초록 ▼

Ⅳ. 연구개발결과
○ 박테리오신 생산균주의 선발 및 박테리오신 상등액 제조법 확립
○ 유방염 원인세균-C. elegans liquid assay 조건 확립
○ 96-well plate를 이용한 초고속 스크리닝 최적화 기술 확립 완료
○ 확립된 초고속 스크리닝 방법을 이용하여 전통식품 및 인체유래의 박테리오신 생산균주를 대상으로 초고속 항균 스크리닝 완료 (최종 3종의 박테리오신 선발)
○ RNA-seq 기술을 이용한 항균 박테리오신의 활성기작 규명
○ 3종의 박테리오신 복합화 조건과 복합박테리오신 특

Ⅳ. 연구개발결과
○ 박테리오신 생산균주의 선발 및 박테리오신 상등액 제조법 확립
○ 유방염 원인세균-C. elegans liquid assay 조건 확립
○ 96-well plate를 이용한 초고속 스크리닝 최적화 기술 확립 완료
○ 확립된 초고속 스크리닝 방법을 이용하여 전통식품 및 인체유래의 박테리오신 생산균주를 대상으로 초고속 항균 스크리닝 완료 (최종 3종의 박테리오신 선발)
○ RNA-seq 기술을 이용한 항균 박테리오신의 활성기작 규명
○ 3종의 박테리오신 복합화 조건과 복합박테리오신 특성 조사
○ 복합박테리오신의 최적 혼합비율 설정 완료
○ 건식분체화 기술을 통한 항균 스펙트럼 최적화 검증 및 현장적용 기술 개발 완료

Abstract ▼

This study was carried out to develop a high-throughput screening (HTS) technology for a novel bacteriocin to control the representative mastitis-causing pathogen in dairy cows, Staphylococcus aureus RF122 using Caenorhabditis elegans in vivo model. Initially, we found that nineteen among sixty-eigh

This study was carried out to develop a high-throughput screening (HTS) technology for a novel bacteriocin to control the representative mastitis-causing pathogen in dairy cows, Staphylococcus aureus RF122 using Caenorhabditis elegans in vivo model. Initially, we found that nineteen among sixty-eight Bacillus licheniformis strains isolated from Korean traditional food resources in the Institute of Sunchang Fermented Soybean Products (Sunchang, Republic of Korea) produced antimicrobial bacteriocins against to pathogen, S. aureus RF122 in vitro by agar well diffusion assay. Next we explored if bacteriocins which had were previously screened on solid agar plates where the anti-staphylococcal C. elegans feeds had selected by agar well diffusion method on a lawn of S. aureus effective in vivo solid assay using the C. elegans-S. aureus infection model. However, agar-based C. elegans in vivo assay was not suitable for large-scale screens for antimicrobial compounds. To overcome such these disadvantages, we had set the liquid assay condition such as the number of dispensed worms in well, the concentration of bacteriocins and media, and the time of exposure to bacteriocin using a 6-well plate and C. elegans before applied the liquid assay using 96-well plate and washing and dispensing machine. As a result of using the liquid assay method on 6-well plate we had set, we were able to screen eight strains among nineteen strains previously found. And then, we tried to have developed a high throughput liquid screening assay that using washing and dispensing instrument to dispense worms to 96-well assay plates and to wash them etc. The assay plates were imaged with an inverted microscope, capturing both transmitted light and fluorescent images with a 2X objective because the use of a 2X objective allows capturing the area of an entirely each well within one image. As using this HTS assay method, we screened strain SRCM 146, the producer of the most enhanced the lifespan of C. elegans. Finally, in validation of the liquid assay in 96-well plate, we were able to rapidly and cost-effectively find three of eleven Bacillus licheniformis strains producing strong bacteriocin by an experiment on C. elegans in vivo model. In further studies, through RNA-seq analyzing of S. aureus RF122 treated and untreated with screened bacteriocin from B. licheniformis SRCM 146 which is the most antimicrobial activity against S. aureus in HTS condition finally selected, we found various genes are transcriptionally or post-transcriptionally regulated by treating with bacteriocin SRCM 146. Moreover, we have founded the bacteriocin whose molecular mass is less than 17.5 kDa on tris SDS-PAGE and which detected protein whose molecular weight was 14050 Da and sequence coverage was 40% (Searched peptide number was 126, Matched peptide number was 51) by HPLC/MS. Using BLAST similarity search, no match (Bacteriocin family protein) with so far described proteins from NCBI database was found. Considering these findings, we named the newly characterized bacteriocin as bacteriocin 146.
Taken in their totality, the liquid C. elegans–pathogen infection model assay described here allows simple, rapid, cost-effective, and reliable screening for potential antimicrobial compounds against pathogen to target.