Cpx signal transduction

The aim of this project was to create a modular bacterial membrane receptor capable of detecting extracellular antigens leading to generation of an intracellular signal. This was approached by fusing an Affinity Body, a modular scaffold protein with high affinity for a specific target, to the Cpx system’s auxiliary inhibitor CpxP. In the presence of the Affinity Body’s target antigen this fusion would bind the target, 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.

Background

The sensing module of the Mantis diagnostic, essential for the generation of a signal upon recognition of target disease antigens, is based on bacterial receptor systems. However, no receptor system exists that can sense such antigens, and it is still challenging with current technology to directly change substrate specificity of such receptors. Therefore, we chose to engineer a system that uses an auxiliary inhibitor protein that influences the receptor without physically being bound to it. The Cpx system (Figure 1) is involved in sensing and responding to stress on the bacterial envelope, and like most systems it consists of a membrane sensor CpxA and a response regulator CpxR, as well as the auxiliary inhibitor CpxP. The latter is able to prevent activation of sensor CpxA by directly binding to it, causing the system to remain in an off-state in normal homeostasis. 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.

Figure 1: Visual representation of the Cpx system in native conditions. Cues associated with envelope stress can activate CpxA, either by CpxP dissociation or due to an unknown mechanism, resulting in phosphorylation of CpxR. Upon dimerization this response regulator can act as a transcription factor for genes involved in the reinstatement of homeostasis.

Approach

As an approach to change the Cpx system to be sensitive for disease antigens we aimed to introduce them as a new substrate for the CpxP protein by fusing the inhibitor to an affinity molecule designed to specifically bind such an antigen. This fusion would allow CpxP to release from the CpxA sensor in case antigens are present in the environment, leading to activation of the Cpx signal and downstream response.

Since antigen specific affinity molecules are produced in , a placeholder affinity molecule specific for IgG was used in the experiments of this subproject. This would allow testing of the sensing module with proven-to-work IgG 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. Without removing this membrane, the antigen place-holder would not be able to reach the Cpx system located in the periplasm. However, spheroplasting is also known to cause activation of the Cpx system due to CpxP, normally free in the periplasm, being titrated away from the cell due to the lack of an outer membrane. Therefore, the inhibitor fusion is tethered to the inner membrane by a transmembrane MBP.

Furthermore, visualization of the Cpx system activation during all experiments is done by a downstream mRFP1 reporter under control of a pCxpR promoter, leading to red fluorescence in case CpxA is being activated. Note that this is not the same visualization module as we envision for the final version of the Mantis device, which would be the faster BifC complementation using CpxR dimerization, as worked on in .
Figure 2 gives a visual representation of the proposed system with its hypothesized behaviour.

Figure 1: Visual representation of the proposed recombinant Cpx system. Presence of disease antigens, such as viral particles, in the environment of the cell can activate the Cpx system by dissociation of CpxP, resulting in downstream mRFP1 gene transcritpion.

Methods

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 composites link), already containing the mRFP1 reporter under control of the pCpxR promoter (biobrick link):

2. Inhibitory capacity assay for CpxP-Affinity Body fusions

The inhibitory capacity of the CpxP – Affinity body fusions was 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 used to inoculate 10 mL M9 cultures with 2 g/L glucose to supress leaky transcription. After 5 hours of growth at 37 °C the cultures were induced with 0.2 mM IPTG to express the fusions. After overnight growth the cells were harvested by centrifugation at 4700 rpm for 5 minutes. Pellets 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 again, washed again in PBS buffer and measured in the platereader for their fluorescence (580/612 excitation/emission) and OD600nm. Resulting fluorescence data (figure 3) indicated that the Cpx system was suppressed in the presence of CpxP – Affinity molecule fusions to the same extent as in the presence of CpxP.

Figure 3: Fluorescence/OD_600nm resulting from expression of CpxP-Affinity molecule variants.

3. Membrane tethering of CpxP – Affinity Body fusions

Membrane tethered variants of the CpxP-Affinity Body fusions were created by fusion to the C-terminus of a MBP misfolder, MalE24-1 (biobrick link). This MBP mutant was created by introduction of a point mutation by PCR, causing the alanine at position 24 of the signal sequence to aspartate. 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)

4. Inhibitory capacity assay for MalE24-1-CpxP-Affinity Body fusions in spheroplasted cells

The inhibitory capacity assay of the MBP-CpxP-Affinity Body fusions were compared to that of the native CpxP gene in spheroplast conditions. 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 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.: 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. 100 μL samples of the cultures were added to 96 wells plates and measured in the plate reader for fluorescence (580/612 excitation/emission) and OD600nm. Results (Figure 4 and 5) indicate that the tethered fusions, as well as the native CpxP, were able to inhibit the system in spheroplast conditions significantly: