Engineering

Overview

    Our strategy of prevention is to neutralize the toxins via outer membrane vesicles (OMVs) with the toxin receptors displayed on the surface. In the experimental workflow, we designed different parts to verify the function of surface display system and the displayed receptors, and finally demonstrated that the receptor displayed both on the surface of engineered bacteria and OMVs can bind to toxins.

Cycle 1

Verifying the function of surface display system ClyA

Design

    ClyA is commonly used as a surface display system which can anchor heterologous protein on the surface of bacteria (1). rLvAPN1 is a truncated form of LvAPN1 (residues 205-591) which has higher affinity to toxins than the receptors on shrimp intestinal epithelial cells (2). In order to neutralize toxins, we tried to fuse rLvAPN1 to the C-terminal of ClyA for displaying the receptor. We constructed the fusion protein ClyA-rLvAPN1-his to verify whether rLvAPN1 could be displayed on the surface of the engineered bacteria or not at first.

Build

    There were four basic parts assembled into the vector: promoter (BBa_I0500), RBS (BBa_B0034), ClyA-rLvAPN1-his coding sequence (BBa_K4195030) and terminator (BBa_B0015). We got the constructed plasmid BBa_K4195131 successfully and transformed it into E. coli BL21(DE3), then the positive transformants were selected through colony PCR (Fig. 1) and sequencing.
Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195131_pSB1C3. Target bands (3879 bp) can be observed at the position between 3000 bp and 5000 bp.

Test

    Immunofluorescence (IF) technology was implemented to test whether the display system is functional or not. BBa_K4195134 (for rLvAPN1-his expression) which has no surface display system was set as negative control, while BBa_K4195131 (for ClyA-rLvAPN1-his expression) was set as positive control. The arabinose-induced overnight culture of the two groups were then incubated with FITC-labeled anti-His-tag antibody and washed for several times, followed by measuring fluorescence intensity and OD600.
Fig. 2 The results of immunofluorescence to characterize the function of ClyA surface display system (p = 0.0092).
    The ratio of fluorescence intensity to OD600 of positive control is higher than that of negative control (p = 0.0092) (Fig. 2), which indicates that our surface display system works well that the cargo (rLvAPN1) is displayed successfully.

Learn

    This result allowed us to conclude that the ClyA can display heterologous proteins on the surface of the engineered bacteria. However, it was not sufficient to prove that the binding affinity of displayed receptor to the toxins is still retained.

Cycle 2

Attempt to verify the function of displayed rLvAPN1 through ClyA-rLvAPN1-his

Design and Build

    After successfully demonstrating the function of ClyA system to display rLvAPN1, we then turned to the verification of the function of rLvAPN1 after displayed. Followed by the Cycle 1, we continued to use ClyA-rLvAPN1-his (BBa_K4195131) to verify the function of displayed rLvAPN1 by testing its ability to bind to toxin PirB.

Test

    IF technology was implemented again to test whether the displayed rLvAPN1 on the engineered bacteria is functional or not. The induced culture with purified his-PirB added was set as positive control while which had no toxins added was set as negative control.
    Unfortunately, we had never got result of significant differences on the ratio of fluorescence intensity to OD600 between positive control and negative control.

Learn

    After several failures, we checked our design carefully. We found that the problem was the displayed receptor and the toxins purified were both fused with His-tag (6×His), so the FITC-labeled anti-His-tag antibody can recognize and bind to the bacteria of both positive control and negative control, resulting in no differences on the ratio of fluorescence intensity to OD600 between the two groups.

Cycle 3

Verifying the retained function of displayed rLvAPN1 through ClyA-rLvAPN1

Design

    After realizing the problem, we constructed the fusion protein ClyA-rLvAPN1 without His-tag to verify the function of displayed receptor rLvAPN1.

Build

    The coding sequence of ClyA-rLvAPN1-his in BBa_K4195131 was replaced by ClyA-rLvAPN1 to create BBa_K4195130. We got the constructed plasmid successfully and transformed it into E. coli BL21(DE3), then the positive transformants were selected through colony PCR (Fig. 3) and sequencing.
Fig. 3 DNA gel electrophoresis of the colony PCR products of BBa_K4195130_pSB1C3. Target bands (3816 bp) can be observed at the position between 3000 bp and 5000 bp.

Test

    1. IF technology was implemented as before to determine the function of displayed rLvAPN1 on the surface of engineered bacteria. Control settings were the same to the last cycle.
Fig. 4 The results of immunofluorescence to probe the binding event on the surface of engineered bacteria. Purified his-PirB was tested to interact with the displayed receptor rLvAPN1 (p = 0.0213).
    The ratio of fluorescence intensity to OD600 of positive control is higher than that of negative control (p = 0.0213) (Fig. 4), which indicates that the displayed rLvAPN1 can bind to the toxins.
    2. Besides, dot blot (3, 4) was further implemented to test whether the displayed rLvAPN1 on OMVs is functional or not. The OMVs extracted from the culture of engineered bacteria harboring BBa_K4195130 was set as positive control while which without rLvAPN1 displayed was set as negative control.
Fig. 5 The imaging results of chemiluminescence (dot blot analysis) to probe the binding event on the surface of OMVs.
    As with his-PirB added, we observed a strong signal from the OMVs fraction derived from the engineered bacteria harboring BBa_K4195130. However, a faint detectable signal was observed from the OMVs fraction derived from the bacteria without rLvAPN1 displayed (INPNC-TTPA in this case) (Fig. 5), suggesting that rLvAPN1 displayed on OMVs can still bind to toxins PirB specifically. For the faint detectable signal observed from the OMVs franction derived from the bacteria without rLvAPN1 displayed, we attributed this to nonspecific adsorption or potential weak cross-reactivity of our FITC-labeled anti-His-tag antibody to INPNC-TTPA.
    For the faint detectable signal observed from the OMVs fraction derived from the bacteria without rLvAPN1 displayed, we attributed this to the potential cross-reactivity of our FITC-labeled anti-His-tag antibody to INPNC-TTPA. Learn more information about this from our Proof of Concept page.

Learn

    The result shows that the ClyA-mediated displayed rLvAPN1 on the surface of engineered bacteria and OMVs can bind to toxins. This was the most important step in proving that our design works.

Conclusion

    Engineering a biological system cannot be easy, we need to put our heart into it and move towards the success through experiment and feedback step by step. Here we have tried to demonstrate how we were able to verify that the receptor displayed both on the surface of engineered bacteria and OMVs can bind to toxins through successive iterations of the DBTL (Design-Build-Test-Learn) cycle (Fig. 6).
Fig. 6 Summary of the different engineering steps completed before reaching the final proof of concept of the retained function of rLvAPN1 displayed.

Reference

      1. K. Murase, Cytolysin A (ClyA): A Bacterial Virulence Factor with Potential Applications in Nanopore Technology, Vaccine Development, and Tumor Therapy. Toxins (Basel). 14, 78 (2022).
      2. W. Luangtrakul et al., Cytotoxicity of Vibrio parahaemolyticus AHPND toxin on shrimp hemocytes, a newly identified target tissue, involves binding of toxin to aminopeptidase N1 receptor. PLoS Pathog. 17, e1009463 (2021).
      3. J. L. Valentine et al., Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies. Cell Chem. Biol. 23, 655-665 (2016).
      4. T. C. Stevenson et al., Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies. Proc. Natl. Acad. Sci. U. S. A. 115, E3106-E3115 (2018).