Part:BBa_K5185001

Human neutrophil peptide 4 (HNP4)

Human Neutrophil Peptide 4 (HNP4) is a member of the α-defensin family of antimicrobial peptides produced by neutrophils. Comprising 33 amino acids and stabilized by three disulfide bonds, HNP4 contributes to the body's first line of defense against microbial infections.

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 HNP4, this part collection includes other HNPs such as HNP1, HBD3, and HD5. By effectually releasing HNP4 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 the human immune system, HNP4 exhibits broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, fungi, and certain enveloped viruses. It disrupts microbial membranes, leading to cell lysis and death of pathogens like Staphylococcus aureus, Escherichia coli, and Candida albicans. Although less abundant than HNP1-3, HNP4 plays a significant role in immune defense and can act synergistically with other defensins to enhance antimicrobial efficacy. The structure of HNP4 is documented in the Protein Data Bank (accession: 1ZMM). Research on HNP4 involves understanding its role in immune modulation and exploring its potential in treating infections, particularly those resistant to conventional antibiotics.

Source

HNP4 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 HNP4. The production of HNP4 was not observed in E. coli.
Subsequently, we decided to use the method of enzymatic cleavage of SUMO tag to release the defensins, including HNP4.

Figure 2: Recombinant expression of HNP4

The molecular weight of the CBM3-SUMO-HNP1 is 33.8kDa.

Figure 3

Figure 4:

Antimicrobial assay of HNP4 showed that the HNP4 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.