In today's society, over one million people die each year from antibiotic-resistant bacteria^[1], which is largely attributed to the excessive use of antibiotics over the past few decades. Moreover, globally, as people's living standards improve, keeping pets has gradually become a common lifestyle. Pets not only provide emotional companionship but also improve the quality of human life to a certain extent. However, as the number of pets continues to grow, health issues related to pets are becoming increasingly prominent, with bacterial infections being particularly notable.

According to the data on pet visits from the World Pet Association (WPA), the top three diseases that pets visit the store for are digestive system diseases, skin diseases, and respiratory system diseases. Most of these three types of diseases are caused by bacterial infections. Therefore, solving the problem of pet diseases caused by bacterial infections has become a major global challenge.

The traditional approach is to use antibiotics, but the difficulty of developing new antibiotics is increasing. Moreover, we have found that in the treatment of pet bacterial infection diseases, the antibiotic-based treatment plan is facing a serious problem of bacterial resistance: due to blind medication by veterinarians, insufficient knowledge, and unreasonable requests for medication by pet owners, the abuse of antibacterial drugs is widespread, resulting in 75% of the isolated strains of bacteria causing pet urinary tract infections being resistant, and over one-third being multi-drug resistant. Common first-line antibiotics such as penicillin and fluoroquinolones frequently fail, not only increasing the risk of treatment failure, prolonging the course of the disease for pets, and raising treatment costs, but also possibly allowing drug-resistant bacteria on pets to be transmitted to humans through contact, breathing, and other means, threatening the health of vulnerable groups such as children and the elderly. This has become the most intractable problem in the treatment of pet infections, which has drawn attention to antimicrobial peptides (AMPs). AMPs exist in various organisms from prokaryotes to humans and have broad-spectrum antibacterial activity.

Figure 1. Canine Clinic Visit Statistics (Source: WPA Website)

Figure 2. Feline Clinic Visit Statistics (Source: WPA Website)

Antimicrobial Peptides (AMPs) are generally defined as small peptide groups consisting of 10 to 50 amino acids, with a positive net charge ranging from +2 to +11^[2,3]. The interaction between positively charged AMPs and negatively charged bacterial membranes triggers the formation of cracks in the membranes, leading to pore formation and ultimately bacterial death^[3]. In addition to pore-forming AMPs, some AMPs can translocate across bacterial membrane barriers through self-promoted uptake and target key cellular processes, such as macromolecular synthesis (DNA, RNA, proteins, and cell walls), protein folding, and enzyme activity^[4]. This multi-mechanism of action not only enhances the efficacy of AMPs but also helps prevent the development of drug resistance.

The neutral net charge of mammalian membranes is attributed to their abundance of zwitterionic phospholipids, which protects mammalian cells from AMP-mediated attack. The hydrophobic interactions between mammalian cell membranes and AMPs are far weaker than the electrostatic interactions between bacterial membranes and AMPs; consequently, AMPs are unable to alter the conformation of mammalian cell membranes^{[5, 6]}. Furthermore, mammalian cell membranes are stabilized by embedded cholesterol, and thus, the activity of AMPs is inhibited in mammalian cell membranes^[7]. Beyond their inherent antimicrobial effects, AMPs also exhibit broad immunomodulatory activities that aid in the clearance of bacteria from the host.

Figure 3. Mechanism of Action of Antimicrobial Peptides (AMPs)^[7]

Cecropin B was originally isolated from the silkworm moth (Bombyx mori); it is a heat-stable and soluble polypeptide that exhibits broad-spectrum antimicrobial activity. It inhibits Gram-positive bacteria, Gram-negative bacteria, and viruses, but has almost no destructive effect on eukaryotic cells. Due to its strong antimicrobial activity, the expression and purification of Cecropin B (CecB) are of great significance for the research on antimicrobial peptides. Currently, Escherichia coli (E. coli) is the most common bacterial strain used for CecB expression. However, while CecB is expressed in E. coli, it also exerts strong antimicrobial activity against the E. coli expression strain itself, thus becoming a key limiting factor for the large-scale expression of CecB.

1. Verification of the Basal Expression of Cecropin B (CecB)

Synthesis of the Cecropin B sequence without a template and testing of its antimicrobial properties

2. Protection of Expression Hosts by Anionic Antioxidant Peptides and the Self-Cleavage System of EDPNG

Antioxidant peptides can inhibit oxidation and the formation of reactive oxygen species (ROS) that cause cell or tissue damage, while anions can neutralize the excessively strong positive charge of antimicrobial peptides. EDPNG is used for cleavage; compared with traditional cleavage methods such as those using TEV protease or enterokinase, this method is faster and more efficient.

3. Enhanced Expression Capability by Signal Peptides

Signal peptides can assist proteins in completing translocation and regulate the translation rate of proteins, providing them with more opportunities to fold correctly and undergo post-processing into active proteins. The so-called secretory expression in Escherichia coli (E. coli) generally refers to the translocation of proteins into the periplasmic space—i.e., the compartment between the outer and inner cell membranes—followed by further translocation into the extracellular medium outside the cell membrane, which constitutes a two-step secretion process.

4. Improvement of Antibacterial Activity via Targeted Mutation

Large language models were used to predict mutation sites, followed by experimental verification.

5. Mining of Novel Antimicrobial Peptide Sequences

Novel antimicrobial peptides are mined using machine learning, followed by their heterologous expression.

6. Enhancement of Expression Efficacy and Antibacterial Activity of Novel Antimicrobial Sequences

The above results are summarized to provide insights for improving the activity of novel antimicrobial peptides.

7. Enhancing Soluble Expression by Overexpressing Chaperones

Chaperones are a class of proteins that maintain protein homeostasis by unfolding, degrading, and labeling misfolded proteins. They can promote the correct folding and soluble expression of proteins, effectively reducing the formation of inclusion bodies, without affecting the activity of the target protein.

1. Cecropin Section

Figure 4. Final Pathway of the Cecropin Section

2. Mutations

The model generated multiple single-point mutation sequences, which are divided into charge-enhanced type and structure-optimized type. Most charge-enhanced mutations involve substitutions with positively charged residues, while most structure-optimized mutations involve substitutions with small-volume hydrophobic residues. The overall sequence maintains the original backbone, and the mutation sites are distributed on the outer side of the peptide chain, which is conducive to interfacial interactions. Additionally, the binding free energy of these mutated sequences to bacterial receptor proteins was calculated.

Table 1. Results of Binding Free Energy

System	ΔVDW	ΔEEL	ΔG_solv	ΔH	-TΔS	ΔG binding
WT + 4RHB	-128.3	-955.1	998.6	-84.8	71.3	-13.5
CecB-E10K + 4RHB	-127.5	-1102.3	1126.3	-103.5	85.0	-18.5
CecB-N15K + 4RHB	-125.9	-1180.7	1205.1	-101.5	80.5	-21.0
CecB-V29W + 4RHB	-129.8	-950.4	996.5	-83.7	71.5	-12.2
WT + 1DPE	-95.6	-680.2	715.3	-60.5	50.1	-10.4
CecB-E10K + 1DPE	-93.8	-695.1	729.4	-59.5	50.0	-9.5
CecB-N15K + 1DPE	-90.1	-705.8	742.5	-53.4	45.0	-8.4
CecB-V29W + 1DPE	-145.2	-675.5	720.8	-99.9	82.3	-17.6

3. Mining of New Peptides

Based on preliminary experiments, Cecropin B didn’t provide substantial benefits at this stage of our investigation. However, leveraging the rapid advancement of artificial intelligence, this study adopted a deep learning process to mine AMP monomers (numbered 7, 4, 2) with brand-new sequence features from an extreme environment bacillus sequence library, and selected the optimal structural tandem configuration through molecular dynamics (MD) simulation. As a result, several monomers with potential performance, namely NJT-Lyy-7, NJT-Lyy-4, and NJT-Lyy-2, were obtained.

Figure 5. Tandem expression cassette of pET-28a-pelB-NJT-LYY-742

1. O'Neill J (2016) Tackling drug-resistant infections globally: final report and recommendations. Government of the United Kingdom
2. Mishra B, Reiling S, Zarena D, Wang G (2017) Host defense antimicrobial peptides as antibiotics: design and application strategies. Curr Opin Chem Biol 38:87–96. https://doi.org/10.1016/j.cbpa.2017.03.014
3. Pasupuleti M, Schmidtchen A, Malmsten M (2012) Antimicrobial peptides: key components of the innate immune system. Crit Rev Biotechnol 32:143–171. https://doi.org/10.3109/07388551.2011.594423
4. Mahlapuu M, Håkansson J, Ringstad L, Björn C (2016) Antimicrobial peptides: an emerging category of therapeutic agents. Front Cell Infect Microbiol 6:194. https://doi.org/10.3389/fcimb.2016.00194
5. Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55:27–55. https://doi.org/10.1124/pr.55.1.2
6. Zasloff M (2002) Antimicrobial peptides of multicellular organisms. Nature 415:389–395. https://doi.org/10.1038/415389a