Novel chimeric lysin with high-level antimicrobial activity against methicillin-resistant Staphylococcus aureus in vitro and in vivo

Abstract

The treatment of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) is a challenge worldwide. In our search for novel antimicrobial agents against MRSA, we constructed a chimeric lysin (named as ClyH) by fusing the catalytic domain of Ply187 (Pc) with the non-SH3b-like cell wall binding domain of phiNM3 lysin. Herein, the antimicrobial activity of ClyH against MRSA strains in vitro and in vivo was studied. Our results showed that ClyH could kill all of the tested clinical isolates of MRSA with higher efficacy than lysostaphin as well as its parental enzyme. The MICs of ClyH against clinical S. aureus strains were found to be as low as 0.05 to 1.61 mg/liter. In a mouse model, a single intraperitoneal administration of ClyH protected mice from death caused by MRSA, without obvious harmful effects. The present data suggest that ClyH has the potential to be an alternative therapeutic agent for the treatment of infections caused by MRSA.

FIG 1

Characteristics of ClyH activity. (A) SDS-PAGE of purified ClyH and Pc-His. (B) SDS-PAGE of purified ClyH-His and Pc-His. M, protein molecular mass markers; ClyH-his, His-tagged ClyH; Pc-his, the catalytic domain of lysin Ply187 fused with a His tag. (C) Lytic activity against S. aureus AB918 in vitro. The decrease in OD₆₀₀ was monitored after addition of ClyH (solid squares) with PBS as a control (open circles). Viability of treated cells measured as log CFU/ml was determined by serial dilution and plating to TSB agar plates (asterisks). (D) The relative activities of ClyH against AB918 cells in buffers at different pHs. (E to G) TEM images of AB918 cells exposed to ClyH. ClyH causes cell wall deformation (E), extrusion and loss of cytoplasmic contents either partly or totally (F), and ultimately formation of a cell “ghost” (G). Bar sizes, 200 nm.

FIG 2

Lytic activity of ClyH (0.5 U) against different strains in vitro. The activity of lysis is defined as the initial velocity of the decrease in OD₆₀₀ over time. Error bars show the standard errors of three independent assays.

FIG 3

Comparison of the activity of ClyH/ClyH-His with that of lysostaphin and Pc-His, respectively. (A) Lytic activity of ClyH (0.16 μM) in comparison with that of lysostaphin at the same concentration. (B) Lytic activity of ClyH-His (1.2 μM) in comparison with that of Pc-His at the same concentration. Error bars represent three independent assays.

FIG 4

Protective effect of ClyH on mice from death caused by MRSA. (A) Curative effects in a mouse model of systemic MRSA infection. Three hours after infection, one group of mice was given 180 U of ClyH, the second group was given 360 U of ClyH, and the third group was given PBS buffer. Meanwhile, another group of mice without MRSA infection were given 540 U of ClyH to test its toxicity. (B) Titers of anti-ClyH antibody induced by repeated injection of ClyH. The control serum is nonimmunized mouse serum. (C) Effect of ClyH-immunized serum on the lytic activity of ClyH against AM025.