Characterization of Modular Bacteriophage Endolysins from Myoviridae Phages OBP, 201φ2-1 and PVP-SE1

Abstract

Peptidoglycan lytic enzymes (endolysins) induce bacterial host cell lysis in the late phase of the lytic bacteriophage replication cycle. Endolysins OBPgp279 (from Pseudomonas fluorescens phage OBP), PVP-SE1gp146 (Salmonella enterica serovar Enteritidis phage PVP-SE1) and 201ϕ2-1gp229 (Pseudomonas chlororaphis phage 201ϕ2-1) all possess a modular structure with an N-terminal cell wall binding domain and a C-terminal catalytic domain, a unique property for endolysins with a Gram-negative background. All three modular endolysins showed strong muralytic activity on the peptidoglycan of a broad range of Gram-negative bacteria, partly due to the presence of the cell wall binding domain. In the case of PVP-SE1gp146, this domain shows a binding affinity for Salmonella peptidoglycan that falls within the range of typical cell adhesion molecules (Kaff = 1.26×106 M−1). Remarkably, PVP-SE1gp146 turns out to be thermoresistant up to temperatures of 90°C, making it a potential candidate as antibacterial component in hurdle technology for food preservation. OBPgp279, on the other hand, is suggested to intrinsically destabilize the outer membrane of Pseudomonas species, thereby gaining access to their peptidoglycan and exerts an antibacterial activity of 1 logarithmic unit reduction. Addition of 0.5 mM EDTA significantly increases the antibacterial activity of the three modular endolysins up to 2–3 logarithmic units reduction. This research work offers perspectives towards elucidation of the structural differences explaining the unique biochemical and antibacterial properties of OBPgp279, PVP-SE1gp146 and 201ϕ2-1gp229. Furthermore, these endolysins extensively enlarge the pool of potential antibacterial compounds used against multi-drug resistant Gram-negative bacterial infections.

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.…”

“…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.…”

“…EDTA), or high hydrostatic pressure in combination with an endolysin have been studied. 21,22,91,92 Although these measures have shown that chemical and/or physical disruption of the outer membrane allows the endolysin to gain access to the protected peptidoglycan, they may not lend themselves well to human therapies. These methods have not always been successful, with some Gram-negative strains remaining insensitive to lysis by endolysins after membrane disruption.…”

“…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.…”

“…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. 21 Currently, it is not known how OBPgp279 transverses the outer membrane. 21 These aforementioned Gram-negative phage endolysins have yet to be tested therapeutically in animal models.…”