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Team:HKUST/Project/Module 1
From 2010.igem.org
Autoinducing Peptide (AIP) Sensor on Lactobacillus Cell Membrane
Background:
1. Two-Component Signaling System (TCS)
In many bacterial species, Two-Component Signaling System (TCS) plays a prominent role in quorum sensing. A classical TCS consists of a transmembrane receptor, which is a histidine protein kinase (HPK), and a cytoplasmic response regulator (RR) [1].When bound by autoinducing peptides (AIP), the transmembrane receptor HPK passes a phosphate group to the downstream cytoplasmic response regulator RR. The cytoplasmic response regulator will then up-regulates or down-regulates various virulence and bacteriocin productions under specific situations [2]. Figure 1 below illustrates the components and transduction pathway of a typical two-component signaling system:
2.AgrBDCA System in Staphylococcus aureus
The agr locus in Staphylococcus aureus consists of two transcription units RNA II and RNA III; they are responsible for self-regulation and effect response respectively [3][4]. RNA II, driven by P2 promoter, contains 4 genes agrB, agrD, agrC and agrA which are responsible for agr quorum sensing in S. aureus. Products of agrB and agrD work cooperatively to produce an AIP, which is later secreted out of the cell and functions as a signaling molecule. Gene products AgrC and AgrA constitute a typical two-component signaling system, in which AgrC, the AIP sensor, consists of two domains, a transmembrane receptor domain and a cytoplasmic histidine protein kinase (HPK) domain. Once the transmembrane receptor domain of AgrC is bound by AIP, its cytoplasmic HPK domain will pass a phosphate group downwards to AgrA, the response regulator. AgrA functions as a transcription factor and activates the two promoters P2 and P3 [5][6]. RNA III, driven by P3 promoter, is related to exotoxin production in S. aureus [7]. Figure 2 below shows the pathways involved in S. aureus AgrBDCA system:
It has been reported that there are altogether 4 types of agr loci; each type of agr locus encodes AgrC and corresponding AIP slightly different from others. AgrC is only activated by AIP belonging to the same group and is usually inhibited by AIP from other groups [8]. Different groups of AgrC and their corresponding AIPs are highly involved in various human diseases or infections. For example, food poisoning caused by staphylococcal enterotoxins is usually associated with agr group I and agr group II S. aureus; toxic shock syndrome toxin 1 (TSST-1), which results in high fever, low blood pressure and malaise of human, is produced by agr group III S. aureus [9].
The characteristics of S. aureus AgrBDCA system aforementioned make it possible to build an engineered AIP sensor on the membrane of a non-pathogenic bacterial species. The engineered AIP sensor, localized on the non-virulent bacteria, could therefore detect the presence of AIP molecules released by S. aureus. Upon receiving this signal, the non-virulent bacteria could be automatically induced to synthesize AIP’s competitor, RNAIII inhibiting peptide (RIP), and hence, repress the activation of AgrC and toxin production in S. aureus.
3. PlnABCD System in Lactobacillus plantarum WCFS1
Several agr-like quorum sensing systems in Lactobacillus plantarum WCFS1 have been identified, among which the PlnABCD system is the best studied [1]. The PlnABCD system, similar to the AgrBDCA system, is a self-regulating system in L. plantarum WCFS1. The transmembrane receptor HPK PlnB, together with two response regulators PlnC and PlnD, constitutes a two-component signaling system (TCS) in L. plantarum WCFS1. PlnA, the inducing peptide (IP) of L. plantarum WCFS1, activates PlnB and thereby phosphoylates PlnC or PlnD. PlnC and PlnD are upregulator and downreuglater of plnA promoter respectively, and therefore, would activate/suppress the transcription initiation of plnABCD. The transcription of plnABCD is usually associated with Lactobacillus plantarum WCFS1 bacteriocin production [10]. Figure 3 below shows the pathways involved in Lactobacillus plantarum WCFS1 PlnABCD system:
Transmembrane signal sensors of both PlnABCD and AgrBDCA systems, i.e. PlnB (of L. plantarum WCFS1) and AgrC (of S.aureus), belong to a same subgroup of histidine protein kinases – the HPK10 subfamily. PlnB and AgrC share a highly homologous cytoplasmic HPK domain regarding amino acid sequence, tertiary structure and biochemical function [6][10]. However, the sequence of PlnB and AgrC’s transmembrane domain are distinct from each other, probably due to the ligand binding specificity required by their corresponding signaling molecules.
Based on the homology between AgrBDCA and PlnABCD at the cytoplasmic HPK domain, we decided to construct a chimeric AIP sensor in Lactobacillus. Such a fusion receptor would link the transmembrane signal sensing domain of AgrCand the cytoplasmic HPK domain of PlnB. By localizing such a fusion protein on Lactobacillus plasma membrane, it is hoped that Lactobacillus can successfully detect the presence of autoinducing peptides (AIPs) produced by S. aureus. Upon the detection of AIP molecules, the chimeric protein kinase will transduce the signal to L. plantarum WCFS1 intrinsic downstream pathways.
Experiment Design:
Construction of Chimeric AIP Receptor
Johnsborg reported in her 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 [10]. 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.
Localization Test of Chimeric Receptor
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:
I.AgrC-PlnB fused with mCherry fluorescence protein. P32 constitutive promoter in shuttle vector pMG36e will drive the transcription process.
II.Complete AgrC fused with mCherry, driven by P32.
The localization test will be performed by observing pMG36e-transformed L. plantarum WCFS1 under fluorescence microscope.
Tests of the Functionality of the Chimeric Receptor
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 1has only the gusA reporter unit were prepared. The construct-inserted shuttle vectors will then be transformed into L. plantarum WCFS1.
I. P32 promoter alone;
II. gusA reporter unit driven by inducible promoter plnA; P32 promoter alone downstream;
III. gusA reporter driven by inducible promoter plnA; P32 promoter driving agrC-plnB AIP receptor
IV. gusA reporter driven by inducible promoter plnA; P32 promoter driving agrC AIP receptor
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.
I. P32 promoter alone, serving as a control group
II. gusA reporter driven by inducible promoter plnA with P32 promoter alone serving as a control group
III. gusA reporter driven by inducible promoter plnA with P32 promoter driving agrC-plnB AIP receptor and plnC
IV. gusA reporter driven by inducible promoter plnA with P32 promoter driving agrC AIP receptor and plnC as a control group to Group III
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.
