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
Department of Biological Sciences, University of North Texas, Denton, Texas, USA.
Microbiol Spectr. 2025 Jan 7;13(1):e0117024. doi: 10.1128/spectrum.01170-24. Epub 2024 Nov 21.
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 phages as only a few of them have been experimentally characterized. Using several bioinformatics tools, we identified nine different functional domain combinations from 250 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 (, , , , , and ). 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 (, , and ). 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 of 2 µg/mL. A higher concentration (≥300 µg/mL) of Nb26lys is needed to inhibit and . 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 phages have potential as possible antimicrobial agents against ESKAPE bacteria.
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 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 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.
由于抗生素耐药性已成为全球主要威胁，世界卫生组织（WHO）紧急呼吁采取替代策略来控制细菌感染。内溶素是一种噬菌体编码蛋白，可降解细菌肽聚糖（PG）并破坏细菌生长。据WHO称，目前仅有三种内溶素产品处于临床阶段开发。在本研究中，由于只有少数噬菌体来源的内溶素经过实验表征，我们探索了新型内溶素。使用多种生物信息学工具，我们从250种噬菌体假定内溶素中鉴定出九种不同的功能域组合。选择了LazerLemon gp35（CHAP；LL35lys）、Nabi gp26（酰胺酶；Nb26lys）和Tribute gp42（PGRP/酰胺酶；Tb42lys）进行实验研究。我们假设：（i）感兴趣的蛋白质将具有降解纯化PG的能力，并且（ii）这些蛋白质将对来自抗生素耐药性方面重要菌属的细菌具有潜在抗菌活性，例如ESKAPE安全相关菌属（粪肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和肠杆菌属）。LL35lys、Nb26lys和Tb42lys在酶谱分析和水解试验中表现出PG降解活性。这些酶（100μg/mL）可将PG浊度降低至32%-40%。杀菌试验表明Tb42lys具有更广泛的抗菌范围（粪肠球菌、金黄色葡萄球菌、肺炎克雷伯菌和鲍曼不动杆菌）。虽然Nb26lys对革兰氏阴性菌的攻击比对革兰氏阳性菌更好，但LL35lys只能降低革兰氏阳性ESKAPE菌株的生长，但其在2μg/mL的低最低抑菌浓度（MIC）下能有效发挥作用。需要更高浓度（≥300μg/mL）的Nb26lys才能抑制肺炎克雷伯菌和鲍曼不动杆菌。从250种假定内溶素中，使用生物信息学方法选择了三种假定内溶素进行克隆和研究：LL35lys、Nb26lys和Tb42lys。所有这些都显示出PG降解活性，这是内溶素的关键功能。LL35lys在低MIC下对革兰氏阳性ESKAPE菌株显示出活性，而Nb26lys和Tb42lys对革兰氏阴性菌有活性。因此，噬菌体来源的内溶素具有作为针对ESKAPE细菌的潜在抗菌剂的潜力。
由于抗生素耐药性已成为全球主要威胁，世界卫生组织（WHO）紧急呼吁采取替代策略来控制细菌感染。内溶素是一种噬菌体编码蛋白，可降解细菌细胞壁中的肽聚糖并破坏细菌生长。据WHO称，目前仅有三种内溶素产品处于临床阶段开发。在本研究中，由于只有少数噬菌体来源的内溶素经过实验表征，我们探索了新型内溶素。使用多种生物信息学工具，我们从250种噬菌体可能的内溶素中鉴定出九种不同的功能酶结构域类型组合。从中选择了三种潜在内溶素进行实验表征。所有三种在降解细胞壁物质和破坏细菌生长方面均显示出阳性结果，表明它们作为潜在抗菌剂的潜力。
