Difference between revisions of "Part:BBa K4990002"

Latest revision as of 10:40, 27 September 2024

mFadA B-domain

Usage in short

You can use it for specific targeting of Fusobacterium nucleatum(Fn) or achieve bacteria-bacteria adhesion.

What you need to know!

Many bacteria have long chain fiber-protein complexes 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.

Our project aims to accomplish bacteria-bacteria targeting. To accomplish this, we intend to utilize the self-assembly property of mFadA. We have used the surface display technology to fuse 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 displaying the fishing rod protein, our engineered bacteria can essentially "fish" for target bacteria, enabling precise bacteria-bacteria targeting.

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:

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).

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.

How does it work?

The following are detailed microscale contacts involved in the self-assembly of mFadA.

Notice

1.The residue numbering is based on the complete mFadA sequence, rather than renumbering after truncation of domain A or B.

2.The protein sequence of mFadA can be referred to in this article: Han YW, Ikegami A, Rajanna C, et al. Identification and characterization of a novel adhesin unique to oral fusobacteria. J Bacteriol. 2005;187(15):5330-5340. doi:10.1128/JB.187.15.5330-5340.2005

3.The information regarding contacts related to self-assembly mentioned above comes from this article: Nithianantham S, Xu M, Yamada M, Ikegami A, Shoham M, Han YW. Crystal structure of FadA adhesin from Fusobacterium nucleatum reveals a novel oligomerization motif, the leucine chain. J Biol Chem. 2009;284(6):3865-3872. doi:10.1074/jbc.M805503200

Two mFadA monomers were sourced from reference [1]. The mFadA protein sequence from Fn ATCC10953 was selected, and structural prediction was conducted using Colabfold. Employing the Rosetta local_docking method, a total of 100,000 rounds of Monte Carlo-based repeated docking were performed, leading to the successful identification of the optimal self-assembly outcome. The obtained assembly closely resembles the binding mode described in reference [2], as illustrated in the diagram below：

We further identified the microscale contacts between the two mFadA monomers. These contacts are categorized into primary and secondary hydrophobic interactions, as well as salt bridge interactions, as depicted in the diagram below

It is worth noting 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.

Saturate mutation of mFadA homodimer and the position 1-17 is B-domain

Wetlab Characterization

The interaction between BTP and mFadA, or between DEH and mFadA.

A. BTP-mFadA complex(~20kDa) and DEH-mFadA complex(~20kDa) could be observed (red squares). And bands at other different molecular weight position appear due to the self-assemble of mFadA and the dimeration of HlpA in DEH(blue squares), whose structures are annotated beside.

B. The structure of BTP.

C. The interaction between BTP and mFadA(C i), or between DEH and mFadA(C ii)

D&E. The complexes formed due to the self-assembly of mFadA(D) and the dimerization of HlpA(E).

In lanes 3-5 of the figure, clear bands appear around 20kDa, evidencing the interaction between BTP (~5kDa) and mFadA (~15kDa). This confirms that BTP can bind with pili monomers and aid in the self-assembly process of pili(Fig 6C(i)), making it an effective method for targeting pathogenic bacteria Fn.

Furthermore, the self-assembly phenomenon of mFadA may result in ladder-like bands at different molecular weight positions. These bands could be due to the interaction of the BTP-mFadA-mFada complex (~35kDa,Fig 6D(i)), and the BTP-mFadA-mFadA-mFadA complex (~50kDa, Fig 6D(ii)). These unexpected bands have indirectly verified the self-assembly of pili monomers, thereby providing further evidence for the reliability of our project.

The figure shows clear bands around 30kDa in lanes 6-8, suggesting that DEH (~15kDa) can bind with pili monomers via interaction with mFadA (~15kDa). This proves that DEH can target pathogenic bacteria Fn by participating in the self-assembly process of pili(Fig 6C(ii)).

Similar to the above, the self-assembly of mFadA can result in the occurrence of unanticipated bands. Furthermore, the breakage of the linker in DEH and the dimerization of target cancer fragments can lead to a more complex band situation. The bands near 30kDa in lanes 6-8 are more prominent than those in lanes 3-5, possibly due to the existence of mFadA dimers in this region. The unexpected band could also formed due to HlpA-DEH-mFadA complex(~40kDa, Fig 6E(i)),DEH-mFadA-mFadA complex(~45kDa, Fig 6E(ii)), DEH-mFadA-mFadA-mFadA complex(~60kDa, Fig 6E(iii)) etc.

In our DEH design, we found thatif using the complete B-domain (①), there is an inability to form a complete α-helix. Therefore, the optimized DEH had the C-terminal five amino acids removed from the B-domain to ensure the stable formation of the α-helix structure at the N-terminus of DEH. As shown in ④, the added GPNG sequence once again played a role, targeting the Fn segment downward and the CRC targeting functional domain upward, effectively achieving "function domain isolation." Therefore, this optimized DEH was selected as our final targeting cytotoxic peptide.

It can do more!

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.

And you can find pilus self-assembly wetlab characterization in https://parts.igem.org/Part:BBa_K4990004

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.

Molecular dynamics analysis results of EC5.| a. RMSD of EC5 protein complexes; b. Combined Rg and RMSD analysis of EC5 protein complexes. c. RMSF of the EC5 protein complex. d. Analysis of protein slewing in the space of EC5 protein complexes.e~f. Hydrogen bonding between the EC5 protein complex and the solvent and internal hydrogen bonding. g. Covariance matrix of EC5 protein complexes. h. Ramachandran diagram of the EC5 protein complex.( The dramatic fluctuations in the figure are caused by the cyclic boundary of the water box.)

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.

Novo MAC method

Although these limitations exist, with the extensive development of microbial genomics and proteomics research, along with a deeper understanding of the genetic background, cell surface structure, and function of environmentally advantageous microbial communities, researchers will continue to develop bacterial surface display systems that cater to various needs.

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.

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.

Reference

[1]Han YW, Ikegami A, Rajanna C, et al. Identification and characterization of a novel adhesin unique to oral fusobacteria. J Bacteriol. 2005;187(15):5330-5340. doi:10.1128/JB.187.15.5330-5340.2005

[2]Nithianantham S, Xu M, Yamada M, Ikegami A, Shoham M, Han YW. Crystal structure of FadA adhesin from Fusobacterium nucleatum reveals a novel oligomerization motif, the leucine chain. J Biol Chem. 2009;284(6):3865-3872. doi:10.1074/jbc.M805503200

[3]Témoin S, Wu KL, Wu V, Shoham M, Han YW. Signal peptide of FadA adhesin from Fusobacterium nucleatum plays a novel structural role by modulating the filament's length and width. FEBS Lett. 2012;586(1):1-6. doi:10.1016/j.febslet.2011.10.047

Usage of This Part by iGEM Aachen 2024

The iGEM Team Aachen 2024 used this part as the foundation for their binding domain within their fusionproteins. The mFadA B-domain has been codon optimized for expression in E. coli.

Additionally the forbidden restriction sites were removed by including silent mutations. This way the resulting biological parts are BioBrick compatible.

For further information, visit our part descriptions:
CDA wild type fusionprotein (BBa_K5033000).

Our mutants of the wild type fusionprotein:
'Alpha' (BBa_K5033001), 'Beta' (BBa_K5033002), 'Gamma' (BBa_K5033003), 'Epsilon' (BBa_K5033004) and 'Theta' (BBa_K5033005). The Gamma mutant has approximately doubled catalytic activity compared to our wild type fusionprotein. Additionally it is expected to have a much higher relative affinity to 5-fluorocytosine over cytosine, making it our most promising candidate.

And our reporter version (BBa_K5033006).

Binding assays with this reporter protein have indicated binding to various fusobacteria strains, but were not particularly conclusive. A priority for the future is the development of more robust assays to clearly quantify the binding. Visit our part description for more details.

Sequence and Features