Antimicrobial Characterization of Site-Directed Mutagenesis of Porcine Beta Defensin 2

Abstract

Porcine β defensin 2 (pBD2) is a small, cationic and amphiphilic antimicrobial peptide. It has broad antimicrobial activities against bacteria and plays an important role in host defense. In order to enhance its antimicrobial activity and better understand the effect of positively charged residues on its activity, we substituted eight amino acid residues with arginine or lysine respectively. All mutants were cloned and expressed in BL21 (DE3) plysS and the mutant proteins were then purified. These mutant versions had higher positive charges but similar structural configurations compared to the wild-type pBD2. Moreover, these mutant proteins showed different antimicrobial activities against E. coli and S. aureus. The mutant I4R of pBD2 had the highest antimicrobial activity. In addition, all the mutants showed low hemolytic activities. Our results indicated that the positively charged residues were not the only factor that influenced antimicrobial activity, but other factors such as distribution of these residues on the surface of defensins might also contribute to their antimicrobial potency.

Characterization of the β-defensin genes in giant panda

“…Indeed, there may be no need for the giant panda to accumulate many nonsynonymous substitutions in its β-defensins for these to be different from their homologs in other carnivores. In some immune peptides, including β-defensins, a single amino acid substitution is sufficient to alter their molecular potency against pathogens 30 . Targeting a broad range of pathogens, Aime-DEFB139 may not need to change significantly under diversifying selection to cope with new pathogens, and an enhanced antimicrobial activity resulting from a point mutation could be sufficient for this coping ability.…”

Antibacterial Activity of Recombinant Porcine β-Defensin 2

“…β-defensins, the evolutionarily oldest defensins, consist of 30-45 residues of amino acid that includes 5-12 positively charged residues such as arginine and lysine. Typical structure of β defensins composed of an alpha helix with three beta sheets (Huang et al, 2015). The βdefensins are either constitutively expressed or induced in the skin and the mucosal surfaces of airways, digestive tract, and urogenital tract for inflammatory and infectious diseases (Elahi et al, 2006;Sang et al, 2006).…”

Antimicrobial Peptides from Venom: Structure, Function and Toxicity

“…These amino acids usually form highly defined cationic domains within the peptide structure (Hicks et al 2013, Lakshmaiah Narayana and. Increasing the charge of AMPs has been demonstrated to increase the antimicrobial activity in numerous studies (Bessalle et al 1992, Huang et al 2015. In parallel, it has also been shown that the elimination of peptide cationic charge drastically reduces antimicrobial activity (Schmidtchen et al 2014).…”

“…A systematic approach to mutagenesis is required to cover as much of the sequence space as possible. Previously, increasing the charge of antimicrobial peptides has been linked with increased antimicrobial activity (Huang et al 2015.…”

“…This methodology was used by (Huang et al 2015) which determined increasing the charge of a porcine antimicrobial peptide resulted in increased antimicrobial activity. As such, this formed the basis for the initial mutagenesis strategy for both Smp 24 and Smp43.…”