SP-CHAP, an endolysin with enhanced activity against biofilm pneumococci and nasopharyngeal colonization

Abstract

Streptococcus pneumoniae (Spn), a Gram-positive bacterium, is responsible for causing a wide variety of invasive infections. The emergence of multi-drug antibiotic resistance has prompted the search for antimicrobial alternatives. Phage-derived peptidoglycan hydrolases, known as endolysins, are an attractive alternative. In this study, an endolysin active against Spn, designated SP-CHAP, was cloned, produced, purified, biochemically characterized, and evaluated for its antimicrobial properties. Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains are widely represented in bacteriophage endolysins but have never previously been reported for pneumococcal endolysins. Here, we characterize the first pneumococcal endolysin with a CHAP catalytic domain. SP-CHAP was antimicrobial against all Spn serovars tested, including capsular and capsule-free pneumococci, and it was found to be more active than the most widely studied pneumococcal endolysin, Cpl-1, while not affecting various oral or nasal commensal organisms tested. SP-CHAP was also effective in eradicating Spn biofilms at concentrations as low as 1.56 µg/mL. In addition, a Spn mouse nasopharyngeal colonization model was employed, which showed that SP-CHAP caused a significant reduction in Spn colony-forming units, even more than Cpl-1. These results indicate that SP-CHAP may represent a promising alternative to combating Spn infections.

Importance: Considering the high rates of pneumococcal resistance reported for several antibiotics, alternatives are urgently needed. In the present study, we report a Streptococcus pneumoniae-targeting endolysin with even greater activity than Cpl-1, the most characterized pneumococcal endolysin to date. We have employed a combination of biochemical and microbiological assays to assess the stability and lytic potential of SP-CHAP and demonstrate its efficacy on pneumococcal biofilms in vitro and in an in vivo mouse model of colonization. Our findings highlight the therapeutic potential of SP-CHAP as an antibiotic alternative to treat Streptococcus pneumoniae infections.

Keywords: CHAP domain; antibiotic resistance; bacteriophage; endolysin; pneumococcus.

Fig 1

Endolysin SP-CHAP is highly active against planktonic-grown pneumococci, and its activity is modulated by capsule polysaccharide availability. Biochemical characterization of optimal conditions for SP-CHAP activity. The role of pH (A), temperature (B), and salt (C) on SP-CHAP lytic activity against Spn DCC1811. Lytic activity of 50 µg/mL Cpl-1 and SP-CHAP on (D) Spn strains TIGR4, D39, DCC1811, 1335, and Lyt4.4 or (E) the commensal staphylococcal bacteria (Staphylococcus hominis and Staphylococcus epidermidis) and commensal streptococcal bacteria (Streptococcus gordonii, Streptococcus intermedius, Streptococcus mitis, and Streptococcus salivarius) as measured by OD₆₀₀. (F) Lytic activity of SP-CHAP and Cpl-1 at various concentrations on Spn Lyt4.4 as measured by the colony-forming unit assay. Data are reported as the log-fold killing compared to untreated controls. (G) Minimum inhibitory concentration (MIC) (μg/mL of endolysin) of SP-CHAP and Cpl-1 on Spn strains TIGR4, DCC1811, DCC1335, Lyt4.4, D39, and R6. (H) Lytic activity of 50 µg/mL Cpl-1 and SP-CHAP on Spn strain Lyt4.4 after incubation with purified pneumococcal capsular polysaccharide. Experiments were done in triplicate, and the error bars represent the standard deviations. For two-way ANOVA with Dunnett’s multiple comparisons test, asterisks denote the level of significance observed: ns, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Fig 2

SP-CHAP reduces pneumococcal biofilm biomass in vitro and nasopharyngeal colonization in vivo. (A) Disruption of Spn Lyt4.4 biofilms by PBS,or 1.56 µg/mL Cpl-1 or SP-CHAP, as visualized by crystal violet staining. (B) Quantification of biomass measured by OD₅₉₅ of crystal violet-stained biofilms. SP-CHAP displayed biofilm eradication ability at all concentrations tested, while Cpl-1 removed biofilms at concentrations as low as 12.5 µg/mL. Statistical analysis denotes differences between PBS and endolysin-treated wells. (C) 630× confocal microscopy consisting of nineteen 0.5 µm slices of Spn biofilms treated with PBS, Cpl-1, or SP-CHAP. Scale bar = 10 µm. (D) Viability of Spn Lyt4.4 biofilms after treatment with 20 µg/mL Cpl-1 or SP-CHAP for 1 h as measured by the CFU assay. Data are reported as the Log CFU/mL of recovered bacteria. (E) Acute systemic toxicity in mice intraperitoneally challenged with vehicle or SP-CHAP. (F) Sketch of mouse colonization model and (G) Log CFU per gram of nasopharyngeal tissue from mice infected with Spn strain TIGR4 and treated with 60 µg of endolysin 48 h post-colonization and samples collected 4 h post-treatment. Samples were tested with two-way ANOVA or one-way ANOVA Kruskal-Wallis test with Dunn’s multiple-comparisons post-test. Asterisks denote the level of significance observed: ns, not significant; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.