Antibacterial activity of Acinetobacter baumannii phage ϕAB2 endolysin (LysAB2) against both Gram-positive and Gram-negative bacteria

Abstract

To investigate the nature and origin of the antibacterial activity of the lytic phage ϕAB2 toward Acinetobacter baumannii, we successfully isolated and characterized a novel phage lysozyme (endolysin) from ϕAB2 and named it LysAB2. To analyze antibacterial activity of LysAB2, the complete LysAB2 and two deletion derivatives were constructed, purified and characterized. Zymographic assays showed that only the intact LysAB2 could lyse the peptidoglycan of A. baumannii and the Staphylococcus aureus cell wall. Antibacterial analysis also showed that only the intact LysAB2 retained the complete bactericidal activity. When applied exogenously, LysAB2 exhibited a broad bacteriolytic activity against a number of Gram-negative and Gram-positive bacteria. Thermostability assays indicated that LysAB2 was stable at 20∼40 °C. Its optimal pH was 6.0, and it was active from pH 4 to 8. Scanning electron microscopy revealed that exposure to 500 μgml(-1) LysAB2 for up to 60 min caused a remarkable modification of the cell shape of the bacteria. Treating bacteria with LysAB2 clearly enhanced permeation of the bacterial cytoplasmic membrane. These results indicate that LysAB2 is an effective lysozyme against bacteria, and they suggest that it is a good candidate for a therapeutic/disinfectant agent to control nosocomial infections caused by multiple drug-resistant bacteria.

Antimicrobial bacteriophage-derived proteins and therapeutic applications

“…However, proximity and orientation of these domains is not universally conserved between endolysins (i.e., a CBD can be located on the N-terminus, mid-protein, C-terminus, or be absent). Although this multi-domain organization is dominant among endolysins from Gram-positive phage, it is not common among endolysins from Gram-negative phage, [20][21][22] which are generally globular and lack CBDs. 23 The number of EADs in the modular endolysins from Gram-positive-infecting phage also varies, with several having two, each encoding a different catalytic activity.…”

“…Truncation of the amphipathic region abolishes this activity, suggesting the region is necessary for LysAB2 to be bacteriolytic of Gram-negative species. 20 However, harboring an amphipathic or highly positively charged amino acid region is not always necessary to destabilize the outer membrane. The endolysin OBPgp279, from Pseudomonas fluorescens phage OBP, can naturally lyse P. aeruginosa, but is devoid of any obvious amphipathic or highly positively charged regions.…”

“…90,94,95 These types of endolysins tend to contain an amphipathic (both hydrophobic and hydrophilic regions) or highly cationic region, which interacts with the negatively charged surface lipopolysaccharides (LPS) to transverse the outer membrane. 20,22,90,94,95 The Gram-negative Acinetobacter baumannii phage endolysin LysAB2, with a predicted Cterminus amphipathic region, has broad lytic activity for both Gram-positive and Gram-negative hosts. Truncation of the amphipathic region abolishes this activity, suggesting the region is necessary for LysAB2 to be bacteriolytic of Gram-negative species.…”

“…92 A few phage endolysins have demonstrated bacteriolytic activity toward Gram-negative cells without the need for physical or chemical outer membrane disruption, or by enzyme modification. 20,21,93 For example, the SPN9CC endolysin naturally lyse only Gram-negative bacteria in the absence of an outer membrane permeabilizer. 93 Likewise, the Gram-positive Bacillus amyloliquefaciens phage endolysin Lys1521 can naturally lyse several P. aeruginosa and E. coli strains.…”

“…A limitation of using native endolysins therapeutically against Gram-negative bacteria is their inability to translocate across the outer membrane. [20][21][22] Yersinia pestis encodes the bacteriocin pesticin, a muramidase that can lyse closely related bacteria. Pesticin misuses the existing outer membrane Ton transport system to be transported into the periplasm and degrade the cell wall peptidoglycan.…”