Introduction

The original concept for the project was to identify specific membrane proteins in P. destructans cells. This idea was quickly abandoned after discovering literature about the secretions of destructin-1 in P. destructans; this enzyme is the main causative agent responsible for the degradation of collagen1. In addition, there are five other serine endopeptidases generated by P. destructans that can contribute to protein degradation. With this, our team sought to prevent the main mechanism of infection through genetic engineering.


Version 1

A series of four circuits were originally developed at the start of the cycle, each producing and secreting its own unique serine protease inhibitor. These inhibitors were Aprotinin, SPINK8, SPINK6, and WAP-4. Each of these proteins on the C-terminus has a Hly-A secretion sequence attached. The sequence permits the inhibitor protein to be recognized and secreted through the system 1 secretion pathway already present in E. coli2. The version was short-lived as our circuit had to include more needs than we originally anticipated, along with some missing parts.


Figure 1: Model of the first four circuits, the serine protease inhibitor arrow represents the individual coding sequence for their respective protease(Aprotinin, SPINK8, SPINK6, and WAP-4).

Version 2

Administrative obstacles (transitioning professors and team members) allowed for more review of the first circuit before testing. Improvements included a histidine tag and linker sequence on the N-terminus of the protease, allowing for future purification while preventing interactions with the protease. The addition of the series to include a sequence for TEVp, an enzyme that cleaves at a specific peptide signal which is placed before the Hly-A secretion signal3. This was done to reduce the amount of interference that could be caused by the Hly-A secretion signal by allowing the sequence to be cleaved after secretion. A small addition includes codon optimization for aprotinin encoding sequence.


Figure 2: Version 2 adds tags for purification and peptide cleavage. In addition, the TEV protease fits in the same model as the Serine Protease circuit series.

Antagonistic Circuit

To characterize if our parts work against their intended target it is necessary to obtain the enzyme we intend on testing our inhibitors on. This led us to create the basic part BBa_K5324001, which encodes for E. coli codon-optimized destructin-1, which we quickly created a circuit for it to be expressed in E. coli. With this composite part, we will be able to develop collagen assays to establish the ability of our generated serine protease inhibitors to prevent degeneration caused by destructin-1. Another advantage of this is we do not have to grow P. destructans in the lab to actively test against the enzyme.


Figure 3: The D1 circuit above produces the destructin-1 serine protease with a histidine tag attached.

Future Ambitions

After characterizing these composite parts we will be able to answer which serine protease inhibitors are the most effective. Then combining those composite parts along with the TEVp circuit to create a level 2 construct that would express multiple enzymes that will work together to inhibit destructin-1 among other endoproteases. In addition, there are other serine protease inhibitors we have the ability to develop based on the effectiveness of the other proteases4.


Figure 4: Above is an example level 2 circuit that would express two serine protease inhibitors along with the TEV protease creating a functioning unit to counter destructin-1.

References

  1. A.J. et al. (2015) ‘Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus Destructans, the causative agent of white-nose syndrome’, Proceedings of the National Academy of Sciences, 112(24), pp. 7478–7483. doi:10.1073/pnas.1507082112.
  2. S., Holland, I.B. and Schmitt, L. (2014) ‘The type 1 secretion pathway — the hemolysin system and beyond’, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1843(8), pp. 1629–1641. doi:10.1016/j.bbamcr.2013.09.017.
  3. M. et al. (2024) ‘Specificity of the mutant tobacco etch virus protease’, Proteins: Structure, Function, and Bioinformatics, 92(9), pp. 1085–1096. doi:10.1002/prot.26693.
  4. T., Chaturvedi, V. and Chaturvedi, S. (2015) ‘Novel trichoderma polysporum strain for the biocontrol of Pseudogymnoascus destructans, the fungal etiologic agent of Bat White-nose syndrome’, PLOS ONE, 10(10). doi:10.1371/journal.pone.0141316.