Regulation of an efflux pump TolCV1 in Vibrio vulnificus = Vibrio vulnificus에서 efflux pump TolCV1의 조절에 관한 연구

다국어 초록 (Multilingual Abstract)

Vibrio vulnificus is a pathogenic marine bacterium posing a serious threat to human health, capable of causing severe sepsis and wound infections. With global warming and rising sea temperatures, the incidence and geographic distribution of V. vulnificus infections continue to expand worldwide. Bacterial efflux pumps play a crucial role in antibiotic resistance and the secretion of virulence factors. TolC is an outer membrane channel protein that interacts with inner membrane transporters to form part of a tripartite efflux pump, promoting the export of multiple substrates. TolCV1, a homolog of TolC in V. vulnificus, is essential for antibiotic resistance, RtxA1 secretion, cytotoxicity to host cells, bile salt resistance, and lethality in mice. In this study, the contribution of the ToxRS regulatory system was examined on TolCV1 secretion and stress adaptation. ToxR is a membrane-associated transcription regulator that, together with ToxS, senses environmental signals such as bile salts, pH, and membrane stress to control the expression of virulence genes, including those encoding outer membrane proteins. In-frame deletion mutants of toxRS were constructed in V. vulnificus MO6-24/O by homologous recombination. Western blot analysis revealed that secretion of TolCV1 was markedly reduced in a toxRS mutant compared with the wild-type, without affecting tolCV1 transcriptional activity. It was confirmed that TolCV1 protein was secreted via outer membrane vesicles (OMVs). The toxRS mutant, similar to the tolCV1 mutant, exhibited impaired growth in the presence of ampicillin (1μg/mL). Moreover, bile salt enhanced TolCV1 expression and secretion in the wild-type, whereas this induction was almost completely abolished by toxRS deletion. Checkerboard assays further demonstrated the synergistic antibacterial activity of ampicillin with the TolCV1 efflux pump inhibitors, phenylalanine-arginine-β-naphthamide (PAβN). In addition, the deletion of toxRS showed reduced cytotoxicity of V. vulnificus in HeLa cells. Collectively, these findings indicate that ToxRS regulates TolCV1 secretion, thereby contributing to antibiotic resistance, bile salt resistance, and host cell cytotoxicity in V. vulnificus.

국문 초록 (Abstract)

Vibrio vulnificus는 패혈증과 상처 감염을 일으킬 수 있는 인체 건강에 심각한 위협을 주는 병원성 해양 박테리아이다. 지구 온난화와 해수 온도 상승으로 인해 V. vulnificus 감염의 발생률과 지리적 분포는 전 세계적으로 계속 확대되고 있다. 박테리아 유출 펌프는 항생제 내성과 독성 인자 분비에 중요한 역할을 한다. TolC는 내막 수송체와 상호 작용하여 tripartite efflux pump의 일부를 형성하여 여러 기질의 유출을 촉진하는 외막 채널 단백질이다. TolC의 V. vulnificus 상동체인 TolCV1은 항생제 내성, RtxA1 분비, 숙주 세포에 대한 세포 독성, 담즙염 저항성 및 쥐의 치사율에 필수적이다. 이 연구에서는 TolCV1 분비 및 스트레스 적응에 대한 ToxRS 조절 시스템의 역할을 연구했다. ToxR은 ToxS와 함께 담즙염, pH, 막 스트레스와 같은 환경 신호를 감지하여 외막 단백질을 암호화하는 것을 포함한 독성 유전자의 발현을 제어하는 막 관련 전사 조절자이다. 먼저, V. vulnificus MO6-24/O에 서 ToxRS의 in-frame 결손 mutant를 제작하였다. 웨스턴 블롯 분석 결과, 야생형에 비해 toxRS mutant에서 TolCV1의 분비가 현저히 감소되었다. 하지만, toxRS mutant 는TolCV1 전사 활성에 영향을 미치지 않는 것으로 나타났다. 또한 TolCV1 단백질은 외막 소포(OMV)를 통해 분비되는 것으로 확인되었다. toxRS mutant는 tolCV1 mutant와 유사하게 암피실린(1 μg/mL)의 존재 하에서 성장이 저해되었다. 또한, 담즙염은 야생형에서 TolCV1 발현과 분비를 증가시켰지만, toxRS mutant에서는 증가되지 않았다. 체커보드 분석 결과 암피실린이 TolCV1 유출 펌프 억제제인 페닐알라닌-아르기닌-β-나프타마이드(PaβN)와 함께 사용되었을때 항균 활성이 상승되었다. 또한, toxRS mutant는 HeLa 세포에서 V. vulnificus의 세포 독성을 감소시키는 것으로 나타났다. 종합적으로, 이러한 발견은 ToxRS가 TolCV1 분비를 조절하여 V. vulnificus에서 항생제 내성, 담즙염 내성 및 숙주 세포 세포 독성에 기여함을 나타낸다.
키워드: Vibrio vulnificus, TolCV1, ToxRS, OMVs, 담즙염 저항성, 암피실린 저항성, 세포 독성

목차 (Table of Contents)

• Abstract 1
• Introduction 3
• 1. Vibrio vulnificus 3
• 2. Efflux pumps 7
• 3. Outer membrane protein TolC 11
• 4. ToxRS regulatory system 21
• Materials and methods 33
• 1. Bacterial Strains, plasmids, and reagents 33
• 2. Cell cultures and reagents 35
• 3. DNA manipulation 35
• 4. toxRS mutations construction and complementation 36
• 5. Western blotting 40
• 6. Purification of V. vulnificus strains' outer membrane vesicles (OMVs) 41
• 7. The effect of toxRS mutation on TolCV1 secretion and expression in the presence of
• ampicillin 41
• 8. The effect of toxRS mutation with bile salt on TolCV1 secretion and expression 42
• 9. Growth determination of toxRS mutation on LB agar plates 42
• 10. Assessment of the growth rate of toxRS mutant strains in the presence of
• ampicillin 43
• 11. Assessment of the growth rates of toxRS mutation in the presence of dose-dependent
• bile salt 44
• 12. Cytotoxicity assay of V. vulnificus strains to host cells 44
• 13. Checkerboard assay 45
• 14. Real-time PCR 46
• 15. Statistical analysis. 47
• Results 48
• 1. Effect of toxRS mutation on the expression of outer membrane protein TolCV1 48
• 2. Effect of toxRS mutations on the transcriptional activities of TolCV1 51
• 3. Effect of toxRS mutation on TolCV1 release through OMVs 53
• 4. Effect of toxRS mutation on V. vulnificus resistance to ampicillin 55
• 5. Effect of toxRS mutation on V. vulnificus resistance to bile salt 58
• 6. Effect of toxRS mutation on V. vulnificus cytotoxicity to host cells 61
• 7. PAβN enhanced the antibacterial effect of ampicillin against V. vulnificus strains 63
• Discussion 67
• References 72
• General abstract (In Korean) 82
• Curriculum vitae 84
• Acknowledgements 86