Team:Wageningen UR/Results/Cpx
Cpx signal transduction
The aim of this project was to create a modular bacterial membrane receptor capable of detecting extracellular antigens leading an intracellular signal. This was attempted by fusing an affinity body to the Cpx system’s auxiliary inhibitor CpxP. Targets present in the medium will be bound by the fusion, leading to alleviation of the Cpx inhibition. This causes the membrane receptor system to become activated, resulting in generation of an intracellular signal, visualized by mRFP1 fluorescence directly coupled to this system.
The sensing module of the Mantis diagnostic is essential for signal generation upon recognition of a disease antigen. Most receptor systems in bacteria are so-called Two-Component Signal Transduction Systems. Their name comes from the fact that they contain a membrane bound sensor and an intracellular response regulator that often acts as a transcription factor. However, none of these systems can sense disease antigens, and it is still challenging with current technology to directly change substrate specificity of such receptors. Instead, we chose to engineer an inhibitor, which influences the receptor. The Cpx system (Figure A) is involved in sensing and responding to stress on the bacterial envelope. It consists of a membrane sensor CpxA and a response regulator CpxR, as well as the auxiliary inhibitor CpxP. The latter is able to inhibit CpxA by protein protein interaction, causing the system to remain in an off-state(ref). This prevents the CpxR regulator from being activated by CpxA, and therefore not act as a transcription factor for genes involved in the alleviation of stress and reinstatement of homeostasis (ref).
Approach
The sensing module of the Mantis diagnostic is required for detection of the antigens and subsequent signal transduction in the bacteria. We approached this by creating a recombinant receptor based on the native Cpx system. This recombinant Cpx system will detect antigens, resulting in downstream signalling. As the Cpx system is normally suppressed by the inhibitor CpxP, we aimed to introduce the antigens as a new substrate for the inhibitor. Thereby, presence of the antigen would result in alleviation of suppression by making CpxP dissociate from the membrane sensor CpxA.
Introduction of antigens as a new substrate for CpxP was approached by fusion to an affinity body. This molecule is designed to bind a specific antigen with high affinity. Since antigen specific affinity bodies are produced in another part of this project, a placeholder affinity molecule specific for IgG was used in this subproject. This would allow testing of the sensing module with proven-to-work IgG (ref) as a placeholder antigen.
To determine whether IgG could induce the system, it is necessary to remove the outer membrane from the E. coli cells by a method called spheroplasting (ref). Without removing the outer membrane, the antigen place-holder would not be able to reach the Cpx system located in the periplasm. However, spheroplasting is known to cause activation of the Cpx system. CpxP is normally free in the periplasm, causing them to be titrated away if cells were spheroplasted (ref). Therefore, the CpxP-affinity body is tethered to the inner membrane by a transmembrane maltose-binding protein (MBP) mutant (ref).
Furthermore, an mRFP1 reporter is placed in the CpxR operon, resulting in red fluorescence if CpxA is activated (Fig. B). In the final version of the Mantis device activation of the Cpx system will be visualized by Bimolecular Fluorescence Complementation (BifC) linked to CpxR dimerization (see the Specific Visualization page).
1. CpxP – affinity body fusions
The CpxP gene, isolated from E. coli K12 genome, was fused together with a synthetic affinity body gene, specific for IgG Fc region, at both the C – and N-terminus. Resulting constructs were named CpxP-Aff and Aff-CpxP. Under control of the tac inducible promoter (biobrick link), these are cloned into the pSB1C3 backbone (biobrick), already containing the mRFP1 reporter under control of the pCpxR promoter (biobrick link). Furthermore, the native CpxP gene was assembled into a similar construct, to act as a control (biobrick link. Figure C gives a visual representation of the created constructs:
2. Inhibitory capacity assay for CpxP – affinity body fusions
The inhibitory capacity of both CpxP – affinity body fusions were compared to that of the native CpxP gene by expressing these protein in an E. coli ΔCpxP knock-out strain (JW5558-1). Strains carrying the different constructs were inoculated in 10 mL M9 cultures containing 2 g/L glucose to suppress leaky transcription. Cells were grown for 5 hours at 37 °C and then induced with 0.2 mM IPTG to express CpxP-affibody fusions. After overnight growth, the cells were harvested by centrifugation at 4700 rpm for 5 minutes. The pellets were resuspended in 1 mL PBS buffer, after which the suspensions were stored at 4 °C to maturate the mRFP1 fluorophores. Finally, cells were harvested, washed in PBS buffer, and measured in the plate reader. A BioTek Synergy Mx Monochromator-based Microplate Reader was used to measure red fluorescence at excitation/emission of 580/612 and Optical Density at 600nm.
Resulting fluorescence data (figure D) indicatesd that the Cpx system was suppressed in the presence of CpxP – affinity body fusions to the same extent as in the presence of CpxP.
3. Membrane tethering of CpxP – affinity bodies
Membrane tethered variants of the CpxP-affinity body fusions were created by C-terminal fusion to MBP misfolder, MalE24-1 (biobrick link). The MBP mutant was created by introducing a point mutation using PCR, causing the alanine at position 24 of the signal sequence to aspartate. This mutation results in localization of the MBP to the inner membrane (ref). The resulting triple fusions, named MalE24-1-CpxP-Aff and MalE24-1-Aff-CpxP (biobrick link), were cloned under control of the ptac promoter in the pSB1C3 backbone (biobrick composites links), already containing the mRFP1 reporter under control of the pCpxR promoter (biobrick link). Furthermore, the MalE24-1-CpxP fusion was also created and assembled into this construct, to act as a control.
4. Inhibitory capacity assay for MalE24-1-CpxP-affinity body fusions in spheroplasts
The inhibitory capacity assay of the MBP-CpxP-affinity body fusions were compared to that of the native CpxP gene in spheroplasts. Strains carrying the constructs were grown in 10 mL LB overnight at 37 °C. The next day, fresh 5 mL cultures were created with 10% inoculum. After growth at 37 °C for 1.5 hours the cultures were induced with 0.2 mM IPTG to express the membrane bound CpxP – affinity body fusions. After another hour of growth the cultures were harvested by 2 minutes of centrifugation at 4700 rpm. Pellets were spheroplasted according to the protocol of Sun et al. (ref) : Pellets were resuspended in 500 μL 0.8 M sucrose, after which the following reagents were added in the mentioned order: 30 μL 1 M Tris-HCl (pH8); 24 μL 0.5 mg/mL lysozyme; 6 μL 5 mg/mL DNase; 6 μL 125 mM EDTA-NaOH (pH8). Suspensions were incubated for 20 minutes at room temperature, after which 100 μL STOP solution (10 mM Tris-HCl (pH8), 0.7 M sucrose, 20 mM MgCl2) was added to stop degradation of the membranes.
After the spheroplasting procedure, cells were kept on ice, spun down and resuspended in 1 mL of M9 medium. Culture samples of a 100 μL samples were added to 96 wells plates and measured in the plate reader for fluorescence (580/612 excitation/emission) and OD600nm. It was found that that the tethered fusions, as well as the native CpxP, were able to inhibit the system in spheroplast conditions (Figure F).
5. IgG sensitivity assay
In this experiment, IgG was added to strains carrying the fusion protein to verify if antigens could induce the system. Similar to experiment 4, cultures were grown, induced and spheroplasted, after which samples were added to 96 wells plates together with varying concentrations of IgG (0.05 - to 0.5 mg/mL). Suspensions were measured in the plate reader for fluorescence (580/610 excitation/emission) and OD600nm over a period of 6 hours. Even though cells only remain spheroplasted for ±5 generations (ref) (±2.5 hours in M9 medium (ref)), it was worthwhile to measure for the extended period of time to account for maturation times of the mRFP1 fluorophores (ref).
In this project a recombinant bacterial receptor was constructed to have affinity for disease antigens. By combining affinity bodies with the E. coli’s native Cpx system’s auxiliary inhibitor CpxP, we attempted to create an antigen sensitive suppressor. This sensor was measured to have a similar inhibitory capacity as the native CpxP protein, indicating that the fusion did in fact not hinder the Cpx functioning. An inner membrane bound variant of this system was created by a triple fusion of misfolded MBP, CpxP and affinity body. This tethered variant was proven to suppress the Cpx system in case the outer membrane was removed, which is necessary for the disease antigens to reach the receptor. To test whether these antigens could actually induce the engineered system, a place-holder antigen IgG was added to the medium. It was shown that no induction resulted from the addition of IgG (figure 1). Therefore we can conclude that the engineered system is not sensitive for antigens under the conditions tested.
The negative outcome of this difficult to interpret as it can be caused by a variety of different factors. First of all, it might be possible that the proposed system is not viable, as the precise functioning of the CpxP inhibitor is not fully understood (ref). This makes it difficult to predict its behaviour, even more so when combined with the Affinity molecule. Furthermore, it might be possible that the Affinity molecule itself does not have proper affinity for the placeholder antigen IgG, as no binding assay was performed in this project due to time constraints. Another factor that makes measurements on the created system difficult is the fragility of spheroplasted cells. These cells only remain in their conformation for ±5 generations. Since expression of the reporter requires active cells, the timeframe of measurement comes down to about 90 minutes.
