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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. [ABSTRACT FROM AUTHOR]

Endolysin enzymes from bacteriophage cause bacterial lysis by degrading the peptidoglycan cell wall. The streptococcal C1 phage endolysin PlyC, is the most potent endolysin described to date and can rapidly lyse group A, C, and E streptococci. PlyC is known to bind the Group A streptococcal cell wall, but the specific molecular target or the binding site within PlyC remain uncharacterized. Here we report for the first time, that the polyrhamnose backbone of the Group A streptococcal cell wall is the binding target of PlyC. We have also characterized the putative rhamnose binding groove of PlyC and found four key residues that were critical to either the folding or the cell wall binding action of PlyC. Based on our results, we suggest that the interaction between PlyC and the cell wall may not be a high‐affinity interaction as previously proposed, but rather a high avidity one, allowing for PlyC's remarkable lytic activity. Resistance to our current antibiotics is reaching crisis levels and there is an urgent need to develop the antibacterial agents with new modes of action. A detailed understanding of this potent endolysin may facilitate future developments of PlyC as a tool against the rise of antibiotic resistance. [ABSTRACT FROM AUTHOR]

Endolysins are bacteriophage-derived peptidoglycan hydrolases that represent an emerging class of proteinaceous therapeutics. While the streptococcal endolysin PlyC has been validated in vitro and in vivo for its therapeutic efficacy, the inherent thermosusceptible structure of the enzyme correlates to transient long-term stability, thereby hindering the feasibility of developing the enzyme as an antimicrobial. Here, we thermostabilized the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain of the PlyCA catalytic subunit of PlyC using a FoldX-driven computational protein engineering approach. Using a combination of FoldX and Rosetta algorithms, as well as visual inspection, a final list of PlyC point mutant candidates with predicted stabilizing G values was assembled and thermally characterized. Five of the eight point mutations were found experimentally to be destabilizing, a result most likely attributable to computationally modeling a complex and dynamic nine-subunit holoenzyme with a corresponding 3.3-Å X-ray crystal structure. However, one of the mutants, PlyC (PlyCA) T406R, was shown experimentally to increase the thermal denaturation temperature by ~2.2°C and kinetic stability 16-fold over wild type. This mutation is expected to introduce a thermally advantageous hydrogen bond between the Q106 side chain of the N-terminal glycosyl hydrolase domain and the R406 side chain of the C-terminal CHAP domain. [ABSTRACT FROM AUTHOR]