BLP-7
ANTIMICROBIAL PEPTIDE AND ANTIMICROBIAL RESISTANCE: why do we use antimicrobial peptide?
Antimicrobial resistance is a multifaceted crisis, imposing a serious threat to global health. According to CDC report, it is estimated that antimicrobial resistance cost $55 billion per year in total in the US [1]. Whats more, due to rapid resistance, novel drug development becomes time consuming and costly. A recent antibiotic combination, ceftazidime/avibactam, developed in resistant isolates within one year of release to market [2]. Therefore, antimicrobial peptide becomes a promising solution. Antimicrobial peptides dig holes on cell membrane with their direct effect to it.Figure 1 shows typical structure antimicrobial peptides. Propionibacterium acnes which leads to acne has been found resistant to lots kinds of antibiotic. Therefore, we hope to find a effective and safe antimicrobial peptide.
![Three-dimensional conformations of natural antimicrobial peptides. Sourced from the Protein Data Bank (A) LL-37 (Homo sapiens) [3]. (B) Magainin-2 (Xenopus laevis) [4]. (C) RTD-1 (Rhesus macaque) [5]. (D) Melittin (Apis mellifera) [6]. (E) TPP3 (Solanum lycopersicum) [7]. (F) Bacteriocin AS-48 (Enterococcus faecalis) [8]](https://static.igem.org/mediawiki/2021/d/de/T--HUST2-China--img--wyk--wyk_1.png)
Figure 1: Three-dimensional conformations of natural antimicrobial peptides. Sourced from the Protein Data Bank (A) LL-37 (Homo sapiens) [3]. (B) Magainin-2 (Xenopus laevis) [4]. (C) RTD-1 (Rhesus macaque) [5]. (D) Melittin (Apis mellifera) [6]. (E) TPP3 (Solanum lycopersicum) [7]. (F) Bacteriocin AS-48 (Enterococcus faecalis) [8]
THE FUNCTION OF BLP-7: how does BLP-7 inhibit bacterias growth?
Bombinin-like peptide 7 (BLP-7), which was determined from Bombina orientalis, has shown to possess certain antibacterial activity. For BLP-7, it has a net positive charge which stabilizes the binding with negative cell membrane via electrostatic interaction. Then, BLP-7 dig holes on cell membrane which leads to a breakdown of membrane potential, an alteration in membrane permeability, and metabolite leakage, ultimately causing bacterial cell death. Study shows that 5M of it inhibits P. acnes growth in vitro [9], but it not effective enough for us. In order to enhance its electrostatic interaction, we increased its positive charge through adding positive amino acid in the peptide (We change valine at the fifth place into arginine) In addition, the structure of peptide influences the bind strength between BLP-7 and membrane. So, we replaced serine at 16th place with proline to increase pole angle.
Figure 2: Membrane discrimination mechanism for cationic AMPs. The polar faces of helical peptides are colored in red and the non-polar faces of helical peptides are colored in green
TLR2 ANTAGONIST
TLR2 AND INFLAMMATION: how does TLR2 induce inflammation?
TLR2 in association with TLR1 or TLR6 is essential for recognizing bacterial lipoproteins and lipopeptides [11-12]. These lipoproteins are anchored to the cell membrane via conserved N termini modified by lipid chains [13] and induce strong proinflammatory signals in macrophages [14]. TLR2-deficient mice do not respond to the lipoproteins and are more susceptible to septicemia due to S. aureus, meningitis due to S. pneumoniae and L. monocytogenes, and infection with M. tuberculosis [15-17]. Human TLR2 with the Arg753Gln polymorphism is significantly less responsive to bacterial lipoproteins derived from B. burgdorferi, T. pallidum, and M. tuberculosis [18-19]. Therefore, TLR2 plays an indispensable role in lipoprotein-induced inflammatory responses.
Figure 3: Inflammation pathway induced by TLR2
TLR2 AND ACNE: What is the relationship between TLR2 and acne?
Propionibacterium acnes has always been considered as a key factor leading to the onset of acne. The interaction between Propionibacterium acnes (P. acnes) and the host is mainly mediated by Toll-like receptors. Studies have shown that TLR2/1 or TLR2/6 heterodimer can be used for specific identification of P. acnes. In human keratinocytes, acne-increased NF-B phosphorylation is inhibited by anti-TLR6 and anti-TLR2 [20]. In addition, the inflammatory response induced by acne will be inhibited by anti-TLR2 neutralizing antibodies and completely blocked by CU-CPT22 [21].
OUR IDEA: How to resolve inflammation?
In order to cut off the immune response due to Propionibacterium acnes, we took a small protein sequence of the binding site of TLR2 and P. acnes surface acylated lipoprotein for site-directed mutation to achieve the irreversible binding effect of TLR2 antagonist to acylated lipoprotein, thereby preventing the combination between acylated lipoprotein and TLR2. On one hand, we increased the hydrophobicity of the hydrophobic pocket bound by the acylated lipoprotein; on the other hand, we contracted the slack -loop sheet. In this way, we not only improved the hydrophobic interaction, but also prevented the bound antagonist from going off-target with the acylated lipoprotein.
Figure 4: TLR2 Antagonist
ELP
ELASTIN LIKE PEPTIDES: what is the mechanism of its aggregation and dispersion?
In previous reports, the cuticle on acne is hard to pass through for drug, especially for huge molecular like protein. In order to cross the skin barrier and gather at the lesion [22,23], we use elastin like peptide (ELP) to regulate our drug's molecular size. ELP is a kind of analogue of elastin protein which exist in nature. Former study [24] shows that ELPs exhibit lower critical solution temperature phase behavior, and above this temperature the ELP forms insoluble, micron-sized aggregates. This process of aggregation and dispersion is reversible and not influenced by linking peptide. So that it is free to cross the cuticle and easy to purify. What's more, elastin like peptide has same properties with elastin exists in human body. Elastin is a major constituent of skin elastic fibers and may be beneficial for dermal regeneration [25]. ELP may have an additional function in acne therapy.
Figure 5: Images of the structural features a kind of ELP changing with temperature
OUR IDEA: how to control molecular size?
We link ELP with ELP-7 and TLR-2 antagonist. ELP is composed of pentapeptide repeating sequence Val-Pro-Gly-Xaa-Gly (Xaa is any amino acid except Pro). Its phase transition temperature of ELP depends on the sequence length, the hydrophobicity of Xaa and the concentration of ELP. The formula to estimate transformation temperature is mentioned in Figure 4. In order to aggregate at 42 degree centigrade, we used Ile as Xaa with a copy number of 27. 42 degree centigrade is a temperature used by acnes heat treatment which may be an adjuvant therapy.
$$ T_t=T_tc+\frac{k}{length}\cdot \ln (\frac{C_c}{conc}) $$
This is a formula to roughly estimate the transformation temperature.
\(\mathrm{Length}\): the chain length of ELP.
\(\mathrm{conc}\): the concentration of ELP (M).
\(\mathrm{T_tc}\), \(\mathrm{k}\), \(\mathrm{C_c}\) are parameter depending on the kind of Xaa.
They all can be calculated by experimental data
References:
[1] Centres for Disease Control and Prevention, US Department of Health and Human Services. Antibiotic resistance threats in the United States. Atlanta: CDC; 2013.
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