Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against Staphylococcus aureus Strains.

Chimeric lysins composed of various combinations of cell wall-lysing (enzymatic) and cell-wall-binding (CWB) domains of endolysins, autolysins, and bacteriocins have been developed as alternatives to or adjuvants of conventional antibiotics. The screening of multiple chimeric lysin candidates for activity via E. coli expression is not cost effective, and we previously reported on a simple cell-free expression system as an alternative. In this study, we sufficiently improved upon this cell-free expression system for use in screening activity via a turbidity reduction test, which is more appropriate than a colony reduction test when applied in multiple screening. Using the improved protocol, we screened and compared the antibacterial activity of chimeric lysin candidates and verified the relatively strong activity associated with the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain of secretory antigen SsaA-like protein (ALS2). ALS2 expressed in E. coli showed two major bands, and the smaller one (subprotein) was shown to be expressed by an innate downstream promoter and start codon (ATG). The introduction of synonymous mutations in the promoter resulted in clearly reduced expression of the subprotein, whereas missense mutations in the start codon abolished antibacterial activity as well as subprotein production. Interestingly, most of the S. aureus strains responsible for bovine mastitis were susceptible to ALS2, but those from human and chicken were less susceptible. Thus, the simple and rapid screening method can be applied to select functional chimeric lysins and define mutations affecting antibacterial activity, and ALS2 may be useful in itself and as a lead molecule to control bovine mastitis.

Development of cationic peptide chimeric lysins based on phage lysin Lysqdvp001 and their antibacterial effects against Vibrio parahaemolyticus: A preliminary study.

Cationic peptide chimeric lysins, Lysqdvp001-5aa, Lysqdvp001-10aa and Lysqdvp001-15aa, were designed based on lysin Lysqdvp001 from Vibrio parahaemolyticus (V. parahaemolyticus) phage qdvp001. These chimeric lysins showed equivalent peptidoglycan hydrolysis activities with Lysqdvp001 and could lyse the bacteria from the outside. The antibacterial activity as well as outer and inner membrane permeabilization of Lysqdvp001 and chimeric lysins against V. parahaemolyticus were Lysqdvp001-15aa>Lysqdvp001-10aa>Lysqdvp001-5aa>Lysqdvp001. Lysqdvp001-15aa exhibited an excellent antibacterial activity with minimum inhibition and bactericidal concentrations (MIC and MBC) of 0.2 and 0.4 mg/mL, respectively, and its antibacterial spectrum was much broader than phage qdvp001. Membrane hyperpolarization and membrane phospholipid exposure of V. parahaemolyticus were observed after Lysqdvp001-15aa treatments. Transmission electron microscope (TEM) showed Lysqdvp001-15aa destroyed structure integrity of V. parahaemolyticus. Besides, MIC and MBC of Lysqdvp001-15aa decreased V. parahaemolyticus counts in oyster by 3.20 and 4.03 log10CFU/g. Lysqdvp001-15aa at MBC eradicated about 50% of V. parahaemolyticus biofilms and inhibited over 90% of the formation of the bacterial biofilms.

Internal cell-penetrating peptide-mediated internalization enables a chimeric lysin to target intracellular pathogens.

Intracellular pathogens pose serious challenges to the public health worldwide. Lysin, peptidoglycan hydrolase from phage, is promising alternative to conventional antibiotics because of its high bactericidal activity and low risk of resistance. However, most proteinaceous lysins cannot penetrate the mammalian cell membrane because of size exclusion. Previously, we reported a broad-spectrum chimeric lysin, ClyR, with a cysteine, histidine-dependent amidohydrolase/peptidase catalytic domain from PlyC lysin and an SH-3b cell-wall binding domain from PlySs2 lysin. Herein, we further report that a novel internal cell-penetrating peptide (CPP) is predicted in the junction region of the two constitutive domains of ClyR, mediated by which ClyR can be internalized by epithelial cells through caveolin-dependent endocytosis to target intracellular pathogens. Residues K153, P154, R169, and R188 of the internal CPP were found to be essential for ClyR-mediated internalization and intracellular killing. RNA-seq analysis further showed that there are minor differences in transcript and metabolic profiles from epithelial cells exposed to 100 µg/ml ClyR for 24 h. Taken together, our findings demonstrate a novel mechanism of internalization by ClyR, providing new insights into the rational designing of the next-generation lysins to target both extracellular and intracellular pathogens.

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

Streptococcus pneumoniae is a leading pathogen for bacterial pneumonia, which can be treated with bacteriophage lysins harboring a conserved choline binding module (CBM). Such lysins regularly function as choline-recognizing dimers. Previously, we reported a pneumococcus-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. A variant of ClyJ with a 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 it binds to choline yet exhibits improved bactericidal activity against multiple pneumococcal strains with different serotypes. In an 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 less clearance and a greater area under curve than ClyJ-3. Taken together, the choline-recognizing monomer ClyJ-3m exhibited enhanced bactericidal activity and improved pharmacokinetic proprieties compared to those of its parental ClyJ-3 lysin. Our study also provides a new way for rational design and programmed engineering of lysins targeting S. pneumoniae.

Construction and characterization of a chimeric lysin ClyV with improved bactericidal activity against Streptococcus agalactiae in vitro and in vivo.

The emergence of antibiotic-resistant beta-hemolytic Streptococcus agalactiae strains poses increasing threat to human beings globally. As an attempt to create a novel lysin with improved activity against S. agalactiae, a chimeric lysin, ClyV, was constructed by fusing the enzymatically active domain (EAD) from PlyGBS lysin (GBS180) and the cell wall binding domain (CBD) from PlyV12 lysin (V12CBD). Plate lysis assay combined with lytic kinetic analysis demonstrated that ClyV has improved activity than its parental enzymatic domain GBS180 against multiple streptococci. Biochemical characterization showed that ClyV is active from pH 7 to 10, with the optimum pH of 9, and is stable under NaCl concentration of < 500 mM. In a S. agalactiae infection model, a single intraperitoneally administration of 0.1 mg/mouse of ClyV protected 100% mice, while it was observed that ~ 29% survive in group that received a single dose of 0.1 mg/mouse of GBS180. Moreover, a high dose of 0.8 mg/mouse ClyV did not show any adverse effects to the health or survival rate of the mice. Considering the robust bactericidal activity and good safety profile of ClyV, it represents a potential candidate for the treatment of S. agalactiae infections.

Linker Editing of Pneumococcal Lysin ClyJ Conveys Improved Bactericidal Activity.

Streptococcus pneumoniae is a leading human pathogen uniquely characterized by choline moieties on the bacterial surface. Our previous work reported a pneumococcus-specific chimeric lysin, ClyJ, which combines the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) enzymatically active domain (EAD) from the PlyC lysin and the cell wall binding domain (CBD) from the phage SPSL1 lysin, which imparts choline binding specificity. Here, we demonstrate that the lytic activity of ClyJ can be further improved by editing the linker sequence adjoining the EAD and CBD. Keeping the net charge of the linker constant, we constructed three ClyJ variants containing different lengths of linker sequence. Circular dichroism showed that linker editing has only minor effects on the folding of the EAD and CBD. However, thermodynamic examination combined with biochemical analysis demonstrated that one variant, ClyJ-3, with the shortest linker, displayed improved thermal stability and bactericidal activity, as well as reduced cytotoxicity. In a pneumococcal mouse infection model, ClyJ-3 showed significant protective efficacy compared to that of the ClyJ parental lysin or the Cpl-1 lysin, with 100% survival at a single ClyJ-3 intraperitoneal dose of 100 µg/mouse. Moreover, a ClyJ-3 dose of 2 µg/mouse had the same efficacy as a ClyJ dose of 40 µg/mouse, suggesting a 20-fold improvement in vivo Taking these results together, the present study not only describes a promising pneumococcal lysin with improved potency, i.e., ClyJ-3, but also implies for the first time that the linker sequence plays an important role in determining the activity of a chimeric lysin, providing insight for future lysin engineering studies.

Kinetics of inactivation of staphylolytic enzymes: Qualitative and quantitative description.

Lysin 2638aR and chimeric Ply187AN-KSH3b fusion protein are capable of lysing antibiotic-resistant strains of Staphylococcus aureus and are promising alternatives to antibiotics. Studies on the stability and structure of lysins 2638aR and Ply187AN-KSH3b are important for assessing the feasibility of their practical use. Both lysins are highly active at physiological pH (7.5) and at low salt content (the concentration of NaCl in the reaction medium is not more than 250 mM). Lysins are inactivated by a monomolecular mechanism and have high stability at 4 °C (storage temperature). The maximum value of the half-inactivation time for lysin 2638aR is 190-200 days (500-1000 mM NaCl, pH 6.0-7.5), for lysin Ply187AN-KSH3b is 320-340 days (10-1000 mM NaCl, pH 6.0). The lysins are pretty stable in human blood serum (the half-inactivation time is 0.5-2 h) at 37 °C. The lysins undergo denaturation in large part due to the destruction of the α-helices at temperatures above 40 °C.

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

Streptococcus pneumoniae is one of the leading pathogens that cause a variety of mucosal and invasive infections. With the increased emergence of multidrug-resistant S. pneumoniae, new antimicrobials with mechanisms of action different from conventional antibiotics are urgently needed. In this study, we identified a putative lysin (gp20) encoded by the Streptococcus phage SPSL1 using the LytA autolysin as a template. Molecular dissection of gp20 revealed a binding domain (GPB) containing choline-binding repeats (CBRs) that are high specificity for S. pneumoniae By fusing GPB to the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain of the PlyC lysin, we constructed a novel chimeric lysin, ClyJ, with improved activity to the pneumococcal Cpl-1 lysin. No resistance was observed in S. pneumoniae strains after exposure to incrementally doubling concentrations of ClyJ for 8 continuous days in vitro In a mouse bacteremia model using penicillin G as a control, a single intraperitoneal injection of ClyJ improved the survival rate of lethal S. pneumoniae-infected mice in a dose-dependent manner. Given its high lytic activity and safety profile, ClyJ may represent a promising alternative to combat pneumococcal infections.

Accurate Detection of Methicillin-Resistant Staphylococcus aureus in Mixtures by Use of Single-Bacterium Duplex Droplet Digital PCR.

Accurate and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is needed to screen MRSA carriers and improve treatment. The current widely used duplex PCR methods are not able to differentiate MRSA from coexisting methicillin-susceptible S. aureus (MSSA) or other methicillin-resistant staphylococci. In this study, we aimed to develop a direct method for accurate and rapid detection of MRSA in clinical samples from open environments, such as nasal swabs. The new molecular assay is based on detecting the cooccurrence of nuc and mecA markers in a single bacterial cell by utilizing droplet digital PCR (ddPCR) with the chimeric lysin ClyH for cell lysis. The method consists of (i) dispersion of an intact single bacterium into nanoliter droplets, (ii) temperature-controlled release of genomic DNA (gDNA) by ClyH at 37°C, and (iii) amplification and detection of the markers (nuc and mecA) using standard TaqMan chemistries with ddPCR. Results were analyzed based on MRSA index ratios used for indicating the presence of the duplex-positive markers in droplets. The method was able to achieve an absolute limit of detection (LOD) of 2,900 CFU/ml for MRSA in nasal swabs spiked with excess amounts of Escherichia coli, MSSA, and other mecA-positive bacteria within 4 h. Initial testing of 104 nasal swabs showed that the method had 100% agreement with the standard culture method, while the normal duplex qPCR method had only about 87.5% agreement. The single-bacterium duplex ddPCR assay is rapid and powerful for more accurate detection of MRSA directly from clinical specimens.

Degradation of methicillin-resistant Staphylococcus aureus biofilms using a chimeric lysin.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for a large number of chronic infections due to its ability to form robust biofilms. Herein, the authors evaluated the anti-biofilm activity of a Staphylococcus specific chimeric lysin ClyH on MRSA biofilms. ClyH is known to be active against planktonic MRSA cells in vitro and in vivo. The minimum concentrations for biofilm eradication (MCBE) of ClyH were 6.2-50 mg l(-1), much lower than those of antibiotics. Scanning electron microscope (SEM) analysis revealed that ClyH eliminated MRSA biofilms through cell lytic activity in a time-dependent manner. Viable plate counts and kinetic analysis demonstrated that biofilms of different ages displayed varying susceptibility to ClyH. Together with previously demonstrated in vivo efficacy of ClyH against MRSA, the degradation efficacy against biofilms of different ages indicates that ClyH could be used to remove MRSA biofilms in vivo.