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 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.
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 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.
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
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
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.
2. Spheroplasting, membrane tethering of CpxP – Affinity molecules and inhibition of system in spheroplasted situation
To determine whether IgG could induce the system by making CpxP dissociate from the CpxA reporter, 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 present in the periplasm, being titrated away from the cell due to the lack of an outer membrane. Therefore, a membrane tethered version of the CpxP-aff fusions was developed, using a misfolded MBP (MalE24-1) fused with the the CpxP-Aff frame, leading to the constructs named MalE24-1-CpxP-Aff and MalE24-1-Aff-CpxP (biobrick link). These were again combined with tac promoter and inserted in the pSB1C3 backbone already containing the mRFP1 reporter.
Expression of the MalE24-1-CpxP-Aff fusions in E. coli ΔCpxP knock-out strain was to indicate whether these fusions could inhibit the activation of the Cpx system to a better extend than the non-tethered native CpxP could. Figure 4 gives a visual representation of the created system and its hypothesized behaviour. To determine the inhibitory capacity, the strains were grown overnight in 10 mL LB liquid cultures, after which they were diluted 10 times in fresh LB cultures of a total volume of 5 mL. These were grown for 1.5 hour at 37 °C, induced with 0.2 mM IPTG, and grown for another hour. Afterwards the cultures were harvested by centrifuging and spheroplasted. Spheroplasted cells were harvested again and resuspended in 1 mL M9 medium, after which their OD600nm and mRFP1 fluoresence was measured over a course of 6 hours. Note that cells only remain in spheroplast formation for ± 5 generations, after which they fully recovered their outer membrane, but mRFP1 levels might be important after this period of 1 hour, since they might take more time to maturate.
Figure 5 shows that the Cpx system was suppressed in spheroplasted strains carrying the membrane tethered CpxP-Aff fusions. However, inhibition can also be seen in strains carrying the non-tethered native CpxP protein, even though the reverse was hypothesized.
3. IgG impact on spheroplasted, membrane tethered CpxP – Affinity molecule fusions.
The final experiment of this project consists of testing the engineered system with the antigen place-holder IgG. Figure 6 shows how the hypothesized behaviour of this system. Strains carrying the membrane tethered CpxP-Aff constructs were grown overnight in 10 mL LB at 37 °C, after which they were used to create fresh 5 mL LB cultures with 10% inoculum. These were grown for 1.5 hours at 37 °C, induced with 0.2 mM IPTG and grown for another hour. Afterwards the cells were harvested by centrifuging and spheroplasting procedure was performed. Spheroplasted cells were harvested again and resuspended in 1 mL M9 medium. In a 96 wells plate cultures were combined with a range of IgG suspensions of different concentration. Measuring the mRFP1 levels over the following 6 hours was to indicate whether IgG could induce the recombinant Cpx system.
As figure 7 indicates, IgG did seemingly not have any effect on the Cpx system, even in the presence of CpxP-Aff carrying strains.
