Design, Overproduction and Purification of the Chimeric Phage Lysin MLTphg Fighting against Staphylococcus aureus

Feng Wang,Yao Zhang,Yan Xiong,Lianbing Lin,Xinyu Ji,Zhengyu Deng,Xiaohang Liu

doi:10.3390/pr8121587

Abstract

With the increasing spread of multidrug-resistant bacterial pathogens, it is of great importance to develop alternatives to conventional antibiotics. Here, we report the generation of a chimeric phage lysin, MLTphg, which was assembled by joining the lysins derived from Meiothermus bacteriophage MMP7 and Thermus bacteriophage TSP4 with a flexible linker via chimeolysin engineering. As a potential antimicrobial agent, MLTphg can be obtained by overproduction in Escherichia coli BL21(DE3) cells and the following Ni-affinity chromatography. Finally, we recovered about 40 ± 1.9 mg of MLTphg from 1 L of the host E. coli BL21(DE3) culture. The purified MLTphg showed peak activity against Staphylococcus aureus ATCC6538 between 35 and 40 °C, and maintained approximately 44.5 ± 2.1% activity at room temperature (25 °C). Moreover, as a produced chimera, it exhibited considerably improved bactericidal activity against Staphylococcus aureus (2.9 ± 0.1 log10 reduction was observed upon 40 nM MLTphg treatment at 37 °C for 30 min) and also a group of antibiotic-resistant bacteria compared to its parental lysins, TSPphg and MMPphg. In the current age of growing antibiotic resistance, our results provide an engineering basis for developing phage lysins as novel antimicrobial agents and shed light on bacteriophage-based strategies to tackle bacterial infections.

Highlights

As a potential antibacterial agent permeating the entire biosphere and existing in substantial numbers, bacterial viruses, or bacteriophages, were estimated to be 1031 particles on the planet, ten times greater than their bacterial counterparts [1]
It has been reported that using the chimeolysin bioengineering to combine lysins of heterologous origins through some designed protein domain linkers could remarkably contribute to the functional enhancement of effectiveness and practicability of chimeric proteins [11,25,26]
Ppahriaptaytphhici pBe,pStiadlmeso,ndeollma saeinr.sEhnutffleriintigd,issi,teS-adlmireocnteeldla mseur.tagTeynpehsiis aanndd aKcloebmsibeilnlaatiponneuomf eonnidaoelyasnindfuGnrcatimon-paol sdiotimveainstsroaifnheotefroSlotagpohuysloocoricgcuinss aaruerecuasp, abthlee ocfopnsroiddeurcaibnlge cehnihmaenocleymsiennstwbiythaitmleparsotvoendeplroogpekritlileinsg[5f,o6,r29M,3L0T].pFhogr leyxsaimn pacleti,vCitsyl2w, aacshoimbseerrivceldytiicn enzyme resulting from combining the catalytic domain (EAD) of Cpl-7 endolysin of pneumococcal phage Cp-7 with the cell binding domain (CBD) of LySMP endolysin of Streptococcus suis phage SMP, exhibited better antibacterial and antibiofilm activity than its parental LysSMP [8]

Summary

Introduction

As a potential antibacterial agent permeating the entire biosphere and existing in substantial numbers, bacterial viruses, or bacteriophages, were estimated to be 1031 particles on the planet, ten times greater than their bacterial counterparts [1]. A phage technology revolution includes phage lytic enzymes (phage lysins), which are usually bacterial cell wall hydrolytic enzymes encoded by the double-stranded DNA phages to rupture susceptible bacteria and release progeny virions They are able to cleave to the peptidoglycan bonds of Gram-positive. Lines of reports have highlighted that the upgraded features and improved properties of phage lysins can be achieved by protein modification and engineering of new derivatives [3], and there are many different modification or engineering strategies, including site-directed mutagenesis, swapping or exchange of functional domains, and a combination of full-length lytic enzymes [4,5,6], which have been utilized to improve specific enzybiotic features of phage lysins Among these engineering approaches, fusion of phage lytic enzymes of heterologous origins have been investigated and the numerous examples of chimeolysins with enhanced characteristics were produced. They showed great improvements in comparison to their parental lysins or to other lytic enzymes [7,8,9]

Methods

Results

Conclusion