Background

Modern antibiotics are often small-molecule inhibitors, a class of drugs composed of chemically-synthesized small organic compounds which bind to and inhibit essential biological targets in bacteria. These medicines, like penicillin, tetracycline, and azithromycin, can easily be produced at scale with an engineerable series of chemical reactions.

The widespread use of antibiotics, however, has encouraged the evolution of antibiotic resistance. Resistant pathogens pose an existential threat to modern medicine, which relies on antibiotics both to treat infections acquired outside the clinic and prevent infections from occurring following invasive procedures.

The scientific community is working to combat this challenge by developing novel antibiotics – but methods that have been fruitful in the past are struggling to keep pace with rapid bacterial evolution. Following a fruitful period of discovery from 1940 - 1960, no novel antibiotic has been discovered since 1987 [8].

Antimicrobial peptides (AMPs) are a promising and largely unexploited alternative to small molecule antibiotics. AMPs are common, naturally occurring peptides that play a critical role in the innate immune system of many organisms, warding off pathogens including bacteria, viruses, and parasites [4]. Thousands of AMPs have been identified with varied structure, mechanism, chemical properties, and efficacy against bacteria, viruses, and parasites. Their impact can range from therapeutics to industrial functions as common antibiotics continue to become more challenging to work with due to resistance.

To date, the large-scale production of AMPs presents significant challenges. Due to their complex structure, chemical synthesis is difficult and expensive, as is common with many proteins. A cheaper and more accessible synthetic strategy could be biosynthesis by established protocols in prokaryotic hosts. This strategy is used for compounds that require mass production such as insulin [9]. However, the desirable antibiotic properties of these compounds make them toxic to common expression organisms, like E. coli. This paradox presents a challenge to using bacteria as expression agents, as the bacteria need to remain alive to best produce the peptides.

A natural solution is to sequester the AMPs in encapsulin nanocompartments to mitigate toxicity. Encapsulins are protein cages found in a range of bacterial species that isolate dangerous chemical reactions and molecular storage from the cytosol [5]. They self-assemble from dozens of subunits into icosahedral geometries, sequestering cargo that presents a short targeting peptide. Literature has shown that fusion of desired cargo to the targeting sequence allows encapsulation [6].

Solution

We propose the development of a high yield, encapsulin-based system to produce AMPs in the very bacteria they are designed to kill. This system must then allow for simple, efficient purification of encapsulated AMPs. Through the use of a targeting peptide, we aim to orient our AMPs inside an encapsulin before the encapsulin self-assembles, shielding the production bacteria from the bactericidal AMPs they are expressing before they cause significant damage. We then plan to purify our encapsulin and isolate the AMP. To separate the AMP from the targeting peptide, we will cleave the protein using a Tobacco Etch Virus (TEV) protease. To accomplish this, we will add a TEV protease site between the AMP and targeting peptide. This system is seen in Figures 1.

Figure 1: Expression of AMP monomer + encapsulin before sequestration of AMP within encapsulin.

Next, we plan to investigate the potential to scale our system through creating AMP polymers with multiple AMPs chained together (Figure 2). During downstream purification, the AMPs will be separated back into monomers through the use of a TEV protease. This will provide insight into the maximum yield we can reasonably expect from our proposed solution.

Figure 2: AMP polymer + encapsulin. TEV: Tobacco Etch Virus Protease Site

Encapsulin Selection

Research performed by the Giessen Lab at the University of Michigan is currently performing research using a T4GALA encapsulin that disassembles at pH 6. This encapsulin is derived from Q. thermotolerans and consists of 240 protein subunits [6]. This encapsulin is conducive to our work because it is a relatively large encapsulin suitable for high AMP yields. It also readily disassembles with pH change, which would enable simple AMP extraction later on as compared to other encapsulin variants which often require extreme pH changes or high levels of urea to disassemble. The Giessen lab generously donated a plasmid construct containing this encapsulin to our team and a graduate student from the lab was willing to offer his advice to our team, thus the T4GALA construct was decided upon.

AMP Selection

We sought an AMP with strong potency against gram negative bacteria like E. coli, a common choice for recombinant protein expression. We were also interested in using a peptide that had been previously tested with an encapsulin carrier protein. Building off research conducted by Tek-Hyung Lee et. al on encapsulins and AMPs, we selected the HMBC2 AMP which is a hybrid compound [5]. One component, melittin, is derived from bee venom and competitively inhibits Ca2+ pump activity, resulting in an increased Ca2+ concentration that is toxic to cells [7]. The other component, cecropin, is derived from moths and disrupts the cell membrane, making it permeable [4]. We differentiated our work from the research done by Tek-Hyung Lee et. al by selecting a different encapsulin than tested in his experimentation and varying our method of AMP and encapsulin fusion through the addition of a targeting peptide.

Novelty

While we used existing research to inform our work [5], our project builds on existing research in numerous ways, including:

1. Using an encapsulin-targeting peptide system engineered to disassemble under mildly acidic conditions
2. Increasing ease-of-use through an easily separable targeting-peptide-based encapsulin-AMP attachment system
3. Working toward stringing AMPs together on the same targeting peptide for maximized yields

References

1. Aslam, B., Wang, W., Arshad, M. I., Khurshid, M., Muzammil, S., Rasool, M. H., Nisar, M. A., Alvi, R. F., Aslam, M. A., Qamar, M. U., Salamat, M., & Baloch, Z. (2018). Antibiotic resistance: a rundown of a global crisis. Infection and drug resistance, 11, 1645–1658. https://doi.org/10.2147/IDR.S173867
2. Thompson T. The staggering death toll of drug-resistant bacteria. Nature. 2022 Jan 31. doi: 10.1038/d41586-022-00228-x. Epub ahead of print. PMID: 35102288.
3. Silver L. L. (2011). Challenges of antibacterial discovery. Clinical microbiology reviews, 24(1), 71–109. https://doi.org/10.1128/CMR.00030-10
4. Huan, Y., Kong, Q., Mou, H., & Yi, H. (2020). Antimicrobial Peptides: Classification, Design, Application and Research Progress in Multiple Fields. Frontiers in microbiology, 11, 582779. https://doi.org/10.3389/fmicb.2020.582779
5. Lee, T. H., Carpenter, T. S., D'haeseleer, P., Savage, D. F., & Yung, M. C. (2020). Encapsulin carrier proteins for enhanced expression of antimicrobial peptides. Biotechnology and bioengineering, 117(3), 603–613. https://doi.org/10.1002/bit.27222
6. Triggered reversible disassembly of an engineered protein nanocage Jesse A. Jones, Ajitha S. Cristie-David, Michael P. Andreas, Tobias W. Giessen bioRxiv 2021.04.19.440480; doi: https://doi.org/10.1101/2021.04.19.440480
7. Ceremuga M, Stela M, Janik E, Gorniak L, Synowiec E, Sliwinski T, Sitarek P, Saluk-Bijak J, Bijak M. Melittin-A Natural Peptide from Bee Venom Which Induces Apoptosis in Human Leukaemia Cells. Biomolecules. 2020 Feb 6;10(2):247. doi: 10.3390/biom10020247. PMID: 32041197; PMCID: PMC7072249.
8. Few antibiotics under development – How did we end up here? (n.d.). ReAct. Retrieved October 8, 2022a, from https://www.reactgroup.org/toolbox/understand/how-did-we-end-up-here/few-antibiotics-under-development/
9. Baeshen, N. A., Baeshen, M. N., Sheikh, A., Bora, R. S., Ahmed, M. M. M., Ramadan, H. A. I., Saini, K. S., et al. (2014). Cell factories for insulin production. Microbial Cell Factories, 13, 141. Retrieved October 8, 2022, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4203937/