Effect of Domain Manipulation in the Staphylococcal Phage Endolysin, Endo88, on Lytic Efficiency and Host Range.

IF 2.4 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Molecular Biotechnology Pub Date : 2025-06-01 Epub Date: 2024-06-21 DOI:10.1007/s12033-024-01216-4

Melvina Krishnan, Hong Yun Tham, Wan Ahmad Kamil Wan Nur Ismah, Khatijah Yusoff, Adelene Ai-Lian Song

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Abstract

The bacteriophage endolysin Endo88 targeting Staphylococcus aureus PS88 consists of the CHAP and Amidase-2 enzymatic domains and one SH3b targeting domain. In this study, the effects of domain manipulations on Endo88 functionality were determined. Three truncated mutants of Endo88 (CHAP, CHAPAmidase and CHAPSH3) and two chimeras (CHAPAmidase-Cpl7Cpl7 and Endo88-Cpl7Cpl7) containing the Cpl7Cpl7 targeting domains of the streptococcal LambdaSa2-ECC endolysin were cloned in E. coli (pET28a), expressed, and then purified. Lytic efficiency and host range were assessed through plate lysis assays and turbidity reduction assays. Endo88 required all domains for maximum functionality, with activity detected against Staphylococcus aureus PS88 (host strain), S. aureus Mu50 (VISA), CoNS (Staphylococcus epidermidis and Staphylococcus hominis), and Enterococcus faecalis. The truncated constructs maintained the original host range but with reduced lytic efficiency. The Amidase-2 and SH3b domains are interdependent in maximizing functionality. The chimera constructs demonstrated reduced functionality, without activity against Streptococcus agalactiae in both assays. This study provides insights into domain function in a staphylococcal endolysin, which could enable the development of prospective engineered antimicrobials against multidrug-resistant pathogens.

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葡萄球菌噬菌体内溶酶 Endo88 的结构域操作对溶解效率和宿主范围的影响

针对金黄色葡萄球菌 PS88 的噬菌体内溶素 Endo88 由 CHAP 和 Amidase-2 酶结构域以及一个 SH3b 靶向结构域组成。本研究确定了结构域操作对 Endo88 功能的影响。将 Endo88 的三个截短突变体（CHAP、CHAPAmidase 和 CHAPSH3）和两个嵌合体（CHAPAmidase-Cpl7Cpl7 和 Endo88-Cpl7Cpl7）克隆到大肠杆菌（pET28a）中，表达并纯化。通过平板裂解试验和浊度降低试验评估了溶解效率和宿主范围。Endo88 需要所有结构域才能发挥最大功能，对金黄色葡萄球菌 PS88（宿主菌株）、金黄色葡萄球菌 Mu50（VISA）、CoNS（表皮葡萄球菌和人葡萄球菌）和粪肠球菌都有活性。截短的构建体保持了原有的宿主范围，但溶解效率有所降低。淀粉酶-2和SH3b结构域在最大限度地发挥功能方面相互依存。嵌合构建体的功能减弱，在两种试验中都没有针对无乳链球菌的活性。这项研究深入揭示了葡萄球菌内溶解素的结构域功能，有助于开发针对耐多药病原体的前瞻性工程抗菌药物。

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来源期刊

Molecular Biotechnology 医学-生化与分子生物学

CiteScore

4.10

自引率

3.80%

发文量

165

审稿时长

6 months

期刊介绍： Molecular Biotechnology publishes original research papers on the application of molecular biology to both basic and applied research in the field of biotechnology. Particular areas of interest include the following: stability and expression of cloned gene products, cell transformation, gene cloning systems and the production of recombinant proteins, protein purification and analysis, transgenic species, developmental biology, mutation analysis, the applications of DNA fingerprinting, RNA interference, and PCR technology, microarray technology, proteomics, mass spectrometry, bioinformatics, plant molecular biology, microbial genetics, gene probes and the diagnosis of disease, pharmaceutical and health care products, therapeutic agents, vaccines, gene targeting, gene therapy, stem cell technology and tissue engineering, antisense technology, protein engineering and enzyme technology, monoclonal antibodies, glycobiology and glycomics, and agricultural biotechnology.