Improving the Lethal Effect of Cpl-7, a Pneumococcal Phage Lysozyme with Broad Bactericidal Activity, by Inverting the Net Charge of Its Cell Wall-Binding Module

Abstract

Phage endolysins are murein hydrolases that break the bacterial cell wall to provoke lysis and release of phage progeny. Recently, these enzymes have also been recognized as powerful and specific antibacterial agents when added exogenously. In the pneumococcal system, most cell wall associated murein hydrolases reported so far depend on choline for activity, and Cpl-7 lysozyme constitutes a remarkable exception. Here, we report the improvement of the killing activity of the Cpl-7 endolysin by inversion of the sign of the charge of the cell wall-binding module (from ؊14.93 to ؉3.0 at neutral pH). The engineered variant, Cpl-7S, has 15 amino acid substitutions and an improved lytic activity against Streptococcus pneumoniae (including multiresistant strains), Streptococcus pyogenes, and other pathogens. Moreover, we have demonstrated that a single 25-g dose of Cpl-7S significantly increased the survival rate of zebrafish embryos infected with S. pneumoniae or S. pyogenes, confirming the killing effect of Cpl-7S in vivo. Interestingly, Cpl-7S, in combination with 0.01% carvacrol (an essential oil), was also found to efficiently kill Gram-negative bacteria such as Escherichia coli and Pseudomonas putida, an effect not described previously. Our findings provide a strategy to improve the lytic activity of phage endolysins based on facilitating their pass through the negatively charged bacterial envelope, and thereby their interaction with the cell wall target, by modulating the net charge of the cell wall-binding modules.

Antimicrobial bacteriophage-derived proteins and therapeutic applications

“…The mortality rate of bacterium-infected embryos was 29% for S. pneumoniae, and 35% for S. pyogenes, whereas survival rates after treatment reached 99% for pneumococcus-and 95.3% for S. pyogenes-infected. 73 Although Cpl-1 was not included in this model, its fourfold increase in potency over Cpl-7S in vitro suggests it may provide better protection at a lower dose. However, a chimeric of Cpl-7S EAD and Cpl-1 CBD provided an enzyme (Cpl-711) even more potent than native Cpl-1.…”

“…72 Most pneumococcal endolysins depend on binding to the choline moieties of teichoic acids for catalytic activity. 68,73 This restricts their lytic spectrum to pneumococcal or related cholinesubstituted species. 73 The pneumococcal endolysin Cpl-7 has a CBD that is not dependent on choline-binding giving it unrestricted activity against not only pneumococcal species but against non-pneumococcal species, including S. pyogenes.…”

“…68,73 This restricts their lytic spectrum to pneumococcal or related cholinesubstituted species. 73 The pneumococcal endolysin Cpl-7 has a CBD that is not dependent on choline-binding giving it unrestricted activity against not only pneumococcal species but against non-pneumococcal species, including S. pyogenes. 73,74 However, bacteriolytic activity of Cpl-7 is significantly lower than that of Cpl-1 and Pal.…”

“…73 The pneumococcal endolysin Cpl-7 has a CBD that is not dependent on choline-binding giving it unrestricted activity against not only pneumococcal species but against non-pneumococcal species, including S. pyogenes. 73,74 However, bacteriolytic activity of Cpl-7 is significantly lower than that of Cpl-1 and Pal. By inverting the Cpl-7 CBD charge from negative to positive (at neutral pH) through 15 amino acid substitutions, lytic activity of the inversion variant Cpl-7S improved fourfold.…”

Aromatic Esters of Bicyclic Amines as Antimicrobials against Streptococcus pneumoniae

“…[11] Them ost appropriate bacterial challenge dose causing 50 %m ortality after 5days was found to be 2.5 10 8 cfu mL À1 for pneumococcal strain D39, when administered by immersion. Seven hours after bacterial challenge,e mbryos were treated with as ingle dose containing different amounts of 31,orphosphate buffer as control, and this operation was repeated for the first three days.E mbryo survival was followed over 5days and the results of three experiments were combined and represented as aM antel-Cox curve (Figure 2).…”