Name: system for efficacy and cytotoxicity screening of inhibitors targeting intracellular mycobacterium tuberculosis
Uploaded: 2017-04-05T14:30:00.000+00:00
Duration: 9 min 57 s
Description: Watch this Scientific Journal Video about System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis at JoVE.com

This work was supported by BC Lung Association and Mitacs.

1. 균주 및 성장 매체 <ol><li> 소 혈청 알부민 25 g, 덱 스트로스 10.0 g, 및 탈 이온수 460 ㎖의 염화나트륨 4.05 g을 가용화하여 알부민 스트로스 염화칼슘 원액 (ADS)을 만든다. 4 ° C에서 ADS 및 저장을 필터 소독. </li><li> 7H9 분말 4.7 g 및 정제수 900 mL의 글리세롤 2 mL를 첨가하여 7H9 국물을 만들기. 10 분 동안 121 ° C에서 7H9 액체 배지를 오토 클레이브가 진행하기 전에 실온으로 냉각시킨다. ADS 100㎖로 7H9 액체 배지 900 ㎖의 트윈 80을 0.5 mL를 첨가하여 7H9ADST합니다. 4 ℃에서 보관하십시오. </li><li> 카나마이신 설페이트 50 mg의 무게를 측정하고 1 ㎖ 탈 이온수에 녹여; 최종 농도 50 ㎎ / ㎖이다. 필터 소독 및 -20 ° C에서 보관. 7H9ADST의 1 L 당 원액을 카나마이신 0.5 ML을 추가합니다. 이 매체는 신선한해야한다, 그래서 문화의 크기에 따라 적절하게 볼륨을 확장 : 참고. </li><li> 7H에서 결핵균 성장9ADST 문화 서에서 카나마이신으로 보충. 매일 문화를 흔들어하고 OD600이 응집을 방지하기 위해 1.0에 도달하기 전에 희석. 주 :이 방법의 개발에 사용되는 결핵균 H37Rv 균주 pJAK2.A 12 플라스미드로 형질 전환 하였다. pJAK2.A은 Hsp60에 프로모터 반딧불이 루시퍼 라제 유전자의 높은 수준의 발현을 허용하고 카나마이신을 사용하여 선택 될 수 pMV361 벡터에 기초하여 통합 플라스미드이다. </li></ol> 2. THP-1 중간 및 유지 보수 <ol><li> RPMI를 완전 배지 (대략 10 % FBS와 2 mM의 글루타민)을 만들기 위해 열 활성화 된 우 태아 혈청 (FBS) 및 RPMI 1640 500 ㎖ 내지 200 mM의 L- 글루타민 5 ㎖ 50 ㎖의 추가. </li><li> 표준 프로토콜에 따라 13 THP-1 세포 배양 물을 유지한다. PAS 사이 배지 1 ㎖ 당 0.2 내지 1 백만의 셀 밀도를 유지하면서 간단하게, 완전 RPMI 배지에서 THP-1 세포를 성장현인. </li></ol> 3. 높은 처리량 세포 내 검사 루시 페라 제 발현 M. 결핵 H37Rv 사용 <ol><li> 600 nm의 파장에서 분광 광도계에서 활발히 성장 세균 현탁액의 광학 밀도를 측정한다. mL를 0.1 OD 600 = 3 × 107 세균의 변환 계수를 이용하여 박테리아의 밀도를 계산한다. </li><li> 새로운 원심 분리 튜브로 1 : 10의 감염 (MOI)의 다수 충분한 박테리아를 피펫. 10 분 동안 3,000 XG에서 펠릿 액체를 흡인. RPMI1640의 450 μL에 인간 혈청 50 μL를 추가합니다. 실험에 대한 값을 적절한하기 위해 볼륨을 확장 할 수 있습니다. </li><li> 박테리아 opsonize 위해 10 % 인간 혈청을 함유하는 RPMI1640 500 μL 당 1 × 108 세균의 농도로 펠렛을 재현 탁. 혼합물을 30 분 동안 37 ℃에서 배양 할 수있다. 혈구와 반전 microsco 카운팅하여 THP-1 세포 배양 물 농도를 결정체육. </li><li> 100 XG에서 멸균 원심 분리 튜브에 세포 펠렛을 10 분간 37 ° C. 상등액을 흡인하고 불완전 mL의 1 개 백만 세포의 밀도로 RPMI에서 세포를 재현 탁. 40 NG / ㎖ 최종 농도 포볼 -12- 미리 스테이트 -13- 아세테이트 (PMA)를 추가한다. 참고 :이이 분화 믹스라고한다. </li><li> 10의 MOI에서 THP-1 분화 믹스 옵 소닌 결핵균 결합 : 1, 96- 웰 평면 바닥 플레이트에서 흰색 웰 당 100 μL의 최종 믹싱 나누어지는. 정기적 균일 성을 보장하기 위해, 혼합물을 교반한다. 분화 및 감염 5 % CO2를 함유하는 가습 인큐베이터에서 37 ℃에서 하룻밤 동안 진행하도록 허용. </li><li> RPMI 각 100 μL로 두 번 우물을 씻으십시오. 완전 RPMI에서 원하는 농도로 희석 된 화합물을 첨가하고 3 일 동안 배양한다. </li><li> 웰로부터의 배지를 흡인. 각 웰 루시퍼 라제 분석 시약 50 μL를 추가한다. t와 플레이트를 밀봉ransparent 접착제 플레이트 씰러. 22 ° C에서 배양 5 분을 허용 한 후 1 초에 루미의 판독을 얻기 웰당. </li></ol> 4. 세포 독성 분석 3- (4,5- 디메틸 티아 졸 -2- 일) -2,5- 디 페닐 테트라 졸륨 브로마이드 (MTT)를 사용하여 검정 (14) <ol><li> RPMI에서 THP-1 세포 분화 불완전 맑은 96 웰 플레이트에서 40 NG PMA / ㎖로 보충. 셀 mL의 1 백만의 밀도와 나누어지는 잘 당 100 μL를 유지한다. 분화가 5 % CO2를 함유하는 가습 인큐베이터에서 37 ℃에서 하룻밤 동안 진행하도록 허용. </li><li> 웰에 완전 RPMI에 희석 된 화합물을 추가 웰로부터 배지를 흡인하고 RPMI 1640 회 씻는다. 3 일 품어. </li><li> 5 ㎎ / ㎖의 스톡 용액을하기 위해 인산 완충 용액 (PBS)을 100 mL의 MTT 0.5 g을 녹이고. 광 거리에서 -20 ° C에서 살균 필터 및 저장; 그것은이 솔루션 신선하게하는 것이 가장 좋습니다. </li><li> 2.5 시간을 befor전자 3 일간의 잠복기의 끝, 각 웰에 MTT 용액 25 μL를 추가하고 잠복기를 완료합니다. </li><li> 50 % N을 준비 N의 디메틸 포름 아미드 (DMF), 탈 이온수 250ml를 DMF 250ml의 혼합함으로써. </li><li> 다음 MTT 추출 버퍼 준비 : 500 ㎖의 병 SDS 100g의 무게를 재고 50 % DMF 300 mL를 첨가. SDS 용해 할 수 있도록 낮은 열을 적용합니다. 순수한 아세트산 10 mL의 1 M 염산 12.5 mL를 넣고. 50 % DMF와 500mL의 마크까지 채우고; 추출 완충액의 최종 조성물은 50 %의 DMF, 20 % SDS, 2.5 % 아세트산, 2.5 %, 1 개 M 염산이다. </li><li> 처리 기간의 마지막에, 각 웰에 (모든 결정을 용해 45 ℃로 가온) 추출 완충액 100 μL를 추가한다. 혼합물을 37 ℃에서 밤새 배양 허용 5 % CO2를 함유하는 가습 인큐베이터에서 C를 °. 570 nm에서 흡광도를 읽어보십시오. 주 : 세포 독성 분석을 가장 잘 인트라 셀에 병렬로 수행THP-1 세포의 동일 배치 나이를 사용하여 울라 화면. </li></ol> 5.에서-국물 활동 분석은 레사 주린 분석 (3)를 사용하여 <ol><li> 미드 로그 상 (- OD 600 0.8 ~ 0.5)로 7H9ADST에서 결핵균 성장. 0.01 OD 600에 7H9ADST로 배양을 희석. 각 웰에 희석 된 화합물을 각각의 시험 농도 및 100 μL 분취를 2 배 7H9ADST의 화합물을 희석한다. </li><li> 각 웰에 희석 된 세균 현탁액 100 ㎕를 옮긴다. 플레이트를 5 일 동안 가습 인큐베이터에서 37 ℃에서 배양 할 수있다. 탈 이온수 및 살균 필터 100 mL에 레사 주린 10 ㎎을 용해. </li><li> 30 μL 레사 주린 용액에 추가하고 48 시간 후 색상 변화를 모니터링; 세균의 성장은 분홍색 파란색에서 색 변환으로 표시됩니다. 주 : 정량 분석도 530에서 여기와 590 nm에서의 형광 중 하나를 측정함으로써 수행 될 수있다 - 560나노 미터 또는 570 nm에서의 흡광도와 600 nm의 15. </li></ol>

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The authors declare no competing financial interests for this work.

이 연구의 목적은 결핵균에 대한 인간의 세포 내 감염 모델을 사용하여 간단하고 비용 효율적인 HTS 방법을 만드는 것이 었습니다. 결핵은 M. 결핵하여 폐포 대 식세포의 감염에 의해 특징 인간의 질병이다. 때문에 바이오 안전성 문제, 박테리아 및 숙주 세포 모두의 생물학적 모델을 포함하는 연구는 과거에 사용되어왔다. 그러나,이 대리 박테리아와 인간이 아닌 모델의 사용은 약물 ...

결핵균, 결핵 (TB)의 원인이되는 에이전트는 전 세계적으로 이환율과 사망률의 주요 원인이다. 약물에 민감한 TB 6 개월의 기간 동안 여러 항생제를 필요로하는 치료가 가능한 질환입니다. 치료 가능한 질병 임에도 불구하고, 결핵 사망률은 2015 일에서 150 만 것으로 추정되었다. 지난 10 년 동안, 약제 내성 결핵균의 유병률에 대한 우려가 증가하고있다. 다제 내성 결핵 (MDR-TB)가 적어도 이소니아지드 (INH) 리팜피신 (RMP), 가장 MDR-TB 균주 따라서 치료 옵션을 제한 두번째 선 TB 약물을 선택하는 것이 내성에 강한 TB로 정의된다 . 약제 내성의 효과는 인간 면역 결핍 바이러스 (HIV)에 감염된 환자의 공동 치료를위한 큰 도전을 생성; 40 환자는 전 세계적으로 2,015 1 HIV 관련 결핵으로 사망. 실망, 미국 식품의 약국 Administ배급은 지난 40 년 2, MDR-TB, bedaquiline에 대한 하나의 새로운 결핵 약을 승인했다. 더 짧은 TB 치료를 찾는의 발전이 시급히 TB와 MDR-TB의 싸움에 앞서 유지하기 위해 필요하다. 전통적으로, TB 약물 스크린 화합물은 성장 배양에 첨가하여 미생물의 박멸에 그 효과는 고체 배지에서 집락 형성 단위 (CFU)을 계산하여 결정함으로써 성장 배지에서 체외 성장 조건 하에서 수행된다. CFU 카운트는 노동 집약적, 시간 소모, 비용이 많이 드는만큼, 다양한 간접 방법이 문제를 완화하기 위해 개발되었습니다. 이러한 방법들은하는, Alamar 블루 생존 분석 3, 녹색 형광 단백질 (GFP) 또는 루시퍼 라제를 발현하는 박테리아에서 형광 발광 5 (4)의 결정 및 총 아데노신 삼인산의 추정 (A 포함TP) 6,7. 전형적인 TB는 박테리아 상주 폐포 대 식세포 (8)의 내부 phagosomes 복제 폐,의 결핵균 감염에 의해 특징된다. 간단한에서 국물 표현형 화면이 세포 외 병원균에 맞게 할 수있다; 그러나, 역사적 관점에서이 방법을 사용하여 식별 결핵균에 대한 화합물은 종종 감염 모델에서 다운 스트림 검증 단계에서 기대에 부응하는 데 실패했다. 우리는 결핵 약물이 가장 세포 내 숙주 세포 감염 모델에서 수행되는 것을 제안한다. 그럼에도 불구하고, 세포 내 모델은 높은 처리량 스크리닝 (HTS) 개발에 많은 기술적, 생물학적 장벽을 가지고있다. 큰 장애물은 여러 단계와 세정 사이에 의하여 세포 외 박테리아의 정교한 제거 예로, 악성 프로세스의 복잡성이다. 두 번째 주요 장애물은 긴 시간 재quirements는 일반적으로 CFU 문화 판에 계산에 의해 수행 성장 탐지으로 완료하는 데 3 주 이상 걸리는 과정이다. CFU 카운트를 대체하는 하나 개의 솔루션은 형광 박테리아와 함께 자동 형광 현미경으로 제공되었습니다. 그러나,이 솔루션은 많은 연구 실험실의 손이 닿지이다 초기 설비 투자를 필요로한다. 간단하고 저렴한 비용, 질병 관련 HTS 방법은 크게 신약 개발 프로세스를 향상시킬 것입니다. 본 연구에서는 세포 내 결핵균에 대한 화합물의 활성을 결정하기에 적합한 빠른, 높은 확장 성이면서 경제적 인 분석을 제공하는 것을 목적으로, 새로운 모듈 형 HTS 시스템에보고한다. (나) 세포, (ⅱ) 세포 독성, 및 (iii)의 국물 분석법 :이 시스템은 세 개의 모듈로 구성된다. 합한 최종 결과는 액션의 전위 모드와 같은 추가 정보, 화합물 성질의 포괄적 인 설명을 제공한다. 이 SC시스템 reening하는 것은 약물 시너지 (9)의 분석을 포함한 행동 모드를 대상으로 다양한 화합물 라이브러리, 여러 프로젝트에서 사용되고, 자식 작용 (10)의 자극, 및 결핵균의 억제 독성 인자 (미공개) -secreted. 행동의 알 수없는 모드의 화합물은 또한 11 연구되고있다. 이 방법의 수정 된 버전은 또한 세포 내 결핵균 (11)에 대해 새로운 화합물을 식별 차 스크리닝 방법으로서의 산업 파트너가 채택되었다.

<table><tbody><tr><td>RPMI 1640</td><td>Sigma-Aldrich</td><td>R5886</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma-Aldrich</td><td>G7513</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher Scientific</td><td>12483020</td><td></td></tr><tr><td>Middlebrook 7H9</td><td>Becton, Dickinson and Company</td><td>271210</td><td></td></tr><tr><td>Tween80</td><td>Fisher Scientific</td><td>T164</td><td></td></tr><tr><td>Albumin, Bovine pH7</td><td>Affymetrix</td><td>10857</td><td></td></tr><tr><td>Dextrose</td><td>Fisher Scientific</td><td>BP350</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358</td><td></td></tr><tr><td>kanamycin sulfate</td><td>Fisher Scientific</td><td>BP906</td><td></td></tr><tr><td>PMA</td><td>Sigma-Aldrich</td><td>P8139</td><td></td></tr><tr><td>MTT</td><td>Sigma-Aldrich</td><td>M2128</td><td></td></tr><tr><td>&lt;em&gt;N,N&lt;/em&gt;-Dimethylformamide (DMF)</td><td>Fisher Scientific</td><td>D131</td><td></td></tr><tr><td>1 M Hydrocholoric acid (HCl)</td><td>Fisher Scientific</td><td>351279212</td><td></td></tr><tr><td>Acetic acid</td><td>Fisher Scientific</td><td>351269</td><td></td></tr><tr><td>SDS</td><td>Fisher Scientific</td><td>BP166</td><td></td></tr><tr><td>Resazurin</td><td>Alfa Aesar</td><td>B21187</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229</td><td></td></tr><tr><td>THP-1</td><td>American Type Culture Collection</td><td>TIB-202</td><td></td></tr><tr><td>&lt;em&gt;M. tuberculosis&lt;/em&gt; H37Rv</td><td></td><td></td><td></td></tr><tr><td>96-well flat bottom white plate</td><td>Corning</td><td>3917</td><td></td></tr><tr><td>95-well flat bottom clear plate</td><td>Corning</td><td>3595</td><td></td></tr><tr><td>Transparent plate sealer</td><td>Thermo Fisher Scientific</td><td>AB-0580</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>Biomate 3</td><td></td></tr><tr><td>Microplate spectrophotometer</td><td>Biotek</td><td>Epoch</td><td></td></tr><tr><td>luminometer</td><td>Applied Biosystems</td><td>Tropix TR717</td><td></td></tr></tbody></table>

system for efficacy and cytotoxicity screening of inhibitors targeting intracellular mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

루시퍼 라제 유전자를 발현 결핵균을 이용하여 높은 처리량 스크리닝 세포 도 2a 및 표 1은 루미 의해 수집 된 원시 데이터, THP-1 세포 안에 결핵균의 TB 약물 리팜피신의 농도 증가의 효과를 나타내는, 상대적 형광 단위 (RLU)에서 발현 함유한다. 도 2a는 리팜피신의 다양한 농도?...

Watch this Scientific Journal Video about System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis at JoVE.com

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

우리는 세포를, 결핵균에 대해 신규 한 화합물을 발견 타겟팅 및 인스 국물 포유 동물 숙주 세포에 대해 박테리아뿐만 아니라 세포 독성이 증가하는 모듈 높은 처리량 스크리닝 시스템을 개발 하였다.

세포 내 타겟팅 억제제의 효능 및 세포 독성 검사를위한 시스템<em&gt; 결핵균</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

system-for-efficacy-cytotoxicity-screening-inhibitors-targeting

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

8.5K 조회수.  University of British Columbia.  우리는 세포를, 결핵균에 대해 신규 한 화합물을 발견 타겟팅 및 인스 국물 포유 동물 숙주 세포에 대해 박테리아뿐만 아니라 세포 독성이 증가하는 모듈 높은 처리량 스크리닝 시스템을 개발 하였다. 

비디오: System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

의 샘플 준비<em&gt; Mycobacterium 결핵</em핵 자기 공명 Metabolomic의 연구&gt; 압축을 풉니 다

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

연구

면역학 및 감염병학

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

고처리량/고함량 스크리닝에 적용된 세포내 진균의 정량화를 위한 현미경 표티픽 분석

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 &#176;C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to &#8805;99% bacterial kill (MBC&#8805;99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

The charcoal agar resazurin assay (CARA) is a semi-quantitative, medium-throughput method to assess activity of test agents against mycobacteria that are replicating, non-replicating, or both. The CARA permits rapid evaluation of time- and concentration-dependent activity and identifies parameters to pursue by colony forming unit (CFU) assays.

마이코 박테리아의 세미 양적, 중간 처리량 열거의 숯 한천 레사 주린 분석의 시각화

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the ...

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

높은 처리량, 실시간, 세포 내 항균 활동의 이중 판독 테스트 및 진핵 세포의 세포 독성

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

높은 처리량을 지원 분석은 대식 세포 계대에 대한 약물 효능을 평가하기<em&gt; 결핵균</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

결핵에 대한 숙주 유도 요법의 전임상 테스트에 사용하기 위한 자동화된 배양 시스템

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

<meta charset="utf8"></head><body>Dual Reporters를 사용한 Mycobacterium abscessus 에 대한 High-Throughput Screening Assay

Assay Development for High-Throughput Drug Screening Against Mycobacteria

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. Treatments are extremely ineffective and based on a multi-drug regimen, resulting in low patient compliance. Consequently, the scientific community is urged to identify new and effective drugs to treat these infections. One of the strategies employed to this end is drug repurposing - the process of identifying new therapeutic opportunities for existing drugs in the market, circumventing the time required to establish pharmacokinetic and safety profiles of new drugs. With most studies on drug development against Mab relying on traditional and time-consuming methods, an assay for high-throughput drug screening was developed against mycobacteria using an in house developed double-reporter strain of Mab. Using liquid-handling robotics, automated microscopy, and analysis, alongside in house developed double reporter strains, bacterial viability can be rapidly measured using two different readouts, luminescence and fluorescence, without adding reagents or performing any extra steps. This reduces time and variability between assays, a major advantage for high-throughput screenings. The described protocol was validated by screening a library of 1280 compounds. The obtained results were corroborated by the literature, with efficient detection of active compounds. Thus, this work fulfilled the aim of supplying the field with a new tool to help fight this extremely drug-resistant bacteria.

<meta charset="utf8"></head><body>저자 스포트라이트: M. abscessus 치료법의 효율적인 발견을 위한 확장 가능한 약물 스크리닝 프로토콜

This study developed an assay for high-throughput screening against Mycobacterium abscessus with a&#160;recently created double reporter strain, using fluorescence and luminescence to assess bacterial viability quickly. This protocol will be relevant for researchers aiming to screen new drugs against this drug-resistant bacteria.

Mycobacteria에 대한 고처리량 약물 스크리닝을 위한 분석 개발

Mycobacterium abscessus (Mab) infections are challenging to treat due to high intrinsic drug resistance, comparable to multidrug-resistant tuberculosis. ...

Micro-CFU 방법을 사용한 결핵 판정 가속화

Micro-Colony Forming Unit Assay for Efficacy Evaluation of Vaccines Against Tuberculosis

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during the 2019-2021 pandemic. The disease is caused by the bacterium Mycobacterium tuberculosis (M.tb). The Mycobacterium bovis strain Bacillus Calmette-Gu&#233;rin (BCG), the only TB vaccine, is the oldest licensed vaccine in the world, still in use. Currently, there are 12 vaccines in clinical trials and dozens of vaccines under pre-clinical development. The method of choice used to assess the efficacy of TB vaccines in pre-clinical studies is the enumeration of bacterial colonies by the colony-forming units (CFU) assay. This time-consuming assay takes 4 to 6 weeks to conclude, requires substantial laboratory and incubator space, has high reagent costs, and is prone to contamination. Here we describe an optimized method for colony enumeration, the micro-CFU (mCFU), that offers a simple and rapid solution to analyze M.tb vaccine efficacy results. The mCFU assay requires tenfold fewer reagents, reduces the incubation period threefold, taking 1 to 2 weeks to conclude, reduces lab space and reagent cost, and minimizes the health and safety risks associated with working with large numbers of M.tb. Moreover, to evaluate the efficacy of a TB vaccine, samples may be obtained from a variety of sources, including tissues from vaccinated animals infected with Mycobacteria. We also describe an optimized method to produce a unicellular, uniform, and high-quality mycobacterial culture for infection studies. Finally, we propose that these methods should be universally adopted for pre-clinical studies of vaccine efficacy determination, ultimately leading to time reduction in the development of vaccines against TB.

저자 스포트라이트: 결핵 백신 효능 및 항원 제시 분석의 효율적인 평가를 위한 CFU 결정 최적화 

The determination of colony-forming units (CFU) is the gold-standard technique for quantifying bacteria, including Mycobacterium tuberculosis which can take weeks to form visible colonies. Here we describe a micro-CFU for CFU determination with increased time efficiency, reduced lab space and reagent cost, and scalability to medium and high throughput experiments.

결핵에 대한 백신의 효능 평가를 위한 Micro-colony Forming Unit Assay

Tuberculosis (TB), the leading cause of death worldwide by an infectious agent, killed 1.6 million people in 2022, only being surpassed by COVID-19 during ...

세포 내 타겟팅 억제제의 효능 및 세포 독성 검사를위한 시스템

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고처리량/고함량 스크리닝에 적용된 세포내 진균의 정량화를 위한 현미경 표티픽 분석

마이코 박테리아의 세미 양적, 중간 처리량 열거의 숯 한천 레사 주린 분석의 시각화

높은 처리량, 실시간, 세포 내 항균 활동의 이중 판독 테스트 및 진핵 세포의 세포 독성

높은 처리량을 지원 분석은 대식 세포 계대에 대한 약물 효능을 평가하기

결핵에 대한 숙주 유도 요법의 전임상 테스트에 사용하기 위한 자동화된 배양 시스템

Mycobacteria에 대한 고처리량 약물 스크리닝을 위한 분석 개발

Mycobacteria에 대한 고처리량 약물 스크리닝을 위한 분석 개발

Mycobacteria에 대한 고처리량 약물 스크리닝을 위한 분석 개발

결핵에 대한 백신의 효능 평가를 위한 Micro-colony Forming Unit Assay