A Choline-Recognizing Monomeric Lysin, ClyJ-3m, Shows Elevated Activity against Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is a leading pathogen for bacterial pneumonia, which could be treated with bacteriophage lysins harboring a conserved choline binding module (CBM). Such kind of lysins regularly function as choline-recognizing dimers. Previously, we reported a pneumococci-specific lysin ClyJ comprising the binding domain from the putative endolysin gp20 from the Streptococcus phage SPSL1 and the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain from the PlyC lysin. And a variant of ClyJ with shortened linker, i.e. ClyJ-3, shows improved activity and reduced cytotoxicity. Resembling typical CBM-containing lysins, ClyJ-3 dimerized upon binding with choline. Herein, we further report a choline-recognizing variant of ClyJ-3, i.e. ClyJ-3m, constructed by deleting its C-terminal tail. Biochemical characterization showed that ClyJ-3m remains a monomer after bound to choline, yet exhibits improved bactericidal activity against multiple pneumococcal strains with different serotypes. In a S. pneumoniae-infected bacteremia model, a single intraperitoneal administration of 2.32 μg/mouse of ClyJ-3m showed 70% protection, while only 20% of mice survived in the group receiving an equal dose of ClyJ-3 (p < 0.05). A pharmacokinetic analysis following single intravenously doses of 0.29 and 1.16 mg/kg of ClyJ-3 or ClyJ-3m in BALB/c mice revealed that ClyJ-3m shows a similar half-life, but smaller Cl (clearance) and higher AUC (area under curve) than ClyJ-3. Taken together, the choline-recognizing monomer ClyJ-3m exhibited enhanced bactericidal activity and improved pharmacokinetic proprieties than its parental ClyJ-3 lysin. Our study also provides a new way for rational design and programmed engineering of lysins targeting on S. pneumoniae.

Structural and Functional Insights Into Skl and Pal Endolysins, Two Cysteine-Amidases With Anti-pneumococcal Activity. Dithiothreitol (DTT) Effect on Lytic Activity

“…Besides, the lysis proceeded somewhat faster at 30 • C but no statistically significant differences were found in the killing capacity after 60 min incubation in a given buffer (Figure 7C). As a whole, our results showed that optimal antipneumococcal activity of Skl compares well with those of Pal and LytA under equivalent conditions, and with those of ClyJ-3 and ClyJ-3m, two variants of a chimera built by fusing the CHAP domain of PlyC and the choline-specific CBD of the gp20 endolysin from the Streptococcus phage SPSL1 (Luo et al, 2020;Yang et al, 2020). Interestingly, the Y264H substitution reduced the bactericidal activity of Skl by several orders of magnitude (Figure 7A), further supporting the relevance of dimerization for the activity.…”

“…Interestingly, the Y264H substitution reduced the bactericidal activity of Skl by several orders of magnitude (Figure 7A), further supporting the relevance of dimerization for the activity. Curiously, the antipneumococcal activity of ClyJ-3 was improved by deletion of the C-terminal tail of the CBD in the ClyJ-3m monomeric variant, which might denote a less favorable interaction of the highly active PlyC domain with the cleavable peptidoglycan bonds upon dimerization of this fully engineered lysin (Luo et al, 2020).…”

Endolysins against Streptococci as an antibiotic alternative

“…Similar to ClyJ, ClyJ-3 forms a dimer conformation upon binding with a choline molecule, while ClyJ-3m forms a monomer upon choline binding. The bactericidal activity was greatly enhanced in ClyJ-3m when compared to the parental enzymes ClyJ-3 and ClyJ ( Luo et al, 2020 ).…”

“…ClyJ exhibited stronger activity against S. pneumoniae than Cpl-1, and it produced no resistance in bacteria after prolonged exposure to the enzyme ( Yang et al, 2019 ). ClyJ also has several derivatives denoted as ClyJ-3 and ClyJ-3m, which are essentially truncated versions of ClyJ ( Luo et al, 2020 ). ClyJ-3 has a shorter linker than the parental ClyJ, whereas ClyJ-3m is a variant of ClyJ-3, which has a truncated C-terminal tail.…”

Gram-Positive Pneumonia: Possibilities Offered by Phage Therapy

“…With the aim of improving its activity, chimeric lysin ClyJ was modified by shortening its linker, which led to increased lytic activity in vivo (20-fold) and reduced cytotoxicity [ 67 ]. Remarkably, a variant of this protein, ClyJ-3m, which remains a monomer after binding choline, exhibited even higher bactericidal activity and improved the pharmacokinetic properties, such as a lesser clearance by the immune system [ 68 ]. Additionally, two new streptococcal endolysins (23TH_48 and SA01_53) have been recently identified in the oral microbiome.…”