Difference between revisions of "Part:BBa K5185028"

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	<partinfo>BBa_K5185028 short</partinfo>		<partinfo>BBa_K5185028 short</partinfo>
		+	CBM3-sumo-HBD3 is a fusion protein with three distinct domains: Human beta-defensin 3 (HBD3, <partinfo>BBa_K3351002</partinfo>), a beta defensin that exhibits antibacterial activity against Gram-positive and Gram-negative bacteria, Carbonhydrate-Binding molecule 3(CBM3, <partinfo>BBa_K4011000</partinfo>) that enhances the ability of enzymes to target and degrade cellulose by attaching to the cellulose surface, and the SUMO tag which improves the solubility, stability, and folding of proteins. This part is part of a collection where the universality of the combined function of the binding domain and defensins is assessed, allowing for a more versatile collection of antibacterial dressings and enhancing the potential of our first aid kit to address more complex situations.
−	HNP1Ala is a modified version of HNP1 through substitution of glycine and tryosine with alanine at the 12th residue and 22nd residue, which is expected to increase the hydrophobicity of HNP1.	+	Our project aims to endow first-aid wound dressings with enhanced antimicrobial functions and a wider and more complex application. By fusing the binding domain CBM3 with the defensin HNP1, we can bestow items such as bandages and antiseptic wipes, specifically those made of cellulose, with properties that facilitate hemostasis and prevents bacterial growth. CBM3 activates hemostatic pathways in the human body, while HNP1 interferes with the normal functionality of bacteria.
−	===Usage and Biology===	+	This is part of a part collection of SUMO linking a binding domain to defensin, which allows defensins to be attached to carbohydrates such as cellulose, chitosan, and collagen. When applied with a SUMO Protease, this fusion protein may effectively release the defensin into the site of injury and therefore achieve the desired antimicrobial effects, acting as a reliable defense against bacterial infections while mitigating the growing concern of antibiotic resistance.
−	α-defensins typically have a triple-stranded beta-sheet structure based on internal disulfide bonds (Lehrer et al. 2012) and further stabilized by other immutable structures. Apart from these residues, point mutations on alpha-defensins tend to have limited effect on the general conformation. Alpha-defensin dimers are formed when amino acids on the second beta-strand interact with each other. Specifically, carboxyl and nitric groups in the amino acid backbone of the two monomers formed 4 hydrogen bonds during dimerization. In addition, cationicity and hydrophobicity are both key determinants in dimerization rates, with the latter showing more significant impact than the former (Rajabi et al. 2012). Thus, our general goal is to create a stable dimer structure while increasing cationicity and hydrophobicity.	+
−	===Modeling===	+	Other than CBM3, this part collection includes other CBMs such as CBM2, CBM5, and VbCBMxx, and also human integrin domains such as α1 and α2. Other than HNP1, other defensins in this part collection include HNP4, HD5, and HBD3. We synthesized the fusion proteins CBM3-sumo-HNP1 (<partinfo>BBa_K5185010</partinfo>), CBM3-sumo-HNP4 (<partinfo>BBa_K5185011</partinfo>),CBM3-sumo-HD5 (<partinfo>BBa_K5185012</partinfo>), and CBM3-sumo-HBD3 (<partinfo>BBa_K5185013</partinfo>) for materials in the first aid kit composed of cellulose, bestowing them with antimicrobial functions. Other fusion proteins we synthesized include CBM5-sumo-HNP1 (<partinfo>BBa_K5185015</partinfo>) which focuses on more enhanced anti-microbial functions and targets especially severe infections, and α2-sumo-HNP1 (<partinfo>BBa_K5185017</partinfo>) which with the use of collagen enables better wound healing, targeting wounds that prioritize wound recovery.  Recognizing this part collection of fusion proteins as effective in treating wounds and achieving antimicrobial needs, we believe the HNPs could each be linked with different wound-dressing materials that would provide an array of approaches and solutions to suit the varied needs of different wounds and circumstances with limited medical resources, such as battlefields and disaster zones.
−	Our models can mainly be categorized in active site amino acid mutations and other amino acid mutations. We sought to stabilize the active site by introducing additional interactions between the two monomers. Upon considerations, we decided attempt creating pi-pi, pi-cation, and hydrogen bonding interactions between amino acid side chains. Aromatic rings composes of conjugate pi-bonds, which causes the pi-electrons to be delocalized and shared across multiple molecules. A pi-pi interaction is when the delocalized electrons are shared between two aromatic rings when placed adjacently, while pi-cation interaction is characterized by an attraction between a positively group and the delocalized pi-electrons. It should be noted that mutagenesis in our models might not accurately reflect the actual configuration of side chain of the modified HNP-1 that are produced in experimentation. Therefore, we would prioritize pi-pi interactions (increase hydrophobicity) and pi-cation (increase hydrophobicity and cationicity) over hydrogen bonding under the same predicted affinity. The tetramer of HNP-1 was obtained from the Protein Data Bank (PDB id: 3GNY), and the dimer molecule was extracted. The amino acids of the active site are T-18, C-19, and I-20 (Amino acid - index), and as C-19 is immutable (as discussed later), we will focus on mutating T-18 and I-20.	+
−	Apart from active site amino acids, other amino acids were also mutated to increase hydrophobicity. It is important to note that there are several immutable that constructs the basic structure of alpha-defensin, namely the cytosine residues (C-2, C-4, C-9, C-19, C-29, C-30) that constitute disulfide bonds, arginine and glutamic acid residues (R-5, E-13) that constitute an essential salt bridge, and an invariant glycine (G-17). Of the remaining amino acids, we selected all non-positive hydrophilic amino acids and mutated them to alanine to increase hydrophobicity without drastically changing the general conformation.	+	===Usage and Biology===
−	Therefore, in this mutation, we first mutated G12 and Y22 to Ala to enhance the hydrophobicity of defensins(Fig. 1A), thus generating the HNP1 mutant HNP1_G12A_Y22A (HNP1Ala).After the alanine substitution of amino acids, the alpha-fold predicted model shows high similarity with the original HNP-1 when aligned in PyMOL (rmsd = 0.482). Hdock result of the final structure HNP-1_Ala gives a confidence score of 0.8058.	+	CBM3-sumo-HBD3 enables controlled release of antimicrobial peptide within the human body, thus achieving sustained antibacterial activity, which makes it suitable for antimicrobial coatings on wound dressings, biodegradable antibacterial materials, or biofilm disruption on cellulose-based surfaces. HBD3 is able to disrupt microbial membranes, leading to cell lysis and death of pathogens like Staphylococcus aureus, Escherichia coli, and Candida albicans, and CBM3 has applications in biotechnological processes, including biomass conversion, biofuel production.
−	We then performed point mutations on amino acids G18 and L20 of the active site to obtain three other HNP1 mutants：HNP1ANF，HNP1AWW(<partinfo>BBa_K5185030</partinfo>) and HNP1AWK(<partinfo>BBa_K5185031</partinfo>).	+
	===Results===		===Results===
−	We assembled HNP1 mutants onto CBM3-SUMO-Defensins modules for characterization(<partinfo>BBa_K51850010</partinfo>)(<partinfo>BBa_K51850011</partinfo>)(<partinfo>BBa_K51850012</partinfo>)(<partinfo>BBa_K51850013</partinfo>)SDS-PAGE electrophoresis identified the expression of HNP1Ala, HNP1AWW and HNP1AWK, and we used them for subsequent antibacterial tests(Fig.1A).	+	The IPTG-induced expression of CBM3-SUMO-defensins was validated in the same manner as the defensins. SDS-PAGE analysis revealed the presence of the correct bands (Fig. 1). Subsequently, we scaled up the fermentation to 400 mL. Once the optical density (OD) reached 0.6–0.8, we induced expression with 0.1 mM IPTG at 20°C for 12 hours. Afterward, we lysed the cells and purified the target protein from the cell lysate using a Ni-NTA affinity chromatography column.
	<html>		<html>
	<img style="display: block;		<img style="display: block;
−	width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/modeling-1.png" text-align="center"><div></html>	+	width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/defensins.jpg" text-align="center"><div>Figure 1: SDS-PAGE analysis of the CBM3-SUMO-Defensins expression in E. coli SHuffle T7</div></html>
−	<br>Figure 1 (A) Sequence of HNP1Ala (B) SDS-PAGE analysis of the recombinant defensins expression in E. coli SHuffle T7	+	The molecular weight of the CBM3-SUMO-Defensins is 33.8kDa，34.0 kDa, 33.9kDa，35.5kDa.
	<html>		<html>
	<img style="display: block;		<img style="display: block;
−	width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/modeling-2.png" text-align="center"> </div></html>	+	width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/hbd3/sumo.png" text-align="center"><div>Figure 2</div></html>
−	<br>Figure 2: The antibacterial activity of HNP1 mutants. Panels A, B, and C display the 12-hour growth curves (left) and 8-hour minimum inhibitory concentration (MIC) determinations (right) for various mutants. CBM3-sumo↓HNP1Ala is shown in Panel B. HNP1Ala still displayed antimicrobial activity despite being not as efficient as HNP1.	+	(a) Verification of SUMO cleavage of CBM3-SUMO-Defensins. -Ulp1 represents CBM3-SUMO-Defensins that have not been treated with Ulp1, +Ulp1 represents CBM3-SUMO↓Defensins cleaved by Ulp1.
		+	<kb> (b) Antimicrobial assays of four types of CBM3-SUMO↓Defensins against Escherichia coli and Staphylococcus aureus
		+
		+	4 types of CBM3-SUMO-Defensins were subjected to salt removal by gradient dialysis, followed by cleavage with recombinant Ulp1. The results from SDS-PAGE electrophoresis showed a slight decrease in the molecular weight of the target protein (Fig. 3a), indicating successful removal of ~4 kDa defensins. Since CBM3-SUMO-Defensins would ultimately be incorporated into wound dressing products in a domain-bound form rather than as individual defensins, we did not further purify the defensins. Instead, we utilized the enzyme-cleaved CBM3-SUMO-Defensins (designated as CBM3-SUMO↓Defensins) for antimicrobial assays. As depicted in Fig. 2b, Escherichia coli and Staphylococcus aureus were selected as representatives of Gram-positive and Gram-negative bacteria, respectively. The CBM3-SUMO↓Defensins cleaved by Ulp1 enzyme exhibited antimicrobial activity against both strains, while the uncleaved CBM3-SUMO-Defensins showed no antimicrobial activity. This suggests that we successfully produced active defensin molecules using the fusion protein cleavage approach.
		+
		+	We utilized the microdilution method to determine the MIC values of four types of CBM3-SUMO-Defensins. For specific details, please refer to our measurement section. Initially, we examined the 24-hour growth curves of Staphylococcus aureus with the addition of CBM3-SUMO↓Defensins. Within the 0–8 hour range, all four types of CBM3-SUMO↓Defensins exhibited antimicrobial activity (Fig. 2a). We selected the 8-hour time point to define the MIC values against Staphylococcus aureus. At this point, the MIC50 values for CBM3-SUMO↓HNP1, CBM3-SUMO↓HNP4, CBM3-SUMO↓HD5, and CBM3-SUMO↓HBD3 were 0.74 μM, 0.368 μM, 1.475 μM, and 1.001 μM, respectively. Additionally, the MIC90 values for CBM3-SUMO↓HNP4/HD5 were 0.735 μM and 1.475 μM, respectively. These values are close to the MIC values reported previously for the four defensins (Wei et al., 2009).
		+
		+	<html>
		+	<img style="display: block;
		+	width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/mic.jpg" text-align="center"><div>Figure 3:</div></html>
		+	Verification of the antibacterial activity of CBM3-SUMO↓Defensins.
		+	(a) The effect of the four CBM3-SUMO↓Defensins on the growth of Staphylococcus aureus within 24 hours.
		+	<kb> (b) The inhibition rate of CBM3-SUMO↓Defensins on Staphylococcus aureus after 8 hours.
		+	<kb> Antimicrobial assay of HBD3 showed that the HBD3 successfully inhibited the  growth of E. coli and S. aureus after enzymatic digestion of the SUMO tag.
		+
		+	It is worth mentioning that when the concentrations of the four types of CBM3-SUMO↓Defensins were reduced to 185 nM, 184 nM, 184 nM, and 125 nM, they were able to promote the growth of Staphylococcus aureus (Fig. 3b). This suggests that the non-defensin portion of CBM3-SUMO↓Defensins may serve as a nutrient for bacteria, providing amino acids upon hydrolysis. After 8 hours, high concentrations of CBM3-SUMO↓Defensins were able to promote the growth of Staphylococcus aureus (Fig. 3a). We speculate that this is due to the short peptide nature of defensins, which makes them susceptible to degradation by proteases, resulting in a shorter effective period. After defensins become inactive after 8 hours, CBM3-SUMO↓Defensins act as nutrients that promote bacterial growth. Therefore, in our antibacterial dressings, a higher concentration is not necessarily better. We believe that in the future, we can choose smaller Binding domains or optimize the sequence of natural Binding domains to increase the proportion of defensin molecules as much as possible while keeping the molar concentration of the fusion protein constant, thereby reducing the non-defensin portion to avoid providing nutrients to bacteria and improving the MIC.
−	The results show that the MIC99 values of the four mutants are all higher than the original version(<partinfo>BBa_K5185000</partinfo>), which means that the point mutations on these amino acids of HNP1 reduce the ability to inhibit Staphylococcus aureus, but the antibacterial activity is not completely lost, and the mutations produce new slightly active substances. Weak HNP1 needs further verification of its antibacterial activity against other pathogenic bacteria.	+	===Reference===
−	However, since protein engineering has diverse applications, our project serves as a promising model for future teams. For example, even though antibacterial activity is reduced, new characteristics are being attributed to HNP-1. When the purpose is changed to, for example, adding aromatic ring structures to form new interactions, our design proves to be successful while maintaining most antibacterial functions.	+	Fu, J., Zong, X., Jin, M., Min, J., Wang, F., & Wang, Y. (2023). Mechanisms and regulation of defensins in host defense. Signal Transduction and Targeted Therapy, 8(1), 300.
		+	<br> Wei, G., de Leeuw, E., Pazgier, M., Yuan, W., Zou, G., Wang, J., ... & Lu, W. (2009). Through the looking glass, mechanistic insights from enantiomeric human defensins. Journal of Biological Chemistry, 284(42), 29180-29192.

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Revision as of 06:13, 2 October 2024

HNP1_G12A_Y22A CBM3-sumo-HBD3 is a fusion protein with three distinct domains: Human beta-defensin 3 (HBD3, BBa_K3351002), a beta defensin that exhibits antibacterial activity against Gram-positive and Gram-negative bacteria, Carbonhydrate-Binding molecule 3(CBM3, BBa_K4011000) that enhances the ability of enzymes to target and degrade cellulose by attaching to the cellulose surface, and the SUMO tag which improves the solubility, stability, and folding of proteins. This part is part of a collection where the universality of the combined function of the binding domain and defensins is assessed, allowing for a more versatile collection of antibacterial dressings and enhancing the potential of our first aid kit to address more complex situations.

Our project aims to endow first-aid wound dressings with enhanced antimicrobial functions and a wider and more complex application. By fusing the binding domain CBM3 with the defensin HNP1, we can bestow items such as bandages and antiseptic wipes, specifically those made of cellulose, with properties that facilitate hemostasis and prevents bacterial growth. CBM3 activates hemostatic pathways in the human body, while HNP1 interferes with the normal functionality of bacteria.

This is part of a part collection of SUMO linking a binding domain to defensin, which allows defensins to be attached to carbohydrates such as cellulose, chitosan, and collagen. When applied with a SUMO Protease, this fusion protein may effectively release the defensin into the site of injury and therefore achieve the desired antimicrobial effects, acting as a reliable defense against bacterial infections while mitigating the growing concern of antibiotic resistance.

Other than CBM3, this part collection includes other CBMs such as CBM2, CBM5, and VbCBMxx, and also human integrin domains such as α1 and α2. Other than HNP1, other defensins in this part collection include HNP4, HD5, and HBD3. We synthesized the fusion proteins CBM3-sumo-HNP1 (BBa_K5185010), CBM3-sumo-HNP4 (BBa_K5185011),CBM3-sumo-HD5 (BBa_K5185012), and CBM3-sumo-HBD3 (BBa_K5185013) for materials in the first aid kit composed of cellulose, bestowing them with antimicrobial functions. Other fusion proteins we synthesized include CBM5-sumo-HNP1 (BBa_K5185015) which focuses on more enhanced anti-microbial functions and targets especially severe infections, and α2-sumo-HNP1 (BBa_K5185017) which with the use of collagen enables better wound healing, targeting wounds that prioritize wound recovery. Recognizing this part collection of fusion proteins as effective in treating wounds and achieving antimicrobial needs, we believe the HNPs could each be linked with different wound-dressing materials that would provide an array of approaches and solutions to suit the varied needs of different wounds and circumstances with limited medical resources, such as battlefields and disaster zones.

Usage and Biology

CBM3-sumo-HBD3 enables controlled release of antimicrobial peptide within the human body, thus achieving sustained antibacterial activity, which makes it suitable for antimicrobial coatings on wound dressings, biodegradable antibacterial materials, or biofilm disruption on cellulose-based surfaces. HBD3 is able to disrupt microbial membranes, leading to cell lysis and death of pathogens like Staphylococcus aureus, Escherichia coli, and Candida albicans, and CBM3 has applications in biotechnological processes, including biomass conversion, biofuel production.

Results

The IPTG-induced expression of CBM3-SUMO-defensins was validated in the same manner as the defensins. SDS-PAGE analysis revealed the presence of the correct bands (Fig. 1). Subsequently, we scaled up the fermentation to 400 mL. Once the optical density (OD) reached 0.6–0.8, we induced expression with 0.1 mM IPTG at 20°C for 12 hours. Afterward, we lysed the cells and purified the target protein from the cell lysate using a Ni-NTA affinity chromatography column.

The molecular weight of the CBM3-SUMO-Defensins is 33.8kDa，34.0 kDa, 33.9kDa，35.5kDa.

(a) Verification of SUMO cleavage of CBM3-SUMO-Defensins. -Ulp1 represents CBM3-SUMO-Defensins that have not been treated with Ulp1, +Ulp1 represents CBM3-SUMO↓Defensins cleaved by Ulp1. <kb> (b) Antimicrobial assays of four types of CBM3-SUMO↓Defensins against Escherichia coli and Staphylococcus aureus

4 types of CBM3-SUMO-Defensins were subjected to salt removal by gradient dialysis, followed by cleavage with recombinant Ulp1. The results from SDS-PAGE electrophoresis showed a slight decrease in the molecular weight of the target protein (Fig. 3a), indicating successful removal of ~4 kDa defensins. Since CBM3-SUMO-Defensins would ultimately be incorporated into wound dressing products in a domain-bound form rather than as individual defensins, we did not further purify the defensins. Instead, we utilized the enzyme-cleaved CBM3-SUMO-Defensins (designated as CBM3-SUMO↓Defensins) for antimicrobial assays. As depicted in Fig. 2b, Escherichia coli and Staphylococcus aureus were selected as representatives of Gram-positive and Gram-negative bacteria, respectively. The CBM3-SUMO↓Defensins cleaved by Ulp1 enzyme exhibited antimicrobial activity against both strains, while the uncleaved CBM3-SUMO-Defensins showed no antimicrobial activity. This suggests that we successfully produced active defensin molecules using the fusion protein cleavage approach.

We utilized the microdilution method to determine the MIC values of four types of CBM3-SUMO-Defensins. For specific details, please refer to our measurement section. Initially, we examined the 24-hour growth curves of Staphylococcus aureus with the addition of CBM3-SUMO↓Defensins. Within the 0–8 hour range, all four types of CBM3-SUMO↓Defensins exhibited antimicrobial activity (Fig. 2a). We selected the 8-hour time point to define the MIC values against Staphylococcus aureus. At this point, the MIC50 values for CBM3-SUMO↓HNP1, CBM3-SUMO↓HNP4, CBM3-SUMO↓HD5, and CBM3-SUMO↓HBD3 were 0.74 μM, 0.368 μM, 1.475 μM, and 1.001 μM, respectively. Additionally, the MIC90 values for CBM3-SUMO↓HNP4/HD5 were 0.735 μM and 1.475 μM, respectively. These values are close to the MIC values reported previously for the four defensins (Wei et al., 2009).

Verification of the antibacterial activity of CBM3-SUMO↓Defensins. (a) The effect of the four CBM3-SUMO↓Defensins on the growth of Staphylococcus aureus within 24 hours. <kb> (b) The inhibition rate of CBM3-SUMO↓Defensins on Staphylococcus aureus after 8 hours. <kb> Antimicrobial assay of HBD3 showed that the HBD3 successfully inhibited the growth of E. coli and S. aureus after enzymatic digestion of the SUMO tag.

It is worth mentioning that when the concentrations of the four types of CBM3-SUMO↓Defensins were reduced to 185 nM, 184 nM, 184 nM, and 125 nM, they were able to promote the growth of Staphylococcus aureus (Fig. 3b). This suggests that the non-defensin portion of CBM3-SUMO↓Defensins may serve as a nutrient for bacteria, providing amino acids upon hydrolysis. After 8 hours, high concentrations of CBM3-SUMO↓Defensins were able to promote the growth of Staphylococcus aureus (Fig. 3a). We speculate that this is due to the short peptide nature of defensins, which makes them susceptible to degradation by proteases, resulting in a shorter effective period. After defensins become inactive after 8 hours, CBM3-SUMO↓Defensins act as nutrients that promote bacterial growth. Therefore, in our antibacterial dressings, a higher concentration is not necessarily better. We believe that in the future, we can choose smaller Binding domains or optimize the sequence of natural Binding domains to increase the proportion of defensin molecules as much as possible while keeping the molar concentration of the fusion protein constant, thereby reducing the non-defensin portion to avoid providing nutrients to bacteria and improving the MIC.

Reference

Fu, J., Zong, X., Jin, M., Min, J., Wang, F., & Wang, Y. (2023). Mechanisms and regulation of defensins in host defense. Signal Transduction and Targeted Therapy, 8(1), 300.
Wei, G., de Leeuw, E., Pazgier, M., Yuan, W., Zou, G., Wang, J., ... & Lu, W. (2009). Through the looking glass, mechanistic insights from enantiomeric human defensins. Journal of Biological Chemistry, 284(42), 29180-29192.

Sequence and Features