Team:HKUST/Project/Experiment Design

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Experiment Design

1. Construction of Chimeric AIP Receptor
2. Localization Test of Chimeric AIP Receptor
3. Functionality Test of the Chimeric AIP Receptor
4. Construction of RIP Production Cassette
5. Secretion Test of Hybrid Inhibiting Peptides
6. Bio-assay of the Reporter Plasmid
7. Functionality Test of Hybrid Inhibiting Peptides
8.References

1. Construction of Chimeric AIP Receptor    Top

Johnsborg, et al.  reported in his study that the transmembrane domain, other than the cytoplasmic domain, of a pheromone receptor would be responsible for the inducing specificity of corresponding signal molecules [1]. A fusion AIP sensor, AgrC (transmembrane domain) - PlnB (cytoplasmic catalytic domain), is designed to sense AIP molecules released by S.aureus and thereby activate Lactobacillus endogenous response regulators.

We fused the transmembrane domain of AgrC (amino acid 1-188, AIP receptor in S.aureus) with the cytoplasmic HPK domain of PlnB (amino acid 212-441, pheromone receptor in L.plantarum WCFS1). The fusion point is Leucine, a conserved amino acid in both AgrC and PlnB (186 in AgrC/212 in PlnB), linking the transmembrane domain and cytoplasmic HPK domain. The AgrC-PlnB chimeric receptor was cloned into the multiple cloning site of e.coli - Lactobacillus shuttle vector pMG36e, which contains a constitutive promoter P32.

2. Localization Test of Chimeric AIP Receptor    Top

To first examine the proper localization of AgrC-PlnB on the membrane of L. plantarum WCFS1, coding sequence of fluorescence protein mCherry was added after the chimeric receptor AgrC-PlnB. Since the N terminal of AgrC and PlnB is putatively associated with membrane localization, mCherry is fused at C terminal of the fusion construct, as denoted below:

The localization test will be performed by observing pMG36e-transformed L. plantarum WCFS1 under fluorescence microscope.

3. Functionality Test of the Chimeric AIP Receptor    Top

Experiment 1: Test in L.plantarum WCFS1

The GusA reporter assay will be used in testing the functionality of the chimeric receptor AgrC-PlnB in L. plantarum WCFS1. A gusA reporter unit consisting of an inducible plnA promoter and a gusA reporter gene was cloned into pMG36e at upstream of P32 promoter; the agrC/agrC-plnB was cloned into the multiple cloning site of pMG36e which is at the downstream of P32 promoter. As for the control groups, an empty pMG36e and a pMG36e which has only the gusA reporter unit were prepared. The construct-inserted shuttle vectors will then be transformed into L. plantarum WCFS1.

Each of the four groups aforementioned will be treated by 1) AIP (inducing peptide for S. aureus) induction 2) IP (inducing peptide for Lactobacillus) induction and 3) control without pheromone induction.

Due to the existence of endogenous pln locus in L. plantarum WCFS1, response regulator PlnC would possibly bind to the plnA promoter in shuttle vector pMG36e as well as the plnA promoter in L. plantarum WCFS1 genomic DNA. PlnA in the genomic DNA would then synthesize inducing peptides IP, which could bind to the original transmembrane sensor PlnB. Consequently, phosphorylated PlnC would result in background noise for testing our designed constructs. In another word, the natural pathway of pln quorum sensing and the introduced pathway of AIP signal transduction would interfere with each other; the gusA expression level could not directly indicate the response from chimeric AIP sensor.

Once synthesized IP, synthesized AIP or placebo was applied, GusA expression level would be examined every 1 hour for consecutive 10 hours. Group 3) is expected to give the highest level of gusA expression among all AIP-induced groups.

Experiment 2: Test in L.sakei Lb790

To reduce the noise brought by the cross talk between the natural pathway of pln quorum sensing and the introduced pathway of AIP signal transduction, modified constructs will be built and transformed into L. sakei Lb790, a Lactobacillus strain which does not have the pln locus. A new part of plnC following RBS is added to the construct to enable the proper signal transduction.

GusA expression level will be measured 3 hours after pheromone induction. Compared with the control groups, the chimeric receptor AgrC-PlnB would be proved to be functional if the GusA expression was comparatively high.

4. Construction of RIP Production Cassette    Top

An expression cassette (Construct b) is designed to express the hybrid peptide DD13-RIP and drive its secretion to extracellular environment. A signal peptide is introduced to the construct to direct the secretion process since there is no evidence showing that DD13-RIP can be automatically secreted out by L. plantarum. Another construct (Construct a), with only P32 promoter and the hybrid peptide DD13-RIP, serves as a control for the test.

Construct b consists of signal peptide lp_0297, a linker and the hybrid peptide DD13-RIP. The whole construct is cloned into plasmid pMG36e, a shuttle vector in E. coli and Lactobacilli [2]. P32 promoter, a constitutive promoter in E. coli and L. plantarum is located at the upstream of this construct [3, 4]. Signal peptide lp_0297 is selected here due to its high efficiency in secreting peptides and proteins in L. plantarum WCFS1 [5]. The linker is to ensure that the separation of signal peptide and hybrid peptide DD13 – RIP operates well and does not affect the normal function of the hybrid peptide. The original terminator in pMG36e will stop the transcription process. The whole construct is under the regulation of P32 promoter and the aforementioned terminator.

5. Secretion Test of Hybrid Inhibiting Peptides    Top

Since the hybrid peptide DD13-RIP is a 21-residue peptide, for which the secretion efficiency of lp_0297 is unknown, a function test is designed to determine the secretion efficiency. Two constructs will be made in this experiment:

In both constructs, FLAG-tag is added before hybrid peptide DD13-RIP. After transformation and selection, L. plantarum containing either Construct a or b will be harvested at the late exponential or early stationary phase. Western blot will be applied to examine the amount of peptide secreted.

6. Bio-assay of the Reporter Plasmid    Top

Plasmid pRN6683, a P3-blaZ fusion plasmid serving as a reporter gene of RNAIII, is commonly used in bio-assay for RNAIII synthesis [6]P3 promoter in AgrBDCA system is the promoter of RNAIII, and blaZ is a S. aureus β-lactamase encoding gene [7] With P3 promoter and blaZ gene, pRN6683 could reflect the activity level of RNAIII by synthesizing β-lactamase. The activity of β-lactamase is measured through its reaction with nitrocefin [8].

Both pRN6683-transformed and normal S. aureus strains are exposed to nitrocefin in early exponential phase [8]. The cells are then harvested for optical density test. Only pRN6683-transformed cells are expected to have strong degradation effect on nitrocefin.

After confirming the positive transformation of reporter plasmid pRN6683, bio-assay of DD13-RIP can be conducted. According to the methods described above, the supernatant of L. plantarum (transformed with Construct 1 and 2) will be added to S. aureus culture (transformed with pRN6683) at their early exponential stage [6, 9]. A standard curve of the concentration decrease of nitrocefin vs. time can be used to estimate the inhibitive effect of DD13-RIP.

7. Functionality Test of Hybrid Inhibiting Peptides    Top

Both Construct 1 and 2 are also designed to test the inhibitive effect on RNAIII of secreted DD13-RIP. These two constructs will determine the secretion and function of DD13-RIP. After replacing P32 promoter with plnA promoter, this new construct will be made into a new construct with the chimeric AIP receptor.

8.References:    Top

1. Johnsborg, O., Diep, D. B. & Nes, N. F. (2003). Structural analysis of the peptide pheromone receptor plnB, a histidine protein kinase from Lactobacillus plantarum. Journal of Bacteriology, 185 (23), 6913–6920.    Back


2. Gov, Y., Bitler, A., Dell'Acqua, G., Torres, J.V., & Balaban, N. (2001). RNAIII inhibiting peptide (RIP), a global inhibitor of Staphylococcus aureus pathogenesis: structure and function analysis. Peptides, 22(10), 1609-1620.    Back


3. Van de Guchte, M., van der Vossen, J.M.B.M, Kok, J., & Venema, G. (1989). Construction of a Lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl Environ Microbiol, 55(1), 224-228.     Back


4. Van Der Lelie, D., Van Der Vossen, J.M.B.M., & Venema, G. (1987). Isolation and characterization of Streptococcus cremoris wg2-specific promoters. Appl Environ Microbiol, 53(10), 2452-2457.    Back


5. Pretzer, G., Snel, J., Molenaar, D.,  Wiersma, A., Bron, P.A., Lambert, J., de Vos, W.M., van der Meer, R., Smits, M.A., & Kleerebezem, M. (2005). Biodiversity-based identification and functional characterization of the mannose-specific adhesin of Lactobacillus plantarum. J Bacteriol, 187(17), 6128-6136.    Back


6. Mathiesen, G., Sveen, A., Brurberg, M.B., Fredriksen, L., Axelsson, L., & Eijsink, V.G. (2009). Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1. BMC Genomics, 10(425).     Back


7. Saunders, C.W., Schmidt, B.J., Mirot, M.S., Thompson, L.D., & Guyer, M.S. (1984). Use of Chromosomal integration in the establishment and expression of blaz, a Staphylococcus aureus β-lactamase gene, in bacillus subtilis. J Bacteriol, 157(3), 718-726.    Back/li>
   
8. Kiran, M.D., Adikesavan, N.V., Cirioni, O., Giacometti, A., Silvestri, C., Scalise, G., Ghiselli, R., Saba, V., Orlandor, F., Shoham, M., & Balaban, N. (2008). Discovery of a quorum-sensing inhibitor of drug-resistant staphylococcal infections by structure-based virtual screening. Molecular Pharmacology, 73(5), 718-726.    Back


9. Balaban, N., & Novick, R.P. (1995). Autocrine regulation of toxin synthesis by Staphylococcus aureus. Proc Natl Acad Sci U S A, 92(5), 1619-1623.    Back