Investigating novel
Streptomyces
bacteriophage endolysins as potential antimicrobial agents
Authors: Maneekul Jindanuch , Chiaha Amanda , Hughes Rachel , Labry Faith , Saito Joshua , Almendares Matthew , Banda Brenda N. , Lopez Leslie , McGaskey Nyeomi , Miranda Melizza , Rana Jenil , Zadeh Brandon R. , Hughes Lee E. 
Published: November 2024

As antibiotic resistance has become a major global threat, the World Health Organization (WHO) has urgently called for alternative strategies for control of bacterial infections. Endolysin, a phage-encoded protein, can degrade bacterial peptidoglycan (PG) and disrupt bacterial growth. According to the WHO, there are only three endolysin products currently in clinical phase development. In this study, we explore novel endolysins from
Streptomyces
phages as only a few of them have been experimentally characterized. Using several bioinformatics tools, we identified nine different functional domain combinations from 250
Streptomyces
phages putative endolysins. LazerLemon gp35 (CHAP; LL35lys), Nabi gp26 (amidase; Nb26lys), and Tribute gp42 (PGRP/amidase; Tb42lys) were selected for experimental studies. We hypothesized that (i) the proteins of interest will have the ability to degrade purified PG, and (ii) the proteins will have potential antimicrobial activity against bacteria from families of importance in antibiotic resistance, such as ESKAPE safe relatives (
Enterococcus raffinosus
,
Staphylococcus epidermidis
,
Klebsiella aerogenes
,
Acinetobacter baylyi
,
Pseudomonas putida
, and
Escherichia coli
). LL35lys, Nb26lys, and Tb42lys exhibit PG-degrading activity on zymography and hydrolysis assay. The enzymes (100 µg/mL) can reduce PG turbidity to 32%–40%. The killing assay suggests that Tb42lys has a broader range (
E. coli
,
P. putida
,
A. baylyi
and
K. aerogenes
). While Nb26lys better attacks Gram-negative than -positive bacteria, LL35lys can only reduce the growth of the Gram-positive ESKAPE strains but does so effectively with a low MIC
90
of 2 µg/mL. A higher concentration (≥300 µg/mL) of Nb26lys is needed to inhibit
P. putida
and
K. aerogenes
. From 250 putative endolysins, bioinformatic methods were used to select three putative endolysins for cloning and study: LL35lys, Nb26lys, and Tb42lys. All have shown PG-degrading activity, a critical function of endolysin. With a low MIC, LL35lys shows activity for the Gram-positive ESKAPE strains, while Nb26lys and Tb42lys are active against the Gram negatives. Therefore, endolysins from
Streptomyces
phages have potential as possible antimicrobial agents against ESKAPE bacteria.

IMPORTANCE

As antibiotic resistance has become a major global threat, the World Health Organization (WHO) has urgently called for alternative strategies for control of bacterial infections. Endolysin, a phage-encoded protein, can degrade bacterial peptidoglycan in the bacterial cell wall and disrupt bacterial growth. According to the WHO, there are only three endolysin products currently in clinical phase development. In this study we explored novel endolysins from
Streptomyces
phages as only a few of them have been experimentally characterized. Using several bioinformatics tools, we identified nine different combinations of functional enzymatic domain types from 250
Streptomyces
bacteriophages possible endolysins. From these, three potential endolysins were selected for experimental characterization. All three showed positive results in degrading cell wall material and disrupting bacterial growth, indicating their potential as possible antimicrobial agents.