The Influence of Dimerization on the Pharmacokinetics and Activity of an Antibacterial Enzyme Lysostaphin

Abstract

The increasing prevalence of antibiotic-resistant strains of pathogenic bacteria is a major healthcare problem. Antibacterial lysins are enzymes that cleave the peptidoglycan of the bacterial cell wall. These proteins hold potential as a supplement or an alternative to traditional antibiotics since they are active against antibiotic resistant strains. However, antibacterial lysins are rapidly eliminated from the systemic circulation, which limits their application. Dimerization of an anti-pneumococcal lysin Cpl-1 has been demonstrated to decrease the clearance rate of this protein in mice. In the present work, we constructed a dimer of an anti-staphylococcal lysin lysostaphin by fusing it with an anti-parallel α-helical dimerization domain. Lysostaphin dimer had a more favorable pharmacokinetic profile with increased terminal half-life and area under the curve (AUC) values compared to monomeric lysostaphin. However, the staphylolytic activity of dimerized lysostaphin was decreased. This decrease in activity was likely caused by the dimerization; since the catalytic efficacy of lysostaphin dimer towards pentaglycine peptide was unaltered. Our results demonstrate that, although dimerization is indeed beneficial for the pharmacokinetics of antibacterial lysins, this approach might not be suitable for all lysins, as it can negatively affect the lysin activity.

Keywords: antibiotic resistance; dimerization; endolysin; lysin; lysostaphin; pharmacokinetics; staphylococcus.

Figure 1

3D model and amino acid sequence of Lst-HDD. (A) A theoretical model of Lst-HDD homodimer. Lysostaphin catalytic domain is colored green, lysostaphin peptidoglycan-binding domain is colored cyan, dimerization domain is colored blue and spacer is colored yellow. The model was constructed in PyMOL (Schrödinger LLC, USA) based on PDB entry 4LXC. (B) The amino acid sequence of Lst-HDD. The color scheme as in (A).

Figure 2

SDS-PAGE and size exclusion chromatography (SEC) of Lst-HDD. (A) SDS-PAGE of purified lysostaphin (lane 1) and Lst-HDD (lane 2); lane M contains molecular weight standards with their molecular weights indicated on the left (B) SEC chromatogram of Lst-HDD. The inlet shows the molecular weight calibration of the column.

Figure 3

Clearing of S. aureus ATCC 29,213 cell suspension due to different concentrations of lysostaphin (A) or Lst-HDD (B). Lysostaphin concentrations are 2 µg/mL (blue), 1 µg/mL (orange), 0.5 µg/mL (grey), 0.25 µg/mL (yellow), and control without lysostaphin (black); Lst-HDD concentrations are 12 µg/mL (blue), 6 µg/mL (orange), 3 µg/mL (grey), 1.5 µg/mL (yellow), 0.75 µg/mL (light blue), 0.38 µg/mL (green), and control without Lst-HDD (black). The mean values of n = 3 (lysostaphin) or n = 2 (Lst-HDD) experiments are shown, error bars represent standard deviation.

Figure 4

Residual concentrations of lysostaphin (blue, circles) and Lst-HDD (orange, triangles) in rat plasma. The mean values over n = 4 rats are shown, error bars represent standard deviation.