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Development of a Bifidobacterium-based Delivery System for Endolysin PHICD111_20024_EAD Targeting Clostridioides difficile

  • 안지은 (An Jieun, 아주대학교 일반대학원)

초록/요약

Antibiotic overuse or misusing can disrupt the gut microbiota, which stimulates an opportunistic pathogen Clostridioides difficile to be dominant and cause C. difficile infection (CDI). Paradoxically, the standard treatment for CDI is antibiotics. Thus, alternative therapies are urgently needed. Phage-derived endolysin is peptidoglycan-degrading enzyme. It has high specificity and low resistance potential. However, endolysins as proteinaceous molecules are vulnerable in the gastrointestinal tract, which is a critical drawback for orally administered drugs. Therefore, an effective delivery system is required. This study explored the potential of Escherichia coli Nissle 1917 (EcN) and Bifidobacterium bifidum BGN4 as a delivery vehicle for an engineered endolysin PHICD111_20024_EAD. Previous studies showed that PHICD111_20024_EAD retained its specificity and catalytic activity under high-salt conditions and complex media, which represent the gut lumen environment. PHICD111_20024_EAD was fused with diverse secretion signal peptides for the vehicle strains to secrete the endolysin in the gut. When EcN was employed as a vehicle, the signal peptide of OmpA was successfully fused with PHICD111_20024_EAD and expressed in the cytosol. However, EcN showed a drastic growth defect, which was probably due to the lytic activity of PHICD111_20024_EAD during its secretion through the peptidoglycan. Instead, a signal peptide S6 was added to the N-terminus of PHICD111_20024_EAD, cloned into an E. coli–Bifidobacterium shuttle vector, pBESGS2, under a constitutive promoter Pgap, and introduced into B. bifidum BGN4. Bifidobacterium producing PHICD111_20024_EAD exhibited clear halos on agar plates containing autoclaved or live C. difficile cells and the spent medium also inhibited the growth of C. difficile, indicating successful secretion of PHICD111_20024_EAD by B. bifidum BGN4. These results demonstrate that B. bifidum BGN4 can serve as an effective delivery platform for endolysins. Key words : CDI, Endolysin, E. coli Nissle 1917, B. bifidum BGN4, delivery carrier

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목차

1. Introduction
2. Materials and Methods
2.1 Bacterial growth conditions
2.2 Construction and Cloning of SPOmpA-PHICD111_20024_EAD & SPLamB-PHICD111_20024_EAD
2.3 Expression and purification of endolysin
2.4 SDS-PAGE & Western blot
2.5 Turbidity assay
2.6 Autoclaved Cell Digestion Plate Assay
2.7 RT-qPCR
2.8 Spent medium viability Test
2.9 Zone of inhibition assay
2.10 Statistical analysis
3. Results
3.1 Expression and secretion of SPOmpA-PHICD111_20024_EAD in E. coli TOP 10
3.2 Expression and secretion of SPOmpA-PHICD111_20024_EAD in EcN
3.3 Expression of SPS6- PHICD111_20024_EAD in B. bifidum BGN 4
3.4 Expression and secretion of SPS6- PHICD111_20024_EAD in B. bifidum BGN 4
3.5 Controlling C. difficile using B. bifidum BGN4 secreting SPS6- PHICD111_20024_EAD
4. Discussion
5. Conclusion
6. Reference

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