Broad Bactericidal Activity of the Myoviridae Bacteriophage Lysins LysAm24, LysECD7, and LysSi3 against Gram-Negative ESKAPE Pathogens

Abstract

The extremely rapid spread of multiple-antibiotic resistance among Gram-negative pathogens threatens to move humankind into the so-called “post-antibiotic era” in which the most efficient and safe antibiotics will not work. Bacteriophage lysins represent promising alternatives to antibiotics, as they are capable of digesting bacterial cell wall peptidoglycans to promote their osmotic lysis. However, relatively little is known regarding the spectrum of lysin bactericidal activity against Gram-negative bacteria. In this study, we present the results of in vitro activity assays of three putative and newly cloned Myoviridae bacteriophage endolysins (LysAm24, LysECD7, and LysSi3). The chosen proteins represent lysins with diverse domain organization (single-domain vs. two-domain) and different predicted mechanisms of action (lysozyme vs. peptidase). The enzymes were purified, and their properties were characterized. The enzymes were tested against a panel of Gram-negative clinical bacterial isolates comprising all Gram-negative representatives of the ESKAPE group. Despite exhibiting different structural organizations, all of the assayed lysins were shown to be capable of lysing Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Salmonella typhi strains. Less than 50 μg/mL was enough to eradicate growing cells over more than five orders of magnitude. Thus, LysAm24, LysECD7, and LysSi3 represent promising therapeutic agents for drug development.

Modulation of Endolysin LysECD7 Bactericidal Activity by Different Peptide Tag Fusion

“…The recombinant LysECD7 was fused to an 8-His tag at the C-terminus (LysECD7-8his) and purified using NiNTA affinity chromatography, as previously characterized by physicochemical and microbiological methods [23]. This molecule showed sufficiently high stability under different conditions and possessed wide bactericidal activity capable of inhibiting the growth of several bacterial species from the ESKAPE group (Pseudomonas, Klebsiella, Acinetobacter, etc).…”

“…Nevertheless, researchers rarely try several tag modifications when purifying the target protein and look for alternative purification methods only if no (or few) biological effects are observed [14,15]. Most of the recombinant endolysins under investigation were purified and tested for antibacterial activity with fused histidine tags [2,6,[16][17][18][19][20][21][22][23]. However, how the activity of these endolysins is modified by polyhistidine tag fusion has not been specifically studied.…”

“…Previously, we described the LysECD7 endolysin, the putative zinc d-alanyl-d-alanine carboxypeptidase (peptidase M15 family) [23] of the lytic bacteriophage ECD7 (NCBI: txid1981499, Escherichia coli), related to the family Myoviridae and infecting the Escherichia species [31]. The ECD7 phage was isolated from chicken excrement in 2012 using a culture of a Shiga-toxin-producing E. coli strain O104:H4, which caused an outbreak of gastroenteritis and hemolytic-uremic syndrome in Germany during 2011 [32].…”

“…LysECD7 could be a promising therapeutic agent for antimicrobial drug development, as it showed a wide spectrum of action against Gram-negative bacteria. However, a number of limitations in the use of the LysECD7-8his molecule were revealed; these limitations may interfere with its further development in preclinical and clinical studies [23]. In particular, it was noted that its activity depends on buffer conditions, the presence of salts, and the pH of the medium.…”

“…Further, the endolysin's bactericidal effect decreased with the addition of human serum to the assay. These limitations were compensated by the addition of EDTA, which may indicate that the problem was in the membrane permeabilization process under certain conditions [23].…”