Part:BBa_K4990002
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 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.
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 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.
It can do more!
We further developed two surface display systems based on bacterial pilus self-assembly.
① Currently, the proteins that can be displayed on the bacterial surface are mostly those with relatively small molecular weights. The efficiency of displaying proteins exceeding 500 amino acids is low.
② The proteins currently displayed are mainly hydrolytic enzymes or simple proteins that do not require complex folding in terms of structure. Proteins with intramolecular disulfide bonds or requiring complex folding often cannot achieve functional display. This is mainly due to the potential structural interference between carrier and passenger proteins. Fusion often disrupts the proper folding of passenger proteins, thereby reducing their activity.
③ Overexpression of exogenous proteins hinders the normal growth and metabolism of host bacteria and may even lead to the death of host bacteria.
④ The current bacterial surface display systems are mostly constructed based on plasmid vectors. The antibiotic resistance genes on these plasmids confer antibiotic resistance to the engineered bacteria, limiting the widespread application of bacterial surface display.
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.
However, our novel surface display system can circumvent all of the aforementioned issues.
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
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 31
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 31
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 31
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 31
- 1000COMPATIBLE WITH RFC[1000]
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