced by adding sub-inhibitory concentrations of nisin (0.1 – 5 ng/ml) to the culture medium.
The plasmid pNZ8148
The plasmid we used, pNZ8148, is a broad host range vector. It carries the chloramphenicol resistant gene and has a PnisA promoter followed by an NcoI restriction site for translational fusions at the ATG sequence. It came originally from the plasmid pSH71 in Lactococcus lactis. The replicon of the vector pNZ8148 contributes to the replication of this plasmid in many Gram-positive bacteria, such as Lactobacillus plantarum and Streptococcus thermophilus. However, its replication in E. coli needs to conduct in a recA+ strain. A recA- strain, for example, DH5α, cannot be used for these vectors because the nisin promoter PnisA is not completely repressed in E. coli, allowing expression of the gene products that can be toxic or lethal. Therefore, to this extent, it is preferred to transform a ligation mixture directly to a Lactococcus lactis strain.
Testing the pathway
The biopart BBa_K515005, which contains an RBS and the sfGFP gene, was assembled at the C terminal of our composite part K2309003 by the conventional restriction digestion and cloning method. Thereafter, by using Gibson Assembly, our composite part was integrated in pNZ8148 at the PstI restriction site. Our detection part was constructed as follows:
Final Assembly
The visualization or detection protocols
Nisin induction of gene expression in Lactococcus lactis
1. Grow 5 ml culture in M17 overnight at 30°C.
2. Dilute 1/25 in 2 x 10 ml fresh medium (30°C).
3. Grow the culture until OD600 ≈ 0.4.
4. Induce one 10 ml culture by culturing with 1 ng/ml nisin and keep the other 10 ml culture as negative control.
5. Incubate 2 - 3 hours, measure OD600 to monitor the growth of the induced and non-induced cultures.
6. Induce one culture with AIP (at a final concentration of 10 µM) and keep the other induced without inducing.
7. Measure the fluorescence signals in the culture by a fluorescence plate reader 3 hours later.
References
George EA, Muir TW (2007) Molecular mechanisms of agr quorum sensing in virulent Staphylococci. ChemBioChem 8: 847–855
Koenig RL, Ray JL, Maleki SJ, Smeltzer MS, Hurlburt BK (2004) Staphylococcus aureus AgrA binding to the RNAIII-agr regulatory region. J Bacteriol 186: 7549–7555
Lyon, G. J., J. S. Wright, T. W. Muir, and R. P. Novick. 2002. Key determinants of receptor activation in the agr autoinducing peptides of Staphylococcus aureus. Biochemistry 41:10095-10104.
Mayville, P., G. Ji, R. Beavis, H. Yang, M. Goger, R. P. Novick, and T. W. Muir. 1999. Structure-activity analysis of synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence. Proc. Natl. Acad. Sci. USA 96:1218-1223.
Pedelacq, J., Cabantous, S., Tran, T. et al. (2015) ‘Engineering and characterization of a superfolder green fluorescent protein’, Nature Biotechnology 24, 79 - 88 (2005) [Online]. DOI: 10.1038/nbt1172
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