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Characterization of PHICD111_20024_EAD as a novel endolysin against Clostridioides difficile

  • 박정석 (아주대학교 일반대학원)

초록/요약

The disruption of the gut microbiome by antibiotics often facilitates the proliferation of Clostridioides difficile, leading to C. difficile infection (CDI). Given the limitations of antibiotics in treating CDI, recent studies have suggested endolysin-based therapies as a promising alternative, due to their specificity in targeting C. difficile without harming beneficial gut microbiota. Based on their strong inhibitory effects against C. difficile, CD27L and PHICD111_20024 were selected and tested under various in vitro conditions to evaluate their antibacterial efficacy. We also evaluated truncated versions, CD27L_EAD and PHICD111_20024_EAD, which lack the cell wall-binding domain, to determine if this modification would enhance lytic activity or stability. These truncated endolysins displayed enhanced lytic activity compared to their full-length forms. Interestingly, despite their high sequence similarity, PHICD111_20024_EAD exhibited greater stability than CD27L_EAD under high-salt and broth conditions, suggesting its potential suitability for use in complex environments like the gut. Structural analysis showed that both truncated endolysins have similar Zn-binding sites. However, the activity of PHICD111_20024_EAD was more vulnerable to EDTA, implying differences in Zn-binding strength, which could impact its catalytic efficiency. In an in vitro model incorporating fecal microbiota, PHICD111_20024_EAD demonstrated superior inhibitory effects against C. difficile, indicating its potential effectiveness in environments resembling the human gut. These findings suggest that PHICD111_20024_EAD is a promising candidate for CDI treatment. Future research will focus on evaluating its efficacy in animal models and optimizing its formulation for potential therapeutic use.

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목차

1. Introduction 1
2. Materials and Methods 4
2.1. Bacterial growth conditions 4
2.2. Expression and purification of endolysins 5
2.3. Bacterial viability test 5
2.4. turbidity reduction test 6
2.5. Growth inhibition assay 6
2.6. Minimum Inhibitiory Concentration (MIC) measurement 6
2.7. Fecal microbiota 7
2.8. Quantitative PCR (qPCR) 7
2.9. Crystal structure analysis 8
2.10. Zinc chelation assay 9
2.11. Statistical analysis 9
Table 1. Primers used in this study 10
3. Results 11
3.1. PHICD111_20024_EAD shows high lytic activity as similar sequence with CD27L_EAD 11
3.2. PHICD111_20024_EAD retains high lytic activity in complex medium conditions 15
3.3. PHICD111_20024_EAD exerts high specificity in fecal microbiota 22
3.4. Structural analysis of PHICD111_20024_EAD and CD27L_EAD 25
3.5. Zn2+ shows double-edged role in lytic activity of PHICD111_20024_EAD 28
4. Discussion 32
5. Conclusion 35
6. Reference 36

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