Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages φKZ and EL

Abstract

SummaryPseudomonas aeruginosa bacteriophage endolysins KZ144 (phage fKZ) and EL188 (phage EL) are highly lytic peptidoglycan hydrolases (210 000 and 390 000 units mg -1 ), active on a broad range of outer membrane-permeabilized Gram-negative species. Site-directed mutagenesis indicates E115 (KZ144) and E155 (EL188) as their respective essential catalytic residues. Remarkably, both endolysins have a modular structure consisting of an N-terminal substrate-binding domain and a predicted C-terminal catalytic module, a property previously only demonstrated in endolysins originating from phages infecting Gram-positives and only in an inverse arrangement. Both binding domains contain conserved repeat sequences, consistent with those of some peptidoglycan hydrolases of Gram-positive bacteria. Fusions of these domains with green fluorescent protein immediately label all outer membrane-permeabilized Gram-negative bacteria tested, isolated P. aeruginosa peptidoglycan and N-acetylated Bacillus subtilis peptidoglycan, demonstrating the broad range of peptidoglycan-binding capacity by these domains. Specifically, A1 chemotype peptidoglycan and fully N-acetylated glucosamine units are essential for binding. Both KZ144 and EL188 appear to be a natural chimeric enzyme, originating from a recombination of a cell wall-binding domain encoded by a Bacillus or Clostridium species and a catalytic domain of an unknown ancestor.

Small but Sufficient: the Rhodococcus Phage RRH1 Has the Smallest Known Siphoviridae Genome at 14.2 Kilobases

“…While the predicted amino acid sequence of Orf5 is most closely related to that of a hypothetical protein in a putative prophage of Rhodococcus erythropolis, it also shares identity with the putative lysin proteins of the Mycobacterium phage Bethlehem and other related Mycobacterium phages (19). In addition, Orf5 contains the pfam01471 motif, composed of three alpha helices and associated with peptidoglycan binding and bacterial cell wall degradation (7). On the basis of these data, we conclude that orf5 encodes a lysin that is an unusual location within the gene module encoding phage structural proteins.…”

ighting antimicrobial resistance in ESKAPE pathogens

“…The potential for lysin resistance in bacteria should be low due to the direction of the highly-conserved peptidoglycan components [32]. Endolysins against Gram-negative pathogens were recently characterised and developed [33][34][35]. In S. aureus, the application of lytic proteins to treat severe infections such as bacteraemia or endocarditis has already been studied.…”

Bioinformatics analysis of bacteriophage and prophage endolysin domains

“…It can be found at the N-or C-terminus of a variety of enzymes involved in bacterial cell wall degradation. The PG binding1 domain is known to bind peptidoglycan experimentally (Briers et al 2007). The E4 sequence type had two catalytic domains positioned C-terminally, VanY endopeptidase and LT GEWL lytic transglycosylase (cd00254).…”

“…But once outer membrane of Gram-negatives is compromised, they also become highly sensitized to enzyme action from outside (much more than Grampositive bacteria) due to the thinness of their peptidoglycan (Briers et al 2007;Lai et al 2011). These enzymes are therefore applicable to eliminate bacterial contaminations from the environment, foods or surfaces, where the presence of antibiotic resistant bacteria is a serious problem.…”