황색포도상구균에 대한 할로겐화 유도체의 항생물막 및 항균 활성

다국어 초록 (Multilingual Abstract)

Staphylococcus aureus remains a clinically important pathogen that is challenging to eradicate due to its capacity for antibiotic resistance and robust biofilm formation, which frequently contributes to chronic and recurrent infections. In this study, the antimicrobial, antibiofilm, and antivirulence properties of halogenated pyrimidines and multi-halogenated indoles were systematically investigated against multiple S. aureus strains, including both methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) isolates.
In the first part of the study, a library of 32 halogenated pyrimidines was screened, leading to the identification of three compounds with pronounced antibiofilm activity: 2,4-dichloro-5-fluoropyrimidine (24DC5FP), 5-bromo-2,4-dichloro-pyrrolo[2,3-d]pyrimidine (24DC5BPP), and 2,4-dichloro-5-iodo-pyrrolo[2,3-d]pyrimidine (24DC5IPP). These compounds inhibited biofilm formation in a concentration-dependent manner while exhibiting bacteriostatic effects. Notably, treatment with 24DC5FP resulted in enlarged cell morphology and wrinkled colony phenotypes. In addition, 24DC5FP markedly reduced hemolytic activity at sub-MIC levels and downregulated the quorum-sensing regulator agrA as well as the virulence-associated genes hla and nuc1, indicating that extensive halogen substitution may contribute to enhanced antibiofilm and antivirulence properties in pyrimidine derivatives.
In the second part, 45 multi-halogenated indoles were evaluated, among which 6-bromo-4-iodoindole and 4-bromo-6-chloroindole showed strong bactericidal activity with MIC values of 20–30 µg/mL, comparable to gentamicin. These compounds effectively suppressed biofilm formation, reduced persister cell populations, and attenuated hemolysis. These effects were accompanied by increased intracellular reactive oxygen species (ROS) levels and reduced expression of agrA, RNAIII, hla, and nuc1. Furthermore, 6-bromo-4-iodoindole displayed pronounced synergistic interactions with the aminoglycosides tobramycin and gentamicin, resulting in substantial reductions in antibiotic MICs. In a 20-day serial passaging assay, gentamicin rapidly selected for resistant populations, whereas resistance development was not observed with the multi-halogenated indoles. Cytotoxicity assays using HepG2 cells and phytotoxicity tests indicated low toxicity. Structure–activity relationship analysis further suggested that the introduction of multiple halogens at the C4–C7 positions of the indole scaffold plays a critical role in enhancing antimicrobial potency.
Taken together, these findings indicate that halogenated pyrimidines and multi-halogenated indoles exert antimicrobial and antibiofilm effects through multiple complementary mechanisms, including inhibition of biofilm formation, suppression of virulence-associated pathways, induction of oxidative stress, and potentiation of conventional antibiotics. This multifunctional profile supports their further investigation as chemical scaffolds for addressing S. aureus infections associated with antibiotic resistance and biofilm formation.

목차 (Table of Contents)

• 1.1. 서론 1
• 1.2. 실험 재료 및 방법 3
• 1.2.1. 박테리아 균주, 배양 조건 및 시약 3
• 1.2.2. 크리스탈 바이올렛을 이용한 생물막 정량 분석 3
• 1.2.3. 세포 성장 및 생존율(CFU) 분석 4
• 1.2.4. 2D·3D 및 주사전자현미경(SEM)을 통한 생물막 관찰 5
• 1.2.5. 슬라임(Slime) 생성 분석 5
• 1.2.6. 콜로니(Colony) 형태 분석 6
• 1.2.7 황색 색소(Staphyloxanthin) 생산 분석 6
• 1.2.8 용혈(Hemolysis) 분석 7
• 1.2.9 RNA 추출 및 qRT-PCR을 이용한 유전자 발현 분석 7
• 1.2.10 통계적 분석 8
• 1.3. 결과 9
• 1.3.1. 다양한 할로겐화 피리미딘 유도체의 항균 및 항생물막 효과 9
• 1.3.2. 활성 물질들의 농도 의존적 생물막 억제 효과 13
• 1.3.3. 활성 피리미딘 유도체가 황색포도상구균의 표현형(Phenotype)에 미치는 영향 15
• 1.3.4. 24DC5FP가 황색포도상구균의 유전자 발현에 미치는 영향 18
• 1.4. 논의 20
• 1.5. 결론 24
• 1.6. 참고문헌 25
• 2.1. 서론 36
• 2.2. 실험 재료 및 방법 38
• 2.2.1. 박테리아 균주, 배양 조건 및 시약 38
• 2.2.2. 크리스탈 바이올렛을 이용한 생물막 정량 분석 39
• 2.2.3. 최소 저해 농도 (MIC) 분석 40
• 2.2.4. 세포 성장 및 생존율(CFU) 분석 41
• 2.2.5.2D·3D 및 주사전자현미경(SEM)을 통한 생물막 관찰 41
• 2.2.6. 퍼시스터(Persister) 세포 분석 42
• 2.2.7. 용혈(Hemolysis) 분석 43
• 2.2.8. 활성산소종 (ROS) 분석 44
• 2.2.9. 항생제 내성 발현 분석 45
• 2.2.10. RNA 추출 및 qRT-PCR을 이용한 유전자 발현 분석 45
• 2.2.11. 항생제와 시너지(synergy) 분석 46
• 2.2.12. 간암 세포 및 식물 모델을 이용한 세포독성 분석 47
• 2.2.13. 3D-QSAR 모델링 및 흡수, 분포, 대사, 및 배설 특성 평가(ADME) 48
• 2.2.14. 통계적 분석 59
• 2.3. 결과 50
• 2.3.1. 다중 할로겐화 인돌의 항생물막 및 항균 효과 50
• 2.3.2. 생물막 억제에 대한 현미경 관찰 결과 59
• 2.3.3. 퍼시스터(persister) 세포 억제 효과 60
• 2.3.4. 병원성 인자 억제, 작용기전 및 약물 내성 분석 62
• 2.3.5. 아미노글리코사이드계 항생제와의 시너지 효과 64
• 2.3.6. HepG2 세포 및 식물 모델을 이용한 독성 평가 66
• 2.3.7. 할로겐화 인돌의 구조-활성 관계 분석 71
• 2.4. 논의 74
• 2.5. 결론 80
• 2.6. 참고문헌 81
• Publications 90
• Award 92
• Conferences 92
• Abstract 93
• 부록 97