Figure 1. Overview of Contribution

Part	Abbreviation	Full Name	Description	Designer	Length (bp)
BBa_K4990001	FK-13	LL-37 truncated peptide	kill both Fn and CRC	Dingding	39
BBa_K4990002	B-domain	mFadA B-domain	targeting Fn	Dingding	81
BBa_K4990003	A-domain	mFadA A-domain	self-assemble with B-domain	Dingding	249
BBa_K4990004	mFadA	mature Fusobacterium adhesin A	self-assemble with itself	Dingding	334
BBa_K4990005	linker A	Self-cleaving linker A	rigid and self-cleaving linker	Dingding	15
BBa_K4990006	linker B	Self-cleaving linker B	reversed rigid and self-cleaving linker	Dingding	15
BBa_K4990007	HlpA	Histone-like protein A	Target CRC	Dingding	273
BBa_K4990008	DEH	Dual-Edged Harpoon	Target and kill both Fn and CRC	Dingding	420
BBa_K4990009	BTP	Bacteria-Targeting Peptide	Target and kill Fn	Dingding	135
BBa_K4990010	TTP	Tumour-Targeting Peptide	Target and kill CRC	Dingding	324
BBa_K4990011	BTR	Bacteria-Targeting Rod	Facilitate BL-Fn adhesion	Dingding	1500
BBa_K4990012	TTR	Tumour-Targeting Rod	Facilitate BL-CRC adhesion	Dingding	1632

Overview
Our contributions are divided into two major sections: "Part Contribution" and "Methodology Contribution."

Based on the self-assembly characteristics of Fn pili, we designed “B-domain” capable of targeting Fn. Building upon this, we developed the "Fishing Rod Protein" as a platform for achieving bacterium-engineered bacteria (bacterium)-bacterium (Fn) targeting through protein-protein interactions. Following the same targeting principles, we also designed a dual-targeting cytotoxic peptide, enabling simultaneous targeting and killing of both Fn and CRC.

Differing from the traditional methods, we have innovatively introduced an entirely new targeting approach that against Fn. Our goal is for this approach to develop into a comprehensive methodology. Building on the self-assembly of pili, we have also introduced a novel Cell-Cell Adhesion Toolkit, a brand-new surface display system, a unique protein scaffold design methodology, and have opened up vast possibilities for the engineering of pilin.

Dual-Edged Harpoon

Figure 2. Title of DEH

DEH possesses the dual capability of targeting and killing Fn and CRC, which is a culmination of our wisdom in peptide drug engineering, pilus self-assembly, fusion protein design, and more. Therefore, it is our No. 1 favorite part. It comprises six components: 1. B-domain for targeting Fn; 2. Linker A for separating functional domains; 3. FK-13 for killing Fn and CRC; 4. Linker B, which not only separates the functional domains but also ensures the positive region of FK-13; 5. HlpA monomer for targeting CRC. These segments have been individually uploaded as parts to the Registry. Of course, you can find the whole information about DEH at Part:BBa K4990008 - parts.igem.org

Figure 3. Subparts of DEH

HlpA
HlpA (Histone-like protein A), as its name suggests, can bind to DNA like histones. Its overall shape resembles that of a crab claw, enabling it to tightly bind non-specifically to DNA grooves using its ribbon-like β-fold regions.

Figure 4. Structure of HlpA

In the case of S. boivs, it secretes HlpA through an unknown mechanism as an anchorless protein. It locates at the surface of S.bovis and facilitates bacterial adhesion to colon tumor cells by connecting to bacterial lipoteichoic acid (LTA) and Heparan Sulfate Proteoglycans (HSPG) on the surface of colon cells.[1]

HlpA monomers are used to design fusion proteins that can bind to HSPG on the surface of colon cancer cells. Therefore, if you want to target colon cancer cells with a protein, you can design a fusion protein with HlpA attached at one end, giving it the ability to target colon cancer cells, essentially creating a "Dual-Edged Harpoon"。[2-3]

Figure 5. Functions of HlpA

FK-13
Fk-13 exhibits antibacterial effects against both Gram-positive and Gram-negative bacteria. Through electrostatic interactions, the positively charged FK-13 connects with negatively charged bacteria, inserting into bacterial cell membranes and leading to the leakage of their contents, resulting in cell death.

Figure 6. Structure of FK-13

Currently, two hypotheses support the mechanism by which FK-13 disrupts cell membranes: the carpet model, based on FK-13's cationic amino acids interacting with the phospholipid head groups in the cell membrane, which can form a carpet-like structure on the membrane surface, ultimately compromising membrane integrity. Changes in helical structure, charge, and hydrophobicity influence its antibacterial activity. On the other hand, the toroidal pore model suggests that due to membrane surface tension and curvature, FK-13 induces the formation of toroidal pores in bacterial membranes, causing leakage of bacterial contents and leading to bacterial death.

Figure 7. Cytotoxic principles of FK-13

The three-dimensional structure below illustrate a linker peptide used by us. It is explicit that the linker peptide is composed of simple α-helices. This linker peptide is commonly used as a rigid linker peptide and is widely incorporated into the design of fusion proteins.

Linker A & B
The three-dimensional structure below illustrate a linker peptide used by us. It is explicit that the linker peptide is composed of simple α-helices. This linker peptide is commonly used as a rigid linker peptide and is widely incorporated into the design of fusion proteins.

Figure 8. Structure of Linker A&B

Modification elevating the positive charge level of FK-13 without altering its spatial structure increases its cytotoxic activity. Therefore, we anticipate that the linkers at both ends of the bactericidal peptide should not reduce the activity of the antimicrobial peptide due to electrostatic interactions. To avoid positioning negatively charged amino acids near the bactericidal peptide, we designed two types of linkers that allow positively charged lysine to enclose the bactericidal peptide. This is advantageous for designing fusion proteins while ensuring bactericidal activity.

Figure 9. two lysine residues enclose the red FK-13, concentrating the positive charge and enhancing the cytotoxic activity.

mFadA B-domain

Figure 10. Title of mFadA B-domain
You need to know!

Many bacteria have long chain fiber-protein on their surfaces, which are called pili or fimbriae. These pili are composed of individual pilus monomers that link together end-to-end in the extracellular environment, self-assembling into long chain fibers with high physical strength.

For Fusobacterium nucleatum, its pili are referred to as Fusobacterium adhesin A (FadA). The monomers that make up these pili come in two forms: ①pre-FadA, which serves as an anchoring structure, attaching the entire pilus to the bacterial inner membrane. ②mature FadA (mFadA), which can link head-to-tail and self-assemble into a long filament.[4]

Figure 11. Brief introduction to FadA

Our project aims to accomplish bacteria-bacteria targeting. To accomplish this, we intend to leverage the self-assembly property of mFadA. We have fused a bacterial pilus monomer onto a membrane protein of the engineered bacterium, which we call the "fishing rod protein" . The membrane protein acts as the fishing rod, the linker serves as the fishing line, and the bacterial pilus monomer functions as the bait. By utilizing surface display techniques to display the fishing rod protein, our engineered bacteria can essentially "fish" for target bacteria, enabling precise bacteria-bacteria targeting.

Figure 12. Bacterial-bacterial targeting through pilus self-assembly

However, displaying the entire bacterial pilus monomer directly on the surface would lead to a range of issues, including steric hindrance, nonspecificity, and metabolic waste. Therefore, we truncated the mFadA to address these concerns.

What it is?
Below is the structure of the mFadA B-domain, which is truncated from the pili monomer of Fn:

Figure 13. Structure of mFadA B-domain

It functions like bait, enticing the Fusobacterium nucleatum to take the hook.

What can it do?
However, not the entire structure of mFadA is involved in self-assembly. Thus, we considered removing unnecessary domains. Upon closer examination of mFadA's structure, we divided it into two domains: Domain A and Domain B. Domain A comprises two anti-parallel α-helical structures, while Domain B consists of a single anti-parallel α-helix. We believe that Domain B is the most crucial. On a microscale, it possesses the function of binding with Domain A, and on a macroscale, it exhibits the capability to target Fn (Fusobacterium nucleatum).

Figure 14. Functions of mFadA domains

Therefore, by displaying the engineered bacteria with the mFadA B-domain on their surface, specific adhesion to Fn can be achieved, enabling bacteria-to-bacteria targeting to become reality.

Figure 15. Cell-cell adhesion through pilus self-assembly

How does it work?
We further identified the microscale contacts between the two mFadA monomers from the best docking result of Rosetta. These interactions s are categorized into primary and secondary hydrophobic interactions, as well as salt bridge interactions, as depicted in the diagram below

Figure 16. interaction between domain A and B

It is noteworthy that while the hydrophobic structure formed by leucine chains makes a significant contribution to the self-assembly, the salt bridge shell formed by four pairs of acidic and basic residues envelops these hydrophobic centers, providing stability to the binding.

Figure 17. hydrophobic centers and salt bridge shell

Reference

[1]O'Neil P, Lovell S, Mehzabeen N, et al. Crystal structure of histone-like protein from Streptococcus mutans refined to 1.9 Å resolution[J]. Acta Crystallographica Section F: Structural Biology Communications, 2016, 72(4): 257-262.

[2] ]Ho C L, Tan H Q, Chua K J, et al. Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention[J]. Nature biomedical engineering, 2018, 2(1): 27-37.

[3] Ou, S., Wang, H., Tao, Y., Luo, K., Ye, J., Ran, S., Guan, Z., Wang, Y., Hu, H., & Huang, R. (2022). Fusobacterium nucleatum and colorectal cancer: From phenomenon to mechanism. Frontiers in Cellular and Infection Microbiology, 12, 1020583. https://doi.org/10.3389/fcimb.2022.1020583

[3] Tang, H., Zhou, T., Jin, W., Zong, S., Mamtimin, T., Salama, E. S., ... & Li, X. (2023). Tumor-targeting engineered probiotic Escherichia coli Nissle 1917 inhibits colorectal tumorigenesis and modulates gut microbiota homeostasis in mice. Life Sciences, 324, 121709.

[4] Témoin, S., Wu, K. L., Wu, V., Shoham, M., & Han, Y. W. (2012). Signal peptide of FadA adhesin from Fusobacterium nucleatum plays a novel structural role by modulating the filament’s length and width. FEBS letters, 586(1), 1-6.

Methodology

Novo targeting method
Diverging from conventional methods, we have innovatively harnessed the self-assembly of bacterial pili for targeting. These protein fibers, formed by monomers connecting end-to-end, are present on the surface of bacteria. However, their variations differ significantly among various bacterial species. Hence, we can capitalize on this shared feature and diversity to craft a tailored, engineered approach for specific bacterial targeting.

Figure 18. Different pilin and its complex

Apart from the FadA we've been using (PDB ID: 3ETW), there are many similar pili structures, such as S. pyogenes pili (PDB ID: 3B2M), Pseudomonas aeruginosa pili - CupE (PDB ID: 8CIO), uropathogenic E. coli - Type I pili (PDB ID: 6Y7S), and E. coli biofilm protein CsgA (PDB ID: ). If we could utilize the self-assembly of these similar pili, it would enable specific targeting of these bacteria.

Figure 19. Novo targeting methodx

Novo display method
Here are 4 problems with common bacterial surface display systems: 1.Passenger protein size is limited; 2.Ukaryotic proteins are difficult to fold correctly in prokaryotic systems; 3. Passenger proteins exert survival pressure on host cells; 4. Limitations of Antibiotic Resistance in Engineering Applications.

However, our novel surface display system can circumvent all of the aforementioned issues. By adding a truncated pilus tag to the passenger proteins, they can be produced by other bacteria, and the chassis bacteria only need to display a single anchor on the surface to achieve the surface display of high-molecular-weight proteins. Similarly, if passenger proteins are produced by eukaryotic cells, it is possible to achieve the surface presentation of eukaryotic proteins on prokaryotic cells. Furthermore, the impact of surface display on the growth of chasis cells is minimized. In addition to this, due to the adhesive properties of the pili, this feature can be utilized for selection instead of relying on antibiotic resistance. Therefore, the two-step surface display method achieved by pili self-assembly can address many traditional issues. However, the application of this method is limited by the strength of binding between pili monomers. Nevertheless, we believe that this can be addressed through protein engineering and directed evolution methods.

Figure 20. Novo surface display method

Novo MAC method
Enzyme immobilization represents a traditional and highly promising method in biocatalysis, surpassing free enzymes in aspects such as pollution control, product separation, enzyme stability, and reusability. And Multi-Enzyme Assembly Cascades, MAC, refers to multiple enzymes are assembled together, enabling them to collaborate and form enzyme cascade reactions to achieve specific biosynthetic or metabolic pathways.

However, the significant challenge in achieving multi-enzyme immobilization stems from compatibility issues between the carrier and target enzymes. To address this, a feasible solution lies in the utilization of naturally immobilized biomolecules within microbial biofilms, with SpyTag-SpyCatcher and SnoopTag-SnoopCatcher serving as ideal linker components. They facilitate irreversible, spontaneous reactions across a wide range of temperature, pH, and organic solvent conditions, while minimizing cleavage and cross-reactivity.

Figure 21. principle of SpyTag and SpyCatcher

SpyTag-SpyCatcher is quite famous, isn't it? But do you know where they come from? In fact, they also originate from pili. When the CnaB2 domain of the S. pyogenes (Streptococcus pyogenes) FbaB protein is split into two segments, the N-terminal peptide is referred to as "SpyTag", and the remaining portion is called "SpyCatcher" [1]. In addition to this, SnoopTag-SnoopCatcher[2] and DogTag-DogCatcher[3], two other MAC tools, also come from pili.

All of these discoveries have given us great confidence and interest. Therefore, we aim to leverage the self-assembly principles of pili in synthetic biology and engineering applications. Pili are commonly found on the surfaces of various bacteria in nature, but their fiber strengths vary, and the engineering challenges differ as well. However, the strong binding between pili monomers and structural differences among different types offer significant prospects for designing orthogonal protein scaffolds in engineering applications.

Hope that future iGEM teams can draw inspiration from this work.