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Construction of Hybrid endolysins Using PHICD111_20024_EAD and LysB4_EAD and Their Application

  • 유시영 (Si-young Yoo, 아주대학교 일반대학원)

초록/요약

Clostridioides difficile is a Gram-positive spore-forming pathogen and the causative agent of C. difficile infection (CDI). With the emergence of antibiotic-resistant strains, this C. difficile has become recognized as one of the major causative agents of hospital-acquired infections (HAIs), creating an urgent need to develop new therapeutics that can serve as alternatives to conventional antibiotics. Bacillus cereus is a Gram-positive foodborne pathogen, which forms spores and biofilms, leading to high resistance to antibiotics and disinfectants. Therefore, B. cereus is a major concern in food- processing industries. Endolysins, derived from bacteriophages, have emerged as a promising alternative to conventional antibiotics, because of their ability to degrade bacterial peptidoglycan with high specificity and low resistance potential. Taking advantage of these beneficial properties of endolysins, we aimed to develop endolysins capable of lysing both pathogens that are difficult to control with conventional antibiotics. We then developed hybrid endolysins by fusing an endolysin that lyses C. difficile with one that lyses B. cereus. We first selected CD27L_EAD and PHICD111_20024_EAD, two endolysins targeting C. difficile that had been characterized in earlier studies. Both enzymes exhibit amidase activity and are derived from the C. difficile phages phiCD27 and phiCD111, respectively, and they showed strong lytic activity against C. difficile. Next, we chose LysB4_EAD, an endopeptidase derived from the B. cereus phage B4, as the enzyme targeting B. cereus. Using these three parental enzymes, we then designed four hybrid endolysins by fusing LysB4_EAD to either the N- or C-terminus of CD27L_EAD and PHICD111_20024_EAD.And four hybrid endolysins showed superior bactericidal activity against both C. difficile and B. cereus to their cognate parent endolysins under buffer conditions. Notably, a hybrid endolysin, L4-GS-C111, maintained lytic activity under a wide range of pH and salt concentrations. However, L4-GS-C111 showed no inhibitory effect on C. difficile in BHI medium, likely because its instability in complex environments results in a loss of lytic activity. This limitation may represent a major drawback to its use as a therapeutic agent for CDI. Therefore, given its stability across a wide pH range, we instead explored whether L4-GS-C111 could be applied in complex food matrices with variable pH conditions. And L4-GS-C111 showed concentration-dependent bacteriolytic activity against B. cereus in soy milk. Furthermore, biofilms of B. cereus were effectively disrupted by L4-GS-C111. These results demonstrate the potential of L4-GS-C111 as a novel bactericidal agent against B. cereus to enhance food safety.

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목차

1. Introduction 1
2. Materials and Methods 4
2.1 Bacterial strains and growth conditions 4
2.2 Hybrid endolysins cloning 6
2.3 Hybrid endolysins expression and purification 8
2.4 Turbidity reduction assay 8
2.5 Growth inhibition test 8
2.6 Antibacterial activity assay in soy milk 9
2.7 Biofilm reduction assay 9
2.8 Confocal laser scanning microscopy of biofilms 9
2.9 Cytotoxicity assay 10
2.10 Statistical analysis 10
3. Results 11
3.1 Construction and expression of hybrid endolysins 11
3.2 Characterization of hybrid endolysins 16
3.3 Evaluation of L4-GS-C111 for treating CDI 27
3.4 Evaluation of L4-GS-C111 for food industry applications 29
4. Discussion 35
5. Conclusion 39
6. Reference 40

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