Date Published: October 11, 2018
Publisher: Public Library of Science
Author(s): Gabriela Contreras, Markus Santhosh Braun, Holger Schäfer, Michael Wink, Simon J Clark.
Antimicrobial peptides (AMPs) are short and generally positively charged peptides found in a wide variety of organisms. CSαβ defensins are a group of AMPs. These defensins are composed of an α-helix and a β-sheet linked by three or four disulphide bridges. In this study, we describe the antimicrobial activity of an anionic CSαβ fungal defensin from Aspergillus fumigatus, AfusinC. AfusinC was recombinantly produced as a fusion protein in Escherichia coli. The tag was removed by proteolytic cleavage, and AfusinC was purified by size exclusion chromatography. About 0.8 mg of recombinant AfusinC was obtained from 1 L of culture. Recombinant AfusinC was active against mainly gram-positive bacteria including human pathogens and a multiresistant-strain of A. aureus. Additionally, AfusinC showed bactericidal effect against Micrococcus luteus.
Overuse of antibiotics has caused the development of new multidrug-resistant microorganisms; as a result, it is estimated that each year 25,000 patients die due to the most common multidrug-resistant bacteria in Europe (i.e. Staphylococcus aureus, Enterobacteriaceae, Pseudomonas aeruginosa) . Hence, it is important to search new antimicrobial agents to overcome this problem. A group of antimicrobial compounds, naturally synthesised by organisms, are antimicrobial peptides (AMPs). AMPs are produced in both prokaryotes and eukaryotes. AMPs are part of the innate immune systems in multicellular organisms , for this reason, they are also called host defence peptides. AMPs are short peptides (12 to 50 amino acids), commonly cationic, and amphipathic. These characteristics enhance selective attachment and insertion of AMPs onto the negatively charged microbial cytoplasmic membranes . This insertion can lead to leaky biomembranes or membrane disruption and consequently to death of microbial cells. AMPs are considered a promising alternative to common antibiotics because they exhibit a broad-spectrum activity against bacteria, fungi, parasites, and viruses. Additionally, the appearance of AMPs-resistant strains is less likely .
AfusinC was recombinantly expressed in E. coli which is the most widely used host for heterologous protein engineering. To prevent the toxicity in the host, a Trx tag was added. Trx promotes target protein solubility and catalyses the formation of disulphide bridges in the E. coli cytoplasm . Trx has been successfully used as a fusion partner to express soluble recombinant cysteine-rich AMPs in E. coli BL21(DE3) [23, 24]. However, tags do not function equally with all partner proteins . In this study, Trx-Afusin was predominantly expressed as inclusion bodies in E. coli BL21(DE3), even at low incubation temperature and IPTG concentration (S2 Fig). Additionally, pET-32c_afuC was transformed into Origami2(DE3)pLysS E. coli, a permissive strain for the formation of disulphide bonds in the cytoplasm due to mutations in both the thioredoxin reductase and glutathione reductase genes . However, the production of Trx-Afusin was only 5 mg/L of the bacterial culture (S3 Fig). Nonetheless, inclusion bodies achieve high yield and purity of recombinant proteins , and in this study, the refolded Trx-AfusinC reached 54 mg/L of the bacterial culture. Furthermore, misfolded AMPs could mask their toxic activity and would not be susceptible to host proteases. Therefore, AfusinC may be feasible to produce without a fusion partner if it formed inclusion bodies, and it would not be lethal to the E. coli host.