Our phage-deployed system approach aims to combat antibiotic resistance in bacteria using lambda phages, a Craspase system, and gasdermin proteins. The system is designed to detect antibiotic-resistant genes in bacteria and trigger cell death via pyroptosis in bacteria presenting genes that match our gRNA sequence. Pyroptosis kills the cells via activated caspases that cleave gasdermin proteins. The cleavage of the gasdermin proteins causes pores to form in the cell membrane, prompting the cell to swell and eventually burst. Our project was initially inspired by Proteus, a project created by McGill University in 2023, which focused on utilizing human gasdermins to target pancreatic cancer. Building from this, our project shifted focus to the use of bacterial gasdermins endogenous to E. coli to target ampicillin-resistance. We first considered using Craspase for a caspase cascade but reverted to the use of gasdermins due to specificity concerns. A caspase cascade could be initiated by a variety of environmental factors which would not have led to a system with the desired specificity. Various alternatives were explored, including mRNA versus highly specific protein cleavage and b-lactamase specific proteases. The final design incorporates a fusion protein of Runella gasdermin, Csx-30, and a Craspase system containing the Csx-30 specific protease Csx-29. The RNA that codes for these systems is to be delivered by lambda phages specific to E. coli. This innovative approach combines several key components to create a highly specific targeting system for combating antibiotic-resistant bacteria.
The components of the project prominently feature Craspase: a protease complex that cleaves target proteins when activated by specific RNA molecules, and gasdermin proteins, which form pores in bacterial membranes, leading to pyroptosis. The fusion protein combines Csx-30 and gasdermin proteins, which are cleaved upon detection of antibiotic-resistant genes by the Csx-29 protease. Modified bacteriophages serve as the delivery method to introduce the system into bacteria. Linker design proved crucial for holding gasdermin halves together based on comparison with human gasdermins. Specificity concerns were addressed by using Csx-30 linkers and engineered E. Coli specific lambda phages. Locating the correct linking area in bacterial gasdermin required comparison with human gasdermin studies, while potential issues with gRNA mutation and unintended protease activity were considered in the design process.
In the future, further experimentation to confirm the correct linking area in bacterial gasdermin and a potential focus on targeting ampicillin-resistant genes in bacteria in clinical settings will be continued project goals. Further refinement of specificity and activation mechanisms are necessary to prevent unintended cell death. The project methodology followed iterative design principles including planning, education, design, development, testing, and analysis. We utilized literature review and online modeling for concept testing and refinement and employed vector building programs for sequence comparison and linker design. This comprehensive approach, combining cutting-edge biotechnology with rigorous scientific methodology, positions the project as a promising avenue for addressing the critical issue of antibiotic resistance in bacteria.