Exploring the impact of the recombinant Escherichia coli strain on defensins antimicrobial activity: BL21 versus Origami strain

Abstract

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a β-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality.

The future of recombinant host defense peptides

“…IBs are an active biomaterial that has already been explored in several applications such as cancer [ 73 ], biocatalysis [ 74 ], tissue regeneration [ 75 ] and immunostimulation [ 76 ], as they are highly stable protein nanoparticles with slow-release properties [ 56 , 57 , 77 ]. Recently, two studies have proven that HDP-based IBs are biologically active against different pathogenic bacteria [ 20 , 37 ]. In the first study, López-Cano et al showed that human α-defensin 5 (HD5) and lingual antimicrobial peptide (LAP) IBs are highly active against MRSA and Pseudomonas aeruginosa, with antimicrobial activities comparable to the soluble counterpart [ 37 ].…”

“…Recently, two studies have proven that HDP-based IBs are biologically active against different pathogenic bacteria [ 20 , 37 ]. In the first study, López-Cano et al showed that human α-defensin 5 (HD5) and lingual antimicrobial peptide (LAP) IBs are highly active against MRSA and Pseudomonas aeruginosa, with antimicrobial activities comparable to the soluble counterpart [ 37 ]. The second study showed the antibiofilm properties of IB-decorated surfaces against a carbapenem resistant Klebsiella pneumoniae [ 20 ], adding to the evidence that antimicrobial IBs can be effectively used against AMR bacteria.…”

“…To maintain antimicrobial activity, constant administration of an antimicrobial that has a short half-life is required, as concentrations under minimum inhibitory concentrations (MIC) will probably happen during treatment, further increasing the appearance of AMRs. This is why a slow-release profile seems to be vital to maintain constant antimicrobial levels for long periods, to get an optimal therapeutic benefit, where HDP-based IBs are promising protein format for antimicrobial applications [ 37 ]. Therefore, antimicrobial IBs can be used as nanopills that display antibacterial activity against different AMR Gram-positive and Gram-negative strains.…”

“…Unlike denaturing protocols, this last strategy allows to obtain soluble protein from IBs without the need to use unfolding and refolding processes. Among these articles, some of them have already successfully tried this approach using IBs formed by antimicrobial proteins [ 20 , 21 , 37 ]. Indeed, solubilized IB proteins show antimicrobial activity against E. coli in a dose-dependent fashion against carbapenem-resistant K. pneumoniae embedded in biofilms [ 20 ].…”

“…Nevertheless, bacteria, have a limited ability to make disulphide-bonds, glycosylation, and other post translational modifications (PTMS) [34][35][36]. These limitations, however, might not be critical for HDP recombinant production [37]. In the absence of a strict need for PTMS, the heterologous expression in bacteria is a reasonable approach for their production with sufficient conformational and functional quality.…”