The prevalence of multidrug-resistant Pseudomonas aeruginosa has stimulated development of alternative therapeutics. Bacteriophage peptidoglycan hydrolases, termed lysins, represent an emerging antimicrobial option for targeting Gram-positive bacteria.

Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.

Bacteriophage tailspike proteins act as primary receptors, often possessing endoglycosidase activity toward bacterial lipopolysaccharides or other exopolysaccharides, which enable phage absorption and subsequent DNA injection into the host. Phage CBA120, a contractile long-tailed Viunalikevirus phage infects the virulent Escherichia coli O157:H7. This phage encodes four putative tailspike proteins exhibiting little amino acid sequence identity, whose biological roles and substrate specificities are unknown. Here we focus on the first tailspike, TSP1, encoded by the orf210 gene. We have discovered that TSP1 is resistant to protease degradation, exhibits high thermal stability, but does not cleave the O157 antigen. An immune-dot blot has shown that TSP1 binds strongly to non-O157:H7 E. coli cells and more weakly to K. pneumoniae cells, but exhibits little binding to E. coli O157:H7 strains. To facilitate structure-function studies, we have determined the crystal structure of TSP1 to a resolution limit of 1.8 Å. Similar to other tailspikes proteins, TSP1 assembles into elongated homotrimers. The receptor binding region of each subunit adopts a right-handed parallel β helix, reminiscent yet not identical to several known tailspike structures. The structure of the N-terminal domain that binds to the virion particle has not been seen previously. Potential endoglycosidase catalytic sites at the three subunit interfaces contain two adjacent glutamic acids, unlike any catalytic machinery observed in other tailspikes. To identify potential sugar binding sites, the crystal structures of TSP1 in complexes with glucose, α-maltose, or α-lactose were determined. These structures revealed that each sugar binds in a different location and none of the environments appears consistent with an endoglycosidase catalytic site. Such sites may serve to bind sugar units of a yet to be identified bacterial exopolysaccharide.

Most skin infections, including those complicating burns, are polymicrobial involving multiple causative bacteria. Add to this the fact that many of these organisms may be antibiotic-resistant, and a simple skin lesion or burn could soon become life-threatening. Membrane-acting cationic peptides from Gram-negative bacteriophage lysins can potentially aid in addressing the urgent need for alternative therapeutics. Such peptides natively constitute an amphipathic region within the structural composition of these lysins and function to permit outer membrane permeabilization in Gram-negative bacteria when added externally. This consequently allows the lysin to access and degrade the peptidoglycan substrate, resulting in rapid hypotonic lysis and bacterial death. When separated from the lysin, some of these cationic peptides kill sensitive bacteria more effectively than the native molecule via both outer and cytoplasmic membrane disruption. In this study, we evaluated the antibacterial properties of a modified cationic peptide from the broad-acting lysin PlyPa01. The peptide, termed PaP1, exhibited potent in vitro bactericidal activity toward numerous high priority Gram-positive and Gram-negative pathogens, including all the antibiotic-resistant ESKAPE pathogens. Both planktonic and biofilm-state bacteria were sensitive to the peptide, and results from time-kill assays revealed PaP1 kills bacteria on contact. The peptide was bactericidal over a wide temperature and pH range and could withstand autoclaving without loss of activity. However, high salt concentrations and complex matrices were found to be largely inhibitory, limiting its use to topical applications. Importantly, unlike other membrane-acting antimicrobials, PaP1 lacked cytotoxicity toward human cells. Results from a murine burn wound infection model using methicillin-resistant Staphylococcus aureus or multidrug-resistant Pseudomonas aeruginosa validated the in vivo antibacterial efficacy of PaP1. In these studies, the peptide enhanced the potency of topical antibiotics used clinically for treating chronic wound infections. Despite the necessity for additional preclinical drug development, the collective data from our study support PaP1 as a potential broad-spectrum monotherapy or adjunctive therapy for the topical treatment of polymicrobial infections and provide a foundation for engineering future lysin-derived peptides with improved antibacterial properties.

Bacteriophage endolysins degrade the bacterial peptidoglycan and are considered enzymatic alternatives to small-molecule antibiotics. In particular, the multimeric streptococcal endolysin PlyC has appealing antibacterial properties. However, a comprehensive thermal analysis of PlyC is lacking, which is necessary for evaluating its long-term stability and downstream therapeutic potential. Biochemical and kinetic-based methods were used in combination with differential scanning calorimetry to investigate the structural, kinetic, and thermodynamic stability of PlyC and its various subunits and domains. The PlyC holoenzyme structure is irreversibly compromised due to partial unfolding and aggregation at 46 °C. Unfolding of the catalytic subunit, PlyCA, instigates this event, resulting in the kinetic inactivation of the endolysin. In contrast to PlyCA, the PlyCB octamer (the cell wall-binding domain) is thermostable, denaturing at ~75 °C. The isolation of PlyCA or PlyCB alone altered their thermal properties. Contrary to the holoenzyme, PlyCA alone unfolds uncooperatively and is thermodynamically destabilized, whereas the PlyCB octamer reversibly dissociates into monomers and forms an intermediate state at 74 °C in phosphate-buffered saline with each subunit subsequently denaturing at 92 °C. Adding folded PlyCA to an intermediate state PlyCB, followed by cooling, allowed for in vitro reconstitution of the active holoenzyme.

Pseudomonas aeruginosa is a leading cause of nosocomial infections, including pneumonia and urinary tract infections, and the primary cause of morbidity and mortality in cystic fibrosis patients. The emergence of multidrug-resistant strains makes these infections life-threatening. To overcome this challenge, lysocins can be employed as novel antipseudomonals. Lysocins use components of the pyocin antimicrobial system to deliver bacteriophage lysins to their peptidoglycan substrate in Pseudomonas . Peptidoglycan cleavage causes membrane destabilization, cytoplasmic leakage, and disruption of the proton motive force, thereby killing the cell. In our previous proof-of-concept study, the PyS2-GN4 lysocin killed only one-third of P. aeruginosa strains due to the targeted receptor. This limitation can now be circumvented by engineering second-generation lysocins that bind and translocate through highly conserved Pseudomonas -specific receptors. One lysocin, PyS5-I-GN4, uses a single domain from pyocin S5 to deliver the GN4 lysin through the conserved ferric pyochelin transporter, consequently killing 95% of multidrug-resistant clinical isolates tested. Importantly, PyS5-I-GN4 displayed antibiofilm properties and was bactericidal in serum and lung surfactant. Serum inactivation observed for lysins is not seen for lysocins, making this approach more effective for treating systemic Gram-negative bacterial infections. Despite its broadened pseudomonal strain coverage, PyS5-I-GN4 demonstrated narrow-spectrum antibacterial activity toward P. aeruginosa only and lacked cytotoxicity toward human cells. A single dose of lysocin was protective and reduced bacteria multiple log ₁₀ -fold in the lungs and secondary organs in a neutropenic murine lung infection model. These findings support lysocins as therapeutics for P. aeruginosa and provide insight into designing future constructs for other Gram-negative pathogens.