PL3 Amidase, a Tailor-made Lysin Constructed by Domain Shuffling with Potent Killing Activity against Pneumococci and Related Species

Abstract

The emergence and spread of antibiotic-resistant bacteria is pushing the need of alternative treatments. In this context, phage therapy is already a reality to successfully fight certain multiresistant bacteria. Among different phage gene products, murein hydrolases responsible of phage progeny liberation (also called lysins or endolysins) are weapons that target specific peptidoglycan bonds, leading to lysis and death of susceptible bacteria when added from the outside. In the pneumococcal system, all but one phage murein hydrolases reported to date share a choline-binding domain that recognizes cell walls containing choline residues in the (lipo)teichoic acids. Some purified pneumococcal or phage murein hydrolases, as well as several chimeric proteins combining natural catalytic and cell wall-binding domains (CBDs) have been used as effective antimicrobials. In this work we have constructed a novel chimeric N-acetylmuramoyl-L-alanine amidase (PL3) by fusing the catalytic domain of the Pal amidase (a phage-coded endolysin) to the CBD of the LytA amidase, the major pneumococcal autolysin. The physicochemical properties of PL3 and the bacteriolytic effect against several pneumococci (including 48 multiresistant representative strain) and related species, like Streptococcus pseudopneumoniae, Streptococcus mitis, and Streptococcus oralis, have been studied. Results have shown that low doses of PL3, in the range of 0.5–5 μg/ml, are enough to practically sterilize all choline-containing strains tested. Moreover, a single 20-μg dose of PL3 fully protected zebrafish embryos from infection by S. pneumoniae D39 strain. Importantly, PL3 keeps 95% enzymatic activity after 4 weeks at 37°C and can be lyophilized without losing activity, demonstrating a remarkable robustness. Such stability, together with a prominent efficacy against a narrow spectrum of human pathogens, confers to PL3 the characteristic to be an effective therapeutic. In addition, our results demonstrate that the structure/function-based domain shuffling approach is a successful method to construct tailor-made endolysins with higher bactericidal activities than their parental enzymes.

ClyJ Is a Novel Pneumococcal Chimeric Lysin with a Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase Catalytic Domain

“…The data are shown as a minimum-to-maximum boxplot, where the box represents the standard deviations, and the medians are marked by a bar. ns, not significant; **, P Ͻ 0.01; ***, P Ͻ 0.001. catalytic domain from Cpl-1 or Cpl-7 (20,22) and the amidase catalytic domain from Pal (41) have been used as potential donors in construction of chimeric lysins targeting S. pneumoniae. Therefore, ClyJ is the first chimeric lysin specific for S. pneumoniae that contains a CHAP domain.…”

Phage Lytic Protein LysRODI Prevents Staphylococcal Mastitis in Mice

“…Most endolysins from phages infecting Gram-positive bacteria have a modular structure (Oliveira et al, 2013), which facilitates protein engineering via domain shuffling to obtain new chimeric proteins with improved lytic activity (Blazquez et al, 2016). Regarding S. aureus, several published reports have shown the effectiveness of phage lytic proteins as therapeutics (Schuch et al, 2014), biopreservatives (Chang et al, 2017) and disinfectants (Gutiérrez et al, 2014).…”

Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins

“…The LysK lysin of S. aureus phage K remained active for at least 60 days of storage at 4°C(38). A fusion derivative of Streptococcus pneumoniae phage endolysin Pal and the LytA S. pneumoniae autolysin retained 95% activity after storage at 37°C for 4 weeks(39). The Cpl-1 lysin of S. pneumoniae phage Cp-1 was stable for more than 6 months at 4°C or 4 weeks at 37°C(34).…”