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-μg 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.

Fig 1

Bacteriolytic and bactericidal effects of different lytic enzymes against S. pneumoniae strain R6. (A) Exponentially growing pneumococci were washed, suspended in PBS at an OD₅₅₀ of ≈0.6, and incubated in the absence or in the presence of the selected enzyme (5 μg · ml⁻¹) at 37°C. Decay of the bacterial suspension OD₅₅₀ was followed for 60 min. Data are representative of four independent experiments. (B) Viable cells were determined on blood agar plates after 60 min of incubation under the same conditions. Data are means from four independent experiments. Error bars represent standard deviations, and asterisks mark results that are statistically significant compared to those for the controls in the absence of enzybiotics (one-way ANOVA with a post hoc Dunnet test; *, P < 0.001).

Fig 2

Modification of the net charge of the CWBM of Cpl-7. (A) Distribution of amino acid substitutions along the sequence of CW_7 repeats. The upper row shows one repeat and linker sequences of Cpl-7, with acidic and basic residues depicted in red and blue, respectively. Positions mutated are underlined, with substitutions indicated below (basic amino acids in blue and neutral polar residues in green). Gray boxes show conserved regions in the CW_7 family (PF08230), and α-helical segments are shown as purple rectangles. (B) Cartoon representation of the three-dimensional structure of the second repeat from the model by Bustamante et al. (17). In blue are residues replaced by basic amino acids, and in green are replacements of aspartic acid residues by asparagines (stick mode representation). (C) Molecular surface of the same repeat colored according to its electrostatic potential in Cpl-7 (left) and Cpl-7S (right) (red for acidic and blue for basic). The binding cavity identified by Fpocket software is shown by the small yellow spheres.

Fig 3

Bacteriolytic and bactericidal effects of phage lytic lysozymes against S. pneumoniae strain R6. (A) Bacterial cells were treated as for Fig. 1, and the time course of bacterial suspension turbidity was followed. Data are representative of four independent experiments. (B) Viable cells were determined on blood agar plates after 60 min of treatment with the enzybiotics. Data are means from four independent experiments. Error bars and asterisks have the same meaning as in Fig. 1. Differences between Cpl-7 and Cpl-7S activities are statistically significant (P < 0.05).

Fig 4

Bacteriolytic and bactericidal effects of pneumococcal phage lysozymes against S. pyogenes and E. faecalis. (A and C) Exponentially growing bacterial cultures were washed, suspended in PBS, and incubated in the absence or presence of the assayed enzyme (5 μg · ml⁻¹) at 37°C. Variation of the OD₅₅₀ of the cultures was followed for 60 min. Data are representative of four independent experiments. (B and D) Bacterial viability was determined after 60 min of treatment with Cpl-1, Cpl-7, and Cpl-7S. Data are means from four independent experiments. Error bars and asterisks have the same meaning as in Fig. 1. Differences between Cpl-7 and Cpl-7S activities are statistically significant (P < 0.05).

Fig 5

Bacteriolytic and bactericidal effects of pneumococcal phage lysozymes against E. coli in the presence of carvacrol. (A) Exponentially growing E. coli DH10B cells were washed, suspended in PBS at an OD₅₅₀ of ≈0.6, and incubated at 37°C in the presence of 0.01% carvacrol and the assayed enzymes (5 μg · ml⁻¹). The time course of bacterial suspension turbidity was followed. Data are representative of four independent experiments. (B) Viable cells were determined on blood agar plates after 60 min of treatment with the same enzymes. Data are means from four independent experiments. Error bars and asterisks have the same meaning as in Fig. 1. Differences between Cpl-7 and Cpl-7S activities are statistically significant (P < 0.05).

Fig 6

Pathogen infection in the zebrafish embryo model. Survival of zebrafish embryos infected with S. pneumoniae or S. pyogenes and treated or not at 7 h after infection with 25 μg Cpl-7S (n = 24 to 36 embryos/condition) is shown. Data are means from four independent experiments. Error bars represent standard deviations, and asterisks mark the results that are statistically significant for the overall comparison of infected or Cpl-7-treated embryos versus the controls (one-way ANOVA with a post hoc Dunnet test; *, P < 0.001).

Fig 7

Localization of S. pneumoniae in infected zebrafish embryos. Representative whole-mount immunofluorescence of zebrafish embryos at 48 h postinfection (5 days postfecundation) with S. pneumoniae was analyzed by CLSM. D39 cells (red) were labeled with a polyclonal antibody recognizing pneumococcal type 2 capsular polysaccharide. Maximal projections from 15 z-stacks were constructed from fluorescence and differential interference contrast confocal images. Top panels, red (bacterial) fluorescence; bottom panels, transmitted light and red (bacterial) fluorescence overlay. (A) Pneumococcus-free embryos at 5 days postfecundation were used as a control (20× objective; details of the gills are shown in Fig. S6 in the supplemental material). (B) Embryo head infected by S. pneumoniae (20× objective). (C) Details of S. pneumoniae infection around the gills (40× objective). Exposure settings were identical for all samples. Bars, 250 μm (A and B) or 25 μm (C).