Studies on the effects of bacteriophages on bacterial antibiotic treatment and cytokine responses in mammalian cells = 박테리오파지의 항생제 병용 치료 및 포유류 세포 내 사이토카인 반응에 미치는 영향 연구

국문 초록 (Abstract)

항생제 내성(AMR)의 증가는 전 세계 공중보건에 심각한 위협을 제기하며, 이를 해결하기 위한 혁신적인 치료 전략이 요구된다. 박테리오파지(파지) 요법은 그 특이성과 효능 덕분에 다제내성 세균 감염을 해결하기 위한 유망한 대안으로 부상하고 있다. 임상적 성공을 보장하기 위해, 파지 요법은 파지의 세포 사멸 효능과 포유류 세포에서의 파지 투여 안전성에 대한 철저한 평가를 요구하고 있다. 이러한 평가는 치료 결과를 최적화하고 파지 투여와 관련된 예기치 못한 위험을 최소화하는 데 필수적이다. 본 연구에서는 파지 요법의 안전성과 효능을 평가하기 위해 항생제와 파지 간의 수용체 특이적 길항 효과와 파지가 포유동물 세포에서 사이토카인 반응에 미치는 자극 효과를 규명하고자 하였다. 아미노글리코사이드 계열 항생제는 감염 초기 단계를 방해하여 박테리오파지 감염을 억제할 수 있다. 본 연구에서는 아미노글리코사이드 계열 항생제인 카나마이신이 비타민 B12 수송체인 BtuB를 수용체로 사용하는 파지에 대해 Escherichia coli 감염에서 강화된 길항 효과를 나타냄을 확인하였다. 카나마이신의 파지 감염 억제 효과가 BtuB, 지질다당류(LPS), 외막 단백질 A(OmpA), 뉴클레오사이드 특이성 포린(Tsx)과 같은 다양한 파지 수용체에 따라 어떻게 달라지는지를 조사하였다. 높은 농도의 카나마이신은 BtuB를 수용체로 사용하는 파지에 의한 E. coli 감염을 억제하였으나, Tsx, OmpA, LPS를 표적으로 하는 다른 파지들에는 영향을 미치지 않았다. 세포벽 및 막을 파괴하거나 단백질 합성을 억제하는 항생제는 수용체에 따른 파지 감염력에 영향을 미치지 않았으며, 이는 카나마이신의 BtuB 표적 파지에 대한 길항 효과를 강조한다. 또한, btuB 유전자의 결손이 카나마이신 내성을 증가시키고 야생형 E. coli에 비해 카나마이신 축적을 감소시킴을 확인하였으며, 이는 카나마이신 흡수를 위해 BtuB가 필요함을 시사한다. BtuB를 표적으로 하는 파지와의 병용 처리는 다른 파지에 비해 카나마이신 내성을 가진 E. coli에서 더 높은 카나마이신 축적을 유도하였으며, 이는 BtuB 표적 파지에 의해 유발된 카나마이신 축적 증가가 강화된 길항 효과의 원인임을 암시한다. 종합적으로, 본 연구는 파지 수용체의 특성이 항생제와 관련된 독특한 파지-항생제 조합 효과를 초래할 수 있음을 보여주며, 효과적인 파지 치료를 위해 최적의 파지-항생제 조합을 설계할 때 수용체 유형의 중요성을 강조한다. 한편, 파지가 유발하는 염증 반응을 이해하는 것은 임상에서의 안전한 사용을 보장하기 위해 필수적이다. 본 연구에서는 이러한 공백을 해결하고자 파지의 포유동물 세포 내 면역 조절 효과를 추가적으로 탐구하였다. 포유동물 세포 내 면역 반응에 대한 파지의 영향을 확인하기 위해 RAW 264.7 마우스 대식세포와 Caco-2 인간 장 상피 세포를 대상으로 10개의 파지를 평가하였다. 연구 결과, Escherichia coli 파지 JEP7은 TNF-α, IL-6, IL-10의 생성을 자극하였으며, Bacillus cereus 파지 PBC2는 IL-10의 생성을 특이적으로 유도하였다. JEP7과 PBC2는 또한 TNF-α, IL-6, IL-10의 발현을 Caco-2 세포에서도 자극하여 지질다당류(LPS)로 자극된 세포에서 관찰된 것과 유사한 수준으로 증가시켰다. 이는 파지가 서로 다른 세포 유형에서 염증 반응을 유도하는 역할을 보여준다. 파지의 내재화, 세포 독성, 바이리온 당화의 비교 분석은 염증성 사이토카인 생성을 유발하는 파지와 그렇지 않은 파지 간의 차이를 나타내지 않았으며, 이는 특정 파지가 포유동물 세포의 면역 반응에 미치는 주요 요인을 밝혀내지 못했음을 시사한다. 추가 연구를 통해 포유류 세포 면역 반응에서 파지의 일반적인 역할과 메커니즘을 규명할 필요가 있다. 종합적으로, 본 연구는 파지 치료에서 수용체 특이적 상호작용과 면역학적 안전성의 중요성을 부각시켰다. 수용체 의존적 파지-항생제 간 병용 처리 효과와 포유동물 세포 내 면역에 미치는 영향을 규명함으로써, 본 연구는 효과적이고 안전한 파지 기반 치료 전략의 최적화를 위한 새로운 통찰을 제공한다.

다국어 초록 (Multilingual Abstract)

The emergence of antimicrobial resistance (AMR) poses a critical threat to global public health, necessitating innovative therapeutic strategies. Bacteriophage (phage) therapy has emerged as a promising alternative to address multidrug-resistant bacterial infections due to its specificity and efficacy. To ensure clinical success, phage therapy requires thorough evaluation of bactericidal efficacy of phages and the safety of phage administration in mammalian cells. Such evaluation is essential for optimizing therapeutic outcomes and minimizing unforeseen risks associated with phage administration. In this study, I investigated two critical aspects of phage therapy that must be assessed for safety and efficacy: receptor-specific antagonistic effect of antibiotics on phage infection, and the stimulatory effects of phages on cytokine responses in mammalian cells. Aminoglycoside antibiotics can inhibit bacteriophage (phage) infection by disrupting the early stage of the infection cycle. Here, I investigated effects of kanamycin on Escherichia coli infection by phages using different receptors such as vitamin B12 transporter (BtuB), lipopolysaccharides (LPS), outer membrane protein A (OmpA), and nucleoside-specific porin (Tsx). High concentrations of kanamycin inhibited bacterial infection by the BtuB-targeting phages but not by the other phages targeting LPS, OmpA, or Tsx on kanamycin-resistant E. coli. The antagonistic effect of kanamycin was specific to the infection of E. coli by BtuB-targeting phages. All the other antibiotics tested did not affect phage infection regardless of the phage receptors. The btuB knock-out mutant showed reduced kanamycin accumulation and increased kanamycin resistance compared to wild-type E. coli, suggesting that BtuB may be involved in kanamycin uptake. Co-treatment of kanamycin with BtuB-targeting phages resulted in higher kanamycin accumulation in kanamycin-resistant E. coli than with other phages. This may be attributed to the previously established link between kanamycin uptake and the up-regulation of btuD, which is induced by the impaired uptake of vitamin B12, a substrate of BtuB, during infection by BtuB-targeting phages. Further investigation using E. coli as a model organism is necessary to elucidate the regulatory relationship between btuB and btuD expression and the role of BtuD in kanamycin transport. Collectively, the antimicrobial activity of co-treatment with antibiotic and phage combinations may be phage receptor-specific, highlighting the importance of consideration of phage receptors when selecting the optimal phage-antibiotic combinations for effective phage therapy. Additionally, understanding the inflammatory responses triggered by phages is critical for ensuring their safety in clinical applications. To date, little is known about the effects of phage on mammalian immunity, which is crucial to safe phage administration in clinical settings. In this study, I investigated the effects of ten phages on inflammatory cytokine production in RAW 264.7 mouse macrophages and Caco-2 human intestinal epithelial cells. I found that the Escherichia coli phage JEP7 stimulated the production of TNF-α, IL-6, and IL-10, while the Bacillus cereus phage PBC2 specifically induced the production of IL-10 in RAW 264.7 cells. JEP7 and PBC2 also induced the expression of TNF-α, IL-6, and IL-10 in Caco-2 cells to the levels comparable to those observed in lipopolysaccharide (LPS)-stimulated cells, demonstrating their role in stimulating cytokine production across different cell types. Comparative analysis of phage internalization, cytotoxicity, and virion glycosylation revealed no differences between groups of the phages that can affect cytokine production and the phages that cannot, suggesting that these may not have a significant impact on the inflammatory response induced by JEP7 and PBC2. These findings demonstrate that phages may play a role in inflammatory response via differential cytokine induction in mammalian cells, although the common factors affecting immune response among the analyzed phages were not identified. Further research is required to explore the general role of phages on mammalian inflammatory responses. Together, this study underscores the dual significance of receptor-specific interactions and immunological safety in phage therapy. By elucidating the receptor-specific antagonism between antibiotics and phages in E. coli and the stimulatory effects of various phages on cytokine responses in mammalian cells, this study can contribute to the optimization of phage-based therapeutic strategies for effective and safe clinical application.

목차 (Table of Contents)

• <!-- Not Allowed Tag Filtered --><Contents>
• Title: Studies on the effects of bacteriophages on bacterial antibiotic treatment and cytokine responses in mammalian cells 1
• Abstract - 1 -
• Contents - 4 -
• List of Figures - 11 -
• List of Tables - 13 -
• I. Introduction - 14 -
• I.1. Key considerations for successful clinical application of phage therapy - 15 -
• I.2. Mechanisms and implications of phage-antibiotic combination effects - 17 -
• I.3. Cytokines as mediators of mammalian inflammatory response - 20 -
• I.4. Purpose of this research - 22 -
• II. Antagonistic Effects of Kanamycin on Bacteriophages Targeting BtuB in Kanamycin-Resistant Escherichia coli - 24 -
• II.1. Introduction - 25 -
• II.2. Materials and Methods - 28 -
• II.2.1. Bacterial strains, plasmids, and phages used in this chapter - 28 -
• II.2.2. Construction of the ΔbtuB mutant and the complemented strain - 28 -
• II.2.3. Bacteriophage genome sequencing, assembly, and annotation - 29 -
• II.2.4. Checkerboard assay - 30 -
• II.2.5. Measurements of kanamycin susceptibility - 30 -
• II.2.6. Phage adsorption assay - 31 -
• II.2.7. Antibiotic uptake measurement by confocal microscopy analysis - 32 -
• II.2.8. Quantification of fluorescence intensity - 32 -
• II.2.9. Statistics analysis - 33 -
• II.3. Results - 37 -
• II.3.1. Kanamycin specifically inhibits bacterial infection by phages targeting BtuB as a receptor - 37 -
• II.3.2. Antibiotics disrupting cell wall or membrane do not impact phage infectivity depending on the phage receptor type - 47 -
• II.3.3. The enhanced antagonistic effect of kanamycin on BtuB-targeting phages is independent of its mode of action - 57 -
• II.3.4. BtuB plays a role in facilitating kanamycin uptake in E. coli - 67 -
• II.3.5. The extent of btuB induction determines the restoration of kanamycin uptake - 73 -
• II.3.6. BtuB does not affect susceptibility and uptake of chloramphenicol and tetracycline in E. coli - 76 -
• II.3.7. Kanamycin inhibits BtuB-targeting phages through increased intracellular accumulation rather than interference with adsorption - 81 -
• II.4. Discussion - 86 -
• III. Cytokine responses triggered by the bacteriophages JEP7 and PBC2 in mammalian cells - 94 -
• III.1. Introduction - 95 -
• III.2. Materials and Methods - 100 -
• III.2.1. Bacterial strains and culture conditions - 100 -
• III.2.2. Phage purification - 100 -
• III.2.3. Cell lines and culture conditions - 101 -
• III.2.4. RNA extraction and quantitative reverse-transcription PCR (qRT-PCR) - 101 -
• III.2.5. Enzyme-linked immunosorbent assay (ELISA) - 102 -
• III.2.6. Phage internalization assay - 103 -
• III.2.7. Cell cytotoxicity assay - 104 -
• III.2.8. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Glycoprotein staining - 104 -
• III.2.9. Statistics analysis - 105 -
• III.3. Results - 109 -
• III.3.1. Phage purification for cytokine response analysis in RAW 264.7 macrophages - 109 -
• III.3.2. JEP7 and PBC2 stimulated inflammatory cytokine production in macrophages - 112 -
• III.3.3. JEP7 and PBC2 also triggered inflammatory cytokine expression in epithelial cells - 120 -
• III.3.4. Phage internalization did not influence cytokine production in macrophages induced by JEP7 and PBC2 - 122 -
• III.3.5. Cytotoxicity of phages is not associated with cytokine production in macrophages induced by JEP7 and PBC2 - 124 -
• III.3.6. Protein glycosylation was not observed in any of the phages tested - 128 -
• III.4. Discussion - 130 -
• IV. Overall conclusion - 135 -
• References - 139 -
• 국문 초록 - 167 -
• <!-- Not Allowed Tag Filtered --><List of Figures>
• Figure II-1. Enhanced antagonistic effects of kanamycin on E. coli infection by the BtuB-targeting phages. - 37 -
• Figure II-2. Interaction plots of phages and kanamycin against kanamycin-resistant E. coli. - 39 -
• Figure II-3. Enhanced antagonistic effects of neomycin on E. coli infection by the BtuB-targeting phages. - 41 -
• Figure II-4. Interaction plots of phages and neomycin against E. coli. - 43 -
• Figure II-5. The effects of ampicillin on E. coli infection by phages. - 47 -
• Figure II-6. Interaction plots of phages and ampicillin against E. coli - 49 -
• Figure II-7. The effects of colistin on E. coli infection by phages. - 51 -
• Figure II-8. Interaction plots of phages and colistin against E. coli. - 53 -
• Figure II-9. The effects of chloramphenicol on E. coli infection by phages. - 57 -
• Figure II-10. Interaction plots of phages and chloramphenicol against E. coli. - 59 -
• Figure II-11. The effects of tetracycline on E. coli infection by phages. - 61 -
• Figure II-12. Interaction plots of phages and tetracycline against E. coli. - 63 -
• Figure II-13. The effect of BtuB on kanamycin susceptibility of E. coli. - 66 -
• Figure II-14. The effect of BtuB on kanamycin accumulation in E. coli. - 67 -
• Figure II-15. The effect of BtuB on ROS generation induced by kanamycin in E. coli. - 69 -
• Figure II-16. Arabinose-induced btuB expression enhances kanamycin accumulation in the complemented strain. - 72 -
• Figure II-17. The effect of BtuB on susceptibility to chloramphenicol and tetracycline in E. coli. - 75 -
• Figure II-18. The effect of BtuB on tetracycline accumulation in E. coli. - 76 -
• Figure II-19. Effect of BtuB-targeting phages on intracellular kanamycin accumulation in E. coli. - 81 -
• Figure II-20. The effect of kanamycin on phage adsorption. - 82 -
• Figure III-1. JEP7 and PBC2 induced mRNA expression of pro-inflammatory cytokines in macrophages. - 110 -
• Figure III-2. JEP7 and PBC2 induced mRNA expression of an anti-inflammatory cytokine in macrophages. - 111 -
• Figure III-3. JEP7 and PBC2 induced the production of pro-inflammatory cytokines in macrophages. - 113 -
• Figure III-4. JEP7 and PBC2 induced the production of anti-inflammatory cytokine in macrophages. - 114 -
• Figure III-5. JEP7 and PBC2 also triggered inflammatory cytokine expression in epithelial cells. - 116 -
• Figure III-6. Phage internalization is independent of cytokine production induced by JEP7 and PBC2 in macrophages. - 118 -
• Figure III-7. Cytotoxicity of phages is not associated with cytokine production induced by JEP7 and PBC2 in macrophages. - 121 -
• Figure III-8. Cytotoxicity of phages against Caco-2 cells. - 122 -
• Figure III-9. Virion glycosylation of phages is not the primary driver of cytokine production induced by JEP7 and PBC2. - 124 -
• <!-- Not Allowed Tag Filtered --><List of Tables>
• Table II-1. Antibiotic-resistant E. coli strains and plasmids used in this chapter - 31 -
• Table II-2. Characteristics of E. coli phages used in this chapter - 32 -
• Table II-3. Primers used in this study - 33 -
• Table III-1. All phage strains used in this chapter - 101 -
• Table III-2. Primers used in this chapter - 103 -
• Table III-3. Endotoxin concentrations of phage preparations after endotoxin removal with Endotrap HD column - 106 -