Structure of an Acinetobacter Broad-Range Prophage Endolysin Reveals a C-Terminal α-Helix with the Proposed Role in Activity against Live Bacterial Cells

Abstract

Proteins that include enzymatic domain degrading the bacterial cell wall and a domain providing transport through the bacterial outer membrane are considered as prospective compounds to combat pathogenic Gram-negative bacteria. This paper presents an isolation and study of an enzyme of this class naturally encoded in the prophage region of Acinetobacter baumannii AB 5075 genome. Recombinant protein expressed in E. coli exhibits an antimicrobial activity with respect to live cultures of Gram-negative bacteria reducing the population of viable bacteria by 1.5–2 log colony forming units (CFU)/mL. However the protein becomes rapidly inactivated and enables the bacteria to restore the population. AcLys structure determined by X-ray crystallography reveals a predominantly α—helical fold similar to bacteriophage P22 lysozyme. The С-terminal part of AcLys polypeptide chains forms an α—helix enriched by Lys and Arg residues exposed outside of the protein globule. Presumably this type of structure of the C-terminal α—helix has evolved evolutionally enabling the endolysin to pass the inner membrane during the host lysis or, potentially, to penetrate the outer membrane of the Gram-negative bacteria.

Modulation of Endolysin LysECD7 Bactericidal Activity by Different Peptide Tag Fusion

“…It is assumed that the positively charged N-or C-terminal domain can interfere with the negatively charged LPS layer, thus allowing endolysins to penetrate through the bacterial outer membrane [16,17]. However, the emergence of an increasing amount of indirect evidence [16,34,36,40] proposes also the complex structure for relatively small endolysins from Gram-negative targeting phages. Thus, the outer membrane permeabilizing activity was previously shown for two highly cationic C-terminal peptides P307 [36] and LysAB2 P3 [16] of phage endolysins.…”

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

“…In contrast, PlyE146 [24] only displayed wide specificity at a concentration of 400 μg/mL. Moreover, similar results were achieved at lower concentrations of this enzyme (below 1 μg/mL) only after cells were partially permeabilized by hypotonic conditions, freezing during storage or after the cells were washed with water [23].…”

“…Some experimental evidence of the broad lytic activity of endolysins against Gram-negative bacteria has come from studies of E. coli and A. baumannii prophages [23,24,27]. An E. coli prophage lysin (PlyE146) has been shown to lyse a wide range of Gram-negative bacteria ( K. pneumoniae , A. baumannii , P. aeruginosa , E. coli , and S. enterica ) [24], and A. baumannii prophage lysin (AcLys) exhibits a similar range of bactericidal activity [23]. Our results show that the wide bactericidal activity of endolysins from Gram-negative-infecting bacteriophages is a much more general phenomenon than was previously appreciated.…”

“…The finding that none of our enzymes required a chemical permeabilizer under some conditions was unexpected. Some lysins are known to possess a natural ability to penetrate the bacterial OM due to positively charged segments that can disorganize or disrupt the OM [13,23], and the recombinant enzymes assayed in this study likely possess such sequences. Specifically, the C-terminal sequences of LysAm24 and LysSi3 contain a positively charged α-helix with nine lysine and arginine residues in 30-aa stretch.…”

“…However, the conserved PG structure of Gram-negative bacteria make the application endolysins potentially useful with respect to their breadth of action [20,21,22]. Recently, additional evidence has demonstrated the activity of specific endolysins toward Gram-negative bacteria [23,24], at least for prophages of Escherichia coli and A. baumannii . However, few studies have described the broad activity of bacteriophage-encoded lysins against Gram-negative bacteria, and growing evidence suggests that many bacteriophage lysins are capable of much more than just acting against parental phage-specific hosts.…”