Difference between revisions of "Part:BBa K5185000"
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<partinfo>BBa_K5185000 short</partinfo>
 
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Human Neutrophil Peptide 1 (HNP1) is a natural antimicrobial peptide belonging to the α-defensin family, produced primarily by neutrophils. It consists of 29 amino acids and is stabilized by three disulfide bonds, which contribute to its robust structure and function. HNP1 exhibits broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and certain enveloped viruses. By using HNP1 or fusing it with other functional proteins, we can enhance antimicrobial properties in various biomedical applications.
 
  
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<br>This part collection can help and inspire other teams we are trying to achieve modification of cellulose membranes using different modification/functionalization proteins.  
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Human Neutrophil Peptide 1 (HNP1, BBa_K5185000) is a natural antimicrobial peptide belonging to the α-defensin family produced primarily by neutrophils. It consists of 29 amino acids and is stabilized by three disulfide bonds, which contribute to its robust structure and function.
  
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Defensins are a family of antimicrobial and cytotoxic peptides thought to be involved in host defense. They are abundant in the granules of neutrophils and also found in the epithelia of mucosal surfaces such as those of the intestine, respiratory tract, urinary tract, and vagina. Members of the defensin family are highly similar in protein sequence and distinguished by a conserved cysteine motif. Several alpha defensin genes are clustered on chromosome 8. This gene differs from other genes of this family by an extra 83-base segment that is apparently the result of a recent duplication within the coding region. The protein encoded by this gene, defensin, α 4, is found in the neutrophils; it exhibits corticostatic activity and inhibits corticotropin stimulated corticosterone production.
  
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==Usage and Biology==
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Human neutrophil peptides (HNPs) are naturally-occuring proteins that can target pathogenic microbes by perforating the cell membranes of Gram-negative bacteria or alternatively inhibiting cell wall synthesis in Gram-positive bacteria where cell walls are thicker and harder to perforate. These antibacterial mechanisms do not easily induce bacterial resistance, as instead of targeting specific cellular structures that can be easily modified by the pathogen, they approach wider and more essential targets such as cell membranes that are less prone to modification. Additionally, HNPs have been shown to enhance the body's immune response by recruiting leukocytes and expressing cytokines, and also take part in wound closure and healing by promoting the proliferation and migration of epithelial and bone-forming cells. Other than HNP1, this part collection includes other HNPs such as HNP4, HBD3, and HD5. By effectually releasing HNP1 into the wound, we can enhance antimicrobial properties in various biomedical applications.
  
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In nature, CBM2s are expressed as a domain of a protein whose functions require being bound to cellulose, such as cellulase. The structure of CBM2 is displayed in the Characterization section (Protein Data Bank accession: 1EXG).  
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This is part of a part collection where we characterize the antimicrobial ability of defensins and their fusion expression with CBM3 binding domains to achieve sustained release using the SUMO enzyme, providing a reliable defense against bacterial infections while mitigating the growing concern of antibiotic resistance. The part collection includes human neutrophil peptides HNP1 (<partinfo>BBa_K5185000</partinfo>), HNP4 (<partinfo>BBa_K5185001</partinfo>), HBD3 (<partinfo>BBa_K3351002</partinfo>), and HD5 (<partinfo>BBa_K3924005</partinfo>) which exhibit antimicrobial properties against Gram-positive and Gram-negative bacteria, fungi, and certain enveloped viruses. When applied to materials in a first aid kit, this part collection can effectively meet antimicrobial needs without having to address concerns of bacterial resistance.
  
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<br>CBM2 fused with spider silk proteins is first done by Mohammadi et al in 2019, where they tested the changes in physical properties on cellulose fibers after mixing with CBM2-spider silk.  
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===Usage and Biology===
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In nature, HNP1 plays a crucial role in the innate immune system by disrupting microbial cell membranes, leading to the lysis and death of pathogens such as Staphylococcus aureus, Escherichia coli, and Candida albicans. Beyond its direct antimicrobial effects, HNP1 has immune-modulatory properties. HNP1 exhibits broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and certain enveloped viruses. It acts as a chemoattractant, recruiting immune cells like monocytes, T cells, and dendritic cells to infection sites, thereby bridging innate and adaptive immunity. The structure of HNP1 is well-characterized (Protein Data Bank accession: 3GNY).
  
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<br>NT2RepCT (2Rep) is water-soluble due to hydrophilic interactions of protein N-terminal and C-terminal. When 2Rep is submerged in a coagulating bath and subjected to a shear force, the repetitive regions will uncoil, form beta-pleated sheet networks and solidify into silk fiber.
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Research into HNP1 includes its potential therapeutic applications, especially in developing new treatments against antibiotic-resistant bacteria.
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<br>The CBM2 connects the 2Rep to our bacterial cellulose membrane (BCM). The 2Rep proteins will also form hydrogen bonds with each another, in the gaps of cellulose fibers in BCM, creating a denser net made of two different materials, thus increasing the physical properties of BCM.  
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===Source===
 
===Source===
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HNP1 is derived from human neutrophils.
  
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CBM2 is derived from <i>Cellulomonas fimi</i>.. NT2RepCT is an artificial spider silk fibroin (Visit <partinfo>BBa_K3264000</partinfo> for more).
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===Results===
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We aim to verify the antimicrobial activity of defensins, including HNP1 and other defensins: HNP4 (<partinfo>BBa_K5185001</partinfo>), HBD3 (<partinfo>BBa_K3351002</partinfo>), and HD5 (<partinfo>BBa_K3924005</partinfo>). The result shows that for the defensins without the pro-segment, only HNP1 were expressed, but only in small amounts. Moreover, we observed that these strains continued to rapidly increase in cell number after IPTG induction, which is contrary to the expected results. If the produced defensin is toxic to E. coli, the growth of cells would be rapidly inhibited. We speculate that there are two reasons why the defensins are not produced and is not toxic. One reason is that the molecular weight of the defensin is too small (<5 kDa), making it easily degraded by endogenous proteases. The second reason is that the addition of 10 amino acids at the N-terminus, including a 6×His tag, affects the correct folding of the defensins. Therefore, we have decided to change our strategy and use protease cleavage to release the defensins, and after N-terminal protease cleavage, it does not contain any amino acid residues.  
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==Characterization==
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In order to modify BCM’s physical properties, we designed and expressed spider silk fibroins fused with cellulose binding matrixes (CBMs; learn more on our description page) to bind to BCM (Fig. 1). For our project, we experimented with CBM3 from Ruminiclostridium thermocellum (Protein Data Bank (PDB) accession: 1NBC; Fig. 1D) (2) and CBM2 from Cellulomonas fimi (PDB accession: 1EXG; Fig. 1C). For our spider silk protein, we chose to use the synthetic mini spider silk protein NT2RepCT (2Rep; first characterized by GreatBaySZ_2019). 2Rep is water-soluble due to hydrophilic interactions of protein N-terminal and C-terminal. When 2Rep is submerged in a coagulating bath and subjected to a shear force, the repetitive regions will uncoil, form beta-pleated sheet networks and solidify into silk fiber (Fig. 1A).
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[[Image:T--LINKS China--Figure 5.png|thumbnail|750px|center|'''Figure 1:'''
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Structure and design of 2Rep and 2Rep fused with CBMs. A) Schematic representing how individual 2Rep proteins becomes silk fibers. B) Design of CBM fused with 2Rep]]
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For adding CBM3 flanking to 2Rep, we synthesized CBM3-BsaI-CBM3 on a pET28a vector. Primers were then used to add BsaI restriction sites in 2Rep to fuse the respective domains together in the synthesized pET28a vector by Golden Gate assembly (Fig. 2A & 2B). After construction, the plasmids were transformed into E. coli BL21(DE3) for IPTG-inducible expression (Fig. 2C).
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===Comparing the solubility of CBM2/3 fused with 2Rep===
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<html>
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<img style="display: block;
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    width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/hbd3/hbd3-1.jpg" text-align="center"><div>Figure 1:</div></html>
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SDS-PAGE analysis of the recombinant defensins expression in E. coli SHuffle T7
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<br> The target proteins have a molecular weight of about 3.5 kDa for HNP1. The result shows that HNP1 was produced in small amounts in E. coli.
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<br> Subsequently, we decided to use the method of enzymatic cleavage of SUMO tag to release the defensins including HNP1.
  
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2Rep was shown to possess good water solubility, but we’re uncertain whether our modification will change this desirable property. To test the solubility, we cultured the modified strains, induced the expression of the constructs, collected the cells and performed SDS-PAGE on cell lysate (Fig. 2). Expression of both constructs, CBM3-2Rep-CBM3 (72kDa) and CBM2-2Rep-CBM2 (65kDa), are observed. CBM2-2Rep-CBM2 was present in the whole cell sample, but absent in the cell lysate supernatant, indicating poor water solubility. In contrast, CBM3-2Rep-CBM3 was present in both whole cell and supernatant. Therefore, CBM3-2Rep-CBM3 was chosen for the rest of the project for its superiority in water solubility.  
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<html>
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<img style="display: block;
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    width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/defensins.jpg" text-align="center"><div>Figure 2:</div></html>
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Recombinant expression of HNP1
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<br> The molecular weight of the CBM3-SUMO-HNP1 is 33.8kDa.
  
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[[Image:T--LINKS China--Figure 2.png|thumbnail|750px|center|'''Figure 2:'''
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<html>
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CBM-2Rep-CBM construction and expression. A) Schematic representing construction of CBM-2Rep-CBM plasmids using golden gate assembly. B) Gel electrophoresis of CBM-CBM and CBM-2Rep-CBM plasmids. C) Schematic representing transformation of CBM2-2Rep-CBM2 and CBM3-2Rep-CBM3 into E. coli DH5α D) SDS-PAGE analysis of CBM3-2Rep-CBM3 and CBM2-2Rep-CBM2. ]]
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<img style="display: block;
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    width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/hbd3/sumo.png" text-align="center"><div>Figure 3:</div></html>
  
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<html>
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<img style="display: block;
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    width: 60%;height: 60%;" src="https://static.igem.wiki/teams/5185/part-org/mic.jpg" text-align="center"><div>Figure 4:</div></html>
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Antimicrobial assay of HNP1 showed that the HNP1 successfully inhibited the  growth of E. coli and S. aureus after enzymatic digestion of the SUMO tag.
  
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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. 2C, 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.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4011009 SequenceAndFeatures</partinfo>
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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. 4a, 4b). 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 and CBM3-SUMO↓HD5 were 0.735 μM and 1.475 μM, respectively. These values are close to the MIC values reported previously for the four defensins.
  
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<!-- Uncomment this to enable Functional Parameter display
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===Reference===
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===Functional Parameters===
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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.
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<partinfo>BBa_K4011009 parameters</partinfo>
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<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 21:29, 1 October 2024

Human neutrophil peptide 1(HNP1)

Human Neutrophil Peptide 1 (HNP1, BBa_K5185000) is a natural antimicrobial peptide belonging to the α-defensin family produced primarily by neutrophils. It consists of 29 amino acids and is stabilized by three disulfide bonds, which contribute to its robust structure and function.

Defensins are a family of antimicrobial and cytotoxic peptides thought to be involved in host defense. They are abundant in the granules of neutrophils and also found in the epithelia of mucosal surfaces such as those of the intestine, respiratory tract, urinary tract, and vagina. Members of the defensin family are highly similar in protein sequence and distinguished by a conserved cysteine motif. Several alpha defensin genes are clustered on chromosome 8. This gene differs from other genes of this family by an extra 83-base segment that is apparently the result of a recent duplication within the coding region. The protein encoded by this gene, defensin, α 4, is found in the neutrophils; it exhibits corticostatic activity and inhibits corticotropin stimulated corticosterone production.

Human neutrophil peptides (HNPs) are naturally-occuring proteins that can target pathogenic microbes by perforating the cell membranes of Gram-negative bacteria or alternatively inhibiting cell wall synthesis in Gram-positive bacteria where cell walls are thicker and harder to perforate. These antibacterial mechanisms do not easily induce bacterial resistance, as instead of targeting specific cellular structures that can be easily modified by the pathogen, they approach wider and more essential targets such as cell membranes that are less prone to modification. Additionally, HNPs have been shown to enhance the body's immune response by recruiting leukocytes and expressing cytokines, and also take part in wound closure and healing by promoting the proliferation and migration of epithelial and bone-forming cells. Other than HNP1, this part collection includes other HNPs such as HNP4, HBD3, and HD5. By effectually releasing HNP1 into the wound, we can enhance antimicrobial properties in various biomedical applications.

This is part of a part collection where we characterize the antimicrobial ability of defensins and their fusion expression with CBM3 binding domains to achieve sustained release using the SUMO enzyme, providing a reliable defense against bacterial infections while mitigating the growing concern of antibiotic resistance. The part collection includes human neutrophil peptides HNP1 (BBa_K5185000), HNP4 (BBa_K5185001), HBD3 (BBa_K3351002), and HD5 (BBa_K3924005) which exhibit antimicrobial properties against Gram-positive and Gram-negative bacteria, fungi, and certain enveloped viruses. When applied to materials in a first aid kit, this part collection can effectively meet antimicrobial needs without having to address concerns of bacterial resistance.

Usage and Biology

In nature, HNP1 plays a crucial role in the innate immune system by disrupting microbial cell membranes, leading to the lysis and death of pathogens such as Staphylococcus aureus, Escherichia coli, and Candida albicans. Beyond its direct antimicrobial effects, HNP1 has immune-modulatory properties. HNP1 exhibits broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and certain enveloped viruses. It acts as a chemoattractant, recruiting immune cells like monocytes, T cells, and dendritic cells to infection sites, thereby bridging innate and adaptive immunity. The structure of HNP1 is well-characterized (Protein Data Bank accession: 3GNY).

Research into HNP1 includes its potential therapeutic applications, especially in developing new treatments against antibiotic-resistant bacteria.

Source

HNP1 is derived from human neutrophils.

Results

We aim to verify the antimicrobial activity of defensins, including HNP1 and other defensins: HNP4 (BBa_K5185001), HBD3 (BBa_K3351002), and HD5 (BBa_K3924005). The result shows that for the defensins without the pro-segment, only HNP1 were expressed, but only in small amounts. Moreover, we observed that these strains continued to rapidly increase in cell number after IPTG induction, which is contrary to the expected results. If the produced defensin is toxic to E. coli, the growth of cells would be rapidly inhibited. We speculate that there are two reasons why the defensins are not produced and is not toxic. One reason is that the molecular weight of the defensin is too small (<5 kDa), making it easily degraded by endogenous proteases. The second reason is that the addition of 10 amino acids at the N-terminus, including a 6×His tag, affects the correct folding of the defensins. Therefore, we have decided to change our strategy and use protease cleavage to release the defensins, and after N-terminal protease cleavage, it does not contain any amino acid residues.

Figure 1:
SDS-PAGE analysis of the recombinant defensins expression in E. coli SHuffle T7
The target proteins have a molecular weight of about 3.5 kDa for HNP1. The result shows that HNP1 was produced in small amounts in E. coli.
Subsequently, we decided to use the method of enzymatic cleavage of SUMO tag to release the defensins including HNP1.

Figure 2:
Recombinant expression of HNP1
The molecular weight of the CBM3-SUMO-HNP1 is 33.8kDa.

Figure 3:

Figure 4:
Antimicrobial assay of HNP1 showed that the HNP1 successfully inhibited the growth of E. coli and S. aureus after enzymatic digestion of the SUMO tag.

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. 2C, 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. 4a, 4b). 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 and CBM3-SUMO↓HD5 were 0.735 μM and 1.475 μM, respectively. These values are close to the MIC values reported previously for the four defensins.

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.