Team:Aalto-Helsinki/Laboratory Theory
Theoretical Background
Antimicrobial Peptides and Dermcidin
New alternatives to antibiotics have received great attention, with an increasing threat of antibiotic resistance 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 been 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 the first line of defense. [2] Mature dermcidin precursor is 110 amino acids long, including a signal peptide. Once the antimicrobial peptide precursor is secreted with sweat to the epidermal surface, the 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 DCD-1L. DCD-1L is one of the most abundant forms of dermcidin-derived peptides. DCD-1L is a 48-amino-acid long anionic peptide active against a wide spectrum of bacteria including Staphylococcus aureus, Escherichia coli, and Propionibacterium acnes. [1, 6] Also, dermcidin has evolved to survive and maintain its activity in the harsh conditions in sweat.
Figure 1. Visualization of a DCD-1L hexamer inserted in a membrane, forming a pore.
Mode of Action
Most AMPs are cationic, taking action by electrostatic interactions with the negatively charged phospholipid bilayer of pathogens. Unlike other AMPs, DCD-1L is anionic, giving it a different mode of action. Although the precise mode of action is not entirely explored, it is thought that DCD-1L forms pores in the bacterial membrane leading to cell death. DCD-1L has an overall net charge of -2 with a cationic N-terminal region and an anionic C-terminal region. The N-terminus of DCD-1L (amino acids 1 to 23), first interacts with the bacterial membrane and allows the placement of peptides on the membrane surface, followed by formation of a hexameric pore that is further stabilized by Zn2+ ions and under low pH sweat conditions. Also, the fact that DCD-1L is amphipathic, supports the alpha helix structure and insertion into the phospholipid bilayer.
Heterologous Production of Dermcidin
In many studies, chemically synthesized dermcidin peptides are used because of difficulties in the recombinant expression caused by toxicity of the peptide to the 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 the form of a fusion protein in a heterologous expression system is an important alternative to be exploited. In previously published methods, DCD-1 is produced recombinantly but CNBr is used to cleave off the fusion tag. [3] CNBr is not the most optimal method to use 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 the perspective of safety, Ulp1 enzyme is utilized in our work to cleave off the His6x-Smt3 tag that is used in expression and purification. Ulp1 is known for its robust and specific proteolytic activity against SUMO fusion proteins, which makes this a promising system for recombinant protein production. [4] the His6x tag in the N-terminus is used for purification with immobilized metal ion affinity chromatography (IMAC) columns designed for histidine tagged proteins.
Employing the Smt3 tag is beneficial to our project in several aspects: it facilitates cleavage of the fusion peptide, it keeps the antimicrobial peptide in an inactive form by blocking its adhesion due to the relatively large size of His6x-Smt3 tag, and it eases the purification step due to the advantageous effects that the SUMO tag has on solubility, preventing the formation of inclusion bodies of the fusion peptides. One other major advantage of this fusion system is that it facilitates detection of the peptide with a conventional method, SDS-PAGE. Our expression system is inducible with addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to the expression culture, since IPTG induces T7 RNA polymerase promoter leading to expression of the gene of interest in the 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 the 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 the wide antibiotic spectrum of DCD-1L, 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 DCD-1L as an antimicrobial coating agent. For this purpose, four different constructs were designed containing a cellulose binding domain (CBM3) from Clostridium thermocellum, to immobilize DCD-1L on cellulose based materials.
Our CBM constructs contain a 22-amino-acid long linker to avoid interference of CBM with hexameric complex formation of the antimicrobial peptides that leads to bacterial cell death. Although, after visits and discussions with healthcare professionals as part of our integrated human practices, we altered the direction of our project to fight acne causing bacteria, P. acnes. We included cellulose to our project since we discovered the 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 that is already present or can easily be incorporated in many applications. [5] It was recently suggested that acne patients have lower expression of dermcidin, leading to progression of the skin condition. With the knowledge of DCD-1L’s activity against P. acnes, acne causing bacteria, we designed an acne product containing DCD-1L 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] 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)
[4] 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.
[5] Ng, V. W., Chan, J. M., Sardon, H., Ono, R. J., García, J. M., Yang, Y. Y., & Hedrick, J. L. (2014). Antimicrobial hydrogels: a new weapon in the arsenal against multidrug-resistant infections. Advanced drug delivery reviews, 78, 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.
