1 Background
Why do we care about acne?
In 2010, the incidence of acne vulgaris accounted for 9.38% of the global population, making it the eighth largest epidemic in the world and affecting ~85% of young adults aged 12~25 years[1]. The global prevalence of acne increased by 5.1% from 2006 to 2016[2]. As a chronic disease, acne has a high incidence and a high recurrence rate, which means it is difficult to cure completely. The social and psychological repercussion of acne also merits attention. In dermatology clinics, approximately 25.2% of patients with acne experience some psychiatric morbidity. The probability of developing major depression disorder(MDD) was 18.5% among patients with acne and 12.0% in the general population without acne, which indicates that patients with acne had a significantly increased risk of developing MDD[3]. The psychosocial impact of acne also has economic consequences to society through detrimental effects on interpersonal relationships, employment opportunities and ability to work[4].
Despite the fact that acne is one of the three skin conditions in the top 10 most prevalent diseases worldwide, the most widely used treatments have changed little in the past 30 years. At present, the topical standard treatment of acne includes the use of benzoyl peroxide and retinoids as well as topical antibiotics. However, benzoyl peroxide and retinoids could irritate the skin and antibiotics may lead to the development of antibiotic resistance and cross-resistance[5]. The usage of isotretinoin(13-cis-retinoic acid) requires vigilant review because of its teratogenic potential. A few cases of isotretinoin-associated rhabdomyolysis, which is the leading cause of acute kidney injury, have been reported in relevant literature to date[6].
![Incidence of acne vulgaris in different age ranges. Image from citation[2]](https://static.igem.org/mediawiki/2021/4/4c/T--HUST2-China--img--Description-image--1.jpg)
Figure 1: Incidence of acne vulgaris in different age ranges. Image from citation[2]
2 Purpose
How does our team deal with acne?
Our project ---- NANO-AI-Cleaner, aims to construct a personalized facial mask, which can kill Propionibacterium acnes(P.acnes) by bombinin-like peptide 7 (BLP-7) and block the local immune storm by TLR2 antagonist (also called TLR2 competitive inhibitor). Elastin-like polypeptide(ELP) connects the two proteins to achieve temperature-controlled aggregation. It can also combine photodynamic therapy and improve the skin microflora, which is beneficial to treat acne. As a promising acne treatment to traditional anti-acne medication, it will help mitigating the risk of teratogenesis caused by isotretinoin and antibiotic abuse. We believe that our project will provide a novel, effective and safe approach to acne treatment.
3 Mechanism:
Why can acne be treated this way?
The pathophysiology is a complex interplay of hormonally stimulated sebum production, abnormal keratinization, P.acnes proliferation and associated inflammation. The P.acnes proliferation and associated inflammation are key factors to acne lesions and sequelae. So, this year we mainly focused on these two key factors, providing a novel, potential and practical treatment strategy for acne.
3.1 BLP-7
What is BLP-7?
BLP-7 is an antimicrobial peptide consisting of 27 amino acids and has been shown to have anti-inflammatory effects and a broad spectrum of antibacterial activity.
Cell experiments show that BLP-7 inhibits the expression of two proinflammatory cytokines interleukin (IL)-8 and granulocyt-macrophage colony-stimulating factor (GM-CSF) in normal human epidermal keratinocytes (NHEKs)[5]. Mice experiments have also shown that BLP-7 can significantly inhibit skin inflammation caused by P.acnes[5]. Like most other antimicrobial peptides (AMPs), BLP-7 can target the lipid bilayer of bacterial cell plasma membrane, dig holes through the membrane and kill bacteria. BLP-7 does not lead to antibiotic resistance and could be used as a potential anti-acne agent.
How about its structure and sequence?
BLP-7 contains one large and one small \(\mathrm{\alpha}\)-helical domain representing 74.07% of the structure, exhibited amphipathic \(\mathrm{\alpha}\)-helical character with the polar residues on the upper side and the hydrophobic residues on the opposite side[7]. It consists of 27 amino acids, "GIGGALLSAGKSALKGLAKGLAEHFAN-NH2", which include four cationic amino acids (Lys, Arg or His) and 18 hydrophobic amino acids[5].
How does it function?
Based on the SMH model, which is used to explain the initial interaction between antimicrobial peptides and bacterial membranes, we can speculate on the mechanism of BLP-7[8]. Relying on the positively charged amino acids lysine, arginine and histidine, BLP-7 is attracted to negatively charged cell membrane. With hydrophobic structure, it can enter the membrane gap, form perforation and destroy the integrity of cell membrane.
What can we do?
Increasing the net positive charge and hydrophobicity of antimicrobial peptides can improve its antibacterial ability[5]. So, we mutated valine into arginine at position 5 to increase the net positive, and serine into proline at position 16 to increase the polar angle. The former sequence from 12 to 16 was retained because it is the main hydrophobicity region. In this way, we expect to obtain a more stable mutant BLP-7 with better antibacterial and anti-inflammatory effects.
Figure 2: BLP-7
3.2 TLR2 antagonist
What is TLR2?
TLR2 is a member of the Toll-like receptor family, which initiates potent immune responses by recognizing diacylated and triacylated lipopeptides. Its ligand specificity is controlled by whether it heterodimerizes with TLR1 or TLR6[9]. P.acnes are mainly recognized by TLR2, and then activate inflammatory factors via the activation of MAPK kinases (MAPKs) and nuclear factor kappa-B [10]. Previous studies have shown that P.acnes attract white blood cells to inflammatory sites by activating TLR2 to release chemokines, and then activate infiltrating cells to release pro-inflammatory cytokines, thus promoting the development of inflammatory acne lesions.
How about its binding site?
TLR2 heterodimerizes with TLR1 or TLR6, and TLR2/1 and TLR2/6 heterodimers form different lipid binding pockets to recognize triacylated and diacylated lipoproteins of P.acnes [9][11]. The ligand-binding site of TLR2 is composed of a large internal pocket formed by hydrophobic residues from the LRR9-12 modules and hydrophilic backbone atoms near the pocket opening located at the domain boundary between the central and the C-terminal domains[9].
What is TLR2 antagonist?
TLR2 antagonist is the binding sites of TLR2 to triacylated lipoproteins, which can inhibit P.acnes surface antigen by binding to it, preventing it from binding to TLR2 on the cell surface and avoiding inflammation. We cut a fragment from TLR2 as TLR2 antagonist and performed some site-directed mutations. The first is to shorten the beta loop to prevent triacylated lipoproteins from missing the target. Secondly, the hydrophobicity of the hydrophobic pocket was enhanced to improve the binding ability of lipoprotein. In this way, we expect to obtain an effective TLR2 antagonist with significant anti-inflammatory effects.
Figure 3: TLR2 antagonist
3.3 ELP
What is ELP?
ELP is derived from the hydrophobic region of tropoelastin, composed of pentapeptide repeat sequence Val-Pro-Gly-Xaa-Gly (Xaa is any amino acid except Pro). Tropoelastin is a soluble 70 kDa precursor of elastin, comprised of two domains--hydrophobic domain and hydrophilic domain.
What are its features?
ELPs have a unique property, inverse temperature phase transition, which allows a temperature dependent reversible change from soluble monomer to insoluble aggregate[3]. When the temperature is higher than a critical temperature, ELPs will aggregate through hydrophobic action, and the hydrophobic tail will be surrounded in the interior while the hydrophilic head will be exposed. But ELPs stay soluble below the certain critical temperature. Its transition temperature can be controlled by regulating the repeat number of ELPs repeat sequence and the amino acid type at Xaa[12].
Due to this special property, ELP is often used for protein purification labels and targeted drug delivery to prolong drug action time and enhance efficacy[12]. The ELP tag purification process is simple, inexpensive and promising, which should be potentially interesting to some biopharmaceutical companies.
As the hydrophobic region of tropoelastin, ELP is biocompatible, non-toxic and non-immunogenic, which means it's totally safe. It can also be conducive to the healing of skin wounds.
How does it function in our project?
In our project, BLP-7 and TLR2 antagonist are co-expressed with ELP respectively. Based on the characteristics of ELP, we can achieve temperature-controlled aggregation to improve local drug concentration and enhance drug efficacy. ELP can also be used as the purification label to help protein purification and help skin wounds heal in the treatment of acne.
![Formation of aggregates by ELP coblock polymers. Figure is a modified version of that drawn by Hassouneh et al. (2015). Copyright (2015) American Chemical Society. From citation [12]](https://static.igem.org/mediawiki/2021/2/2e/T--HUST2-China--img--Description-image--4.jpg)
Figure 4: Formation of aggregates by ELP coblock polymers. Figure is a modified version of that drawn by Hassouneh et al. (2015). Copyright (2015) American Chemical Society. From citation [12]
3.4 NANO-AI-Cleaner
Under the temperature higher than critical temperature, ELP monomers connected with BLP-7 or TLR2 antagonist will aggregate together through hydrophobic interaction to form ellipsoidal polymers, which can increase the concentration of local administration and prolong the retention time of the drug, achieving better bactericidal and better anti-inflammatory effect. We call these ellipsoidal polymers NANO-AI-Cleaner.
3.5 Synthesis pathway
What is the phage lambda pL and pR promoters?
The temperature inducible expression system, based on the pL and pR phage lambda promoters, has been widely used to produce recombinant proteins in prokaryotic cells[13]. The gene cloned downstream of the promoters can be efficiently regulated by the thermolabile Tcl repressors. Gene expression is inhibited at culture temperatures below 37 degree centigrade (normally in the range of 28-32 degree centigrade) whereas the normal expression is ensured upon inactivation of the mutant repressor by increasing the temperature above 37 degree centigrade.
What are its advantages?
The promotor commonly employed for heterologous protein expression can present different shortcomings. For chemical inducers, such as IPTG and anti-biotics, can be expensive and toxic. For nutrient exhaustion, such as depletion of an amino acid to form the culture, can be difficult to balance cell metabolism and protein production. And pH-inducible expression systems can be immature and depart from the optimal pH for physiological conditions. Many of the shortcomings can be overcome when using the phage lambda pL and pR promoters. There is no doubt that it is cheaper, safer and more convenient. Its ability to combine photothermal therapy is also a significant advantage.
How does it function in our project?
Our target gene was cloned downstream of the pL and pR promoters. When cultured at 37 degree centigrade, the expression of target gene is inhibited by Tcl and the target protein is hardly expressed. Under the induction of temperature at 45 degree centigrade or infrared light which increases temperature above 37 degree centigrade, Tcl degrades and the inhibition of the promoter releases. So, the expression of target protein is initiated[13]. The expression of the target protein will be under the control of temperature to aggregate into ellipsoidal polymers, which will play the anti-acne role.
Figure 5: The Synthetic pathway
4 The future vision
Our project provides a novel strategy to improve skin health with synthetic biology. In the future, we can develop different types of products, including masks, cream and spray, for users of different conditions. We can also use probiotics as the chassis organisms and achieve controllable production of more types of beneficial products, which will improve skin microecology. Bacteria firmly combined in the mask can prevent leakage and ensure biological safety. According to the user's specific situation, personalized and precise treatment can be used to treat various skin diseases including acne from all directions and multiple targets, regulate skin conditions on an overall level, and inhibit the occurrence of skin diseases from the source. We hope it will contribute more to human skin health in the future.
References
[1] Vos T, Flaxman AD, Naghavi M et al. Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990-C2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 2012; 380:2163-C96. doi:10.1016/S0140-6736(12)61729-2
[2] Lynn DD, Umari T, Dunnick CA, et al. The epidemiology of acne vulgaris in late adolescence. Adolesc Health Med Ther. 2016;7:13-C25. doi:10.2147/AHMT.S55832
[3] Vallerand IA, Lewinson RT, Parsons LM, et al. Risk of depression among patients with acne in the U.K.: a population-based cohort study. Br J Dermatol. 2018;178(3):e194-e195. doi: 10.1111/bjd.16099
[4] Layton AM, Thiboutot D, Tan J. Reviewing the global burden of acne: how could we improve care to reduce the burden?. Br J Dermatol. 2021;184(2):219-225. doi:10.1111/bjd.19477
[5] Wu Y, Qiang Y, Cao K, et al. Inhibitory effect of the antimicrobial peptide BLP-7 against Propionibacterium acnes and its anti-inflammatory effect on acne vulgaris. Toxicon. 2020;184:109-115. doi:10.1016/j.toxicon.2020.06.006.
[6] Dasgupta K, Lim P, Reedstorm H. A Common Drug With a Dangerous Side Effect: Acute Rhabdomyolysis Caused by the Synergistic Effect of Isotretinoin and Exercise in an Adolescent. Cureus. 2020;12(10):e10929. Published 2020 Oct 13. doi: 10.7759/cureus.10929
[7] Zhou C, Wang Z, Peng X, et al. Discovery of two bombinin peptides with antimicrobial and anticancer activities from the skin secretion of Oriental fire-bellied toad, Bombina orientalis. Chem Biol Drug Des. 2018;91(1):50-61. doi: 10.1111/cbdd.13055
[8] Baltzer SA, Brown MH. Antimicrobial peptides: promising alternatives to conventional antibiotics. J Mol Microbiol Biotechnol. 2011;20(4):228-235. doi: 10.1159/000331009
[9] Kang JY, Nan X, Jin MS, et al. Recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer. Immunity. 2009;31(6):873-884. doi: 10.1016/j.immuni.2009.09.018
[10] Fang F, Xie Z, Quan J, Wei X, Wang L, Yang L. Baicalin suppresses Propionibacterium acnes-induced skin inflammation by downregulating the NF-\(\kappa\)B/MAPK signaling pathway and inhibiting activation of NLRP3 inflammasome. Braz J Med Biol Res. 2020;53(12):e9949. Published 2020 Oct 21. doi: 10.1590/1414-431X20209949
[11] Su Q, Grabowski M, Weindl G. Recognition of Propionibacterium acnes by human TLR2 heterodimers. Int J Med Microbiol. 2017;307(2):108-112. doi: 10.1016/j.ijmm.2016.12.002
[12] Yeboah A, Cohen RI, Rabolli C, et al. Elastin-like polypeptides: A strategic fusion partner for biologics. Biotechnol Bioeng. 2016;113(8):1617-1627. doi: 10.1002/bit.25998
[13] Valdez-Cruz NA, Caspeta L, Perez NO, et al. Production of recombinant proteins in E. coli by the heat inducible expression system based on the phage lambda pL and pR promoters. Microb Cell Fact. 2010;9:18.