Team:Aalto-Helsinki/Laboratory Theory
Theoretical Background
Antimicrobial Peptides and Dermcidin
New alternatives to antibiotics have been receiving great attention, with increasing threat of antibiotic resistant to commonly used antibiotics. Antimicrobial peptides emerge as a promising alternative due to their wide range of spectrum. Since the 70’s, several antimicrobial peptides from various species have discovered. Antimicrobial peptides are known for their important role in natural self-defense mechanism for many species, including bacteria, fungi and primates.
Dermcidin is an antimicrobial peptide (AMP) found in primates with no homology to other know AMPs. (1) It is expressed in a constitutive manner in eccrine sweat glands and secreted to epidermal surface as a part of first line of defense. (2) Mature dermcidin precursor is 110 amino acids long, including signal peptide. Once antimicrobial peptide precursor is secreted with sweat to epidermal surface, 19 amino acid long signal peptide is cleaved, and it goes under further proteolytic processing leading to several dermcidin derived peptides such as DCD1 and DCD1L. DCD1L is one of the most abundant form of dermcidin derived peptide. DCD1L is a 48-amino-acid long anionic peptide active against wide spectrum of bacteria including Staphylococcus aureus, Escherichia coli, and Propionibacterium acnes. (1,6) Also, dermcidin is evolved to survive and maintain its activity in harsh conditions that is in sweat.
Figure 1. Visualization of DCD-1L hexamer inserted in a membrane, forming a pore.
Mode of Action
Most AMPs are cationic, taking action by electrostatic interactions with negatively charged phospholipid bilayer of pathogens. Unlike other AMPs, DCD1L is anionic, giving it a different mode of action. Although the precise mode of action is not entirely explored, it is thought that[KME1] forms pores on bacterial membrane leading to cell death. DCD1L has overall net charge -2 with cationic N-terminal region and anionic C-terminal region. N-terminal of DCD1L (1 to 23), first interacts with bacterial membrane initially and allows peptides placement on membrane surface, followed by formation of a hexameric pore that further stabilized by Zn+2 ions and under low pH sweat conditions. Also, the fact that DCD1L is amphipathic, supports alpha helix structure and insertion into phospholipid bilayer.
Heterologous Production of Dermcidin
In many studies, chemically synthesized dermcidin peptides are used because of difficulties in recombinant expression caused by toxicity of peptide to expression host due to its antimicrobial nature. However, chemical synthesis is expensive, costing several hundreds for mg quantities. Therefore, production of the antimicrobial peptide in form of a fusion protein in a heterologous expression system is an important alternative to be exploited. In previous published methods, DCD-1 is produced recombinantly but to cleave off the fusion tag CNBr is used. (4) CNBr was not the most optimal method to be used not only due to its volatile and toxic nature, especially in safety level 1 premises, but also because of possible side reactions in the presence of serine and threonine following methionine. With perspective of safety, in our work, Ulp1 enzyme, known for its robust and specific proteolytic activity against SUMO fusion proteins, is utilized to cleave of Hisx6-Smt3 tag is used for expression and purification. (3) 6xHis tag in N-terminus is used for purification with immobilized metal ion affinity chromatography (IMAC) columns designed for histidine tagged proteins.
Employing of Smt3 tag is beneficial in several aspects in our project; to facilitate cleavage of fusion peptide, to keep antimicrobial peptide in inactivation form by blocking its adhesion due to relatively large size of His-Smt3 tag, and advantages due to SUMO tag is its effect on solubility and preventing inclusion bodies of fusion peptide significantly easing purification step. One other major advantage of the fusion system used is that it facilitated easier detect of the peptide with a conventional method, SDS-PAGE. Our expression system is inducible with addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to expression culture, since IPTG induces T7 RNA polymerase promoter leading to expression of gene of interest in plasmid.
Immobilization and CBM
Bacterial adhesion to surfaces and biofilm formation is one of the major reasons for spreading bacterial infections especially in public places such as hospital environment. Preventing pathogen colonization on surfaces in such environments, is crucial. Therefore, immobilization of antimicrobial peptides can be a good alternative to other bactericidal agents because of wide antibiotic spectrum of DCD1L given that immobilization is a known way of increasing stability and resilience of peptides. Thus, throughout our project one of our goals was to immobilize DCD1L as an antimicrobial coating agent. For this purpose, four different constructs are designed containing a cellulose binding domain (CBM3) from Clostridium thermocellum, to immobilize DCD1L on cellulose based materials.
Our CBM constructs contain 22 amino acid long linker, to avoid interference with hexameric complex formation that leads cell death. Although, after visits and discussions with healthcare professionals as a part of our integrated human practices efforts we altered the direction of our project to fight acne causing bacteria, P. acnes. We included cellulose to our project since we discovered versatile nature of cellulosic materials, expanding possible applications.
Figure 2. Visualization of the function of dermcidin bound to cellulose. This image was made for the postcard collaboration which we participated in.
Cellulose based materials can be used in various forms from plastic-like hard materials to hydrogels. Also, cellulose is a medically safe, ecofriendly, and abundant material, already present or can easily be incorporated in many applications. [KME2] (5) It is recently suggested that acne patients have lower expression of dermcidin, leading to progression of the skin condition. With the knowledge of DCD1L’s activity against P. acnes, acne causing bacteria, we designed an acne product containing DCD1L peptide with cellulose hydrogel aiming to combat P. acnes. (6) You can read more from about the application from our Applied design page.
References
[1] Schittek, B., Hipfel, R., Sauer, B., Bauer, J., Kalbacher, H., Stevanovic, S., ... & Rassner, G. (2001). Dermcidin: a novel human antibiotic peptide secreted by sweat glands. Nature immunology, 2(12), 1133-1137.
[2] Burian, M., & Schittek, B. (2015). The secrets of dermcidin action. International Journal of Medical Microbiology, 305(2), 283-286.
[3] Malakhov, M. P., Mattern, M. R., Malakhova, O. A., Drinker, M., Weeks, S. D., & Butt, T. R. (2004). SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins. Journal of structural and functional genomics, 5(1), 75-86.
[4] Cipáková, I., Gasperík, J., & Hostinová, E. (2006). Expression and purification of human antimicrobial peptide, dermcidin, in Escherichia coli. Protein expression and purification, 45(2), 269-274.5)
[5] Ng, Victor WL, et al. "Antimicrobial hydrogels: a new weapon in the arsenal against multidrug-resistant infections." Advanced drug delivery reviews 78 (2014): 46-62.
[6] Nakano, T., Yoshino, T., Fujimura, T., Arai, S., Mukuno, A., Sato, N., & Katsuoka, K. (2015). Reduced expression of dermcidin, a peptide active against Propionibacterium acnes, in sweat of patients with acne vulgaris. Acta dermato-venereologica, 95(7), 783-786.
