Name: system for efficacy and cytotoxicity screening of inhibitors targeting intracellular mycobacterium tuberculosis
Uploaded: 2017-04-05T14:30:00
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 액...

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>N,N-Dimethylformamide (DMF)</td>
			<td>Fisher Scientific</td>
			<td>D131</td>
			<td></td>
		</tr>
		<tr>
			<td>1M 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></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1</td>
			<td>American Type Culture Collection</td>
			<td>TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>M. tuberculosis H37Rv</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></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></td>
			<td></td>
			<td></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 ...

The overall goal of this protocol is to provide multidimensional assessment of how compounds affect M.tuberculosis survival inside human macrophages. This method is centered on a Luciferase-based efficacy assay. When combined with cytotoxicity an MIC assay can provide useful information into modification of HIT compounds in TB drug development.The main advantage of our method is that it is time and labor saving. It is replacing a CFU colony forming unit assay with a Luciferase based assay. Generally individuals new to this technique will struggle with consistency with manual pipetting instead of automation.A consistent pipetting technique is crucial. Grow THP1 cells in RPMI in complete medium. While maintaining a cell density of 0.2 to one million cells per milliliter of medium between passages.Use a spectrophotometer to measure the OD600 of an actively growing bacterial suspension. Then calculate the bacterial density using the conversion factor of 0.1 OD600 equals three times 10 to the seventh bacteria per milliliter. Pipette out sufficient bacteria for an MOI of 10 to one into a new centrifuge tube.Then pellet the bacteria at 3, 000 times G for 10 minutes. To opsonize the bacteria, add 50 microliters of human serum to 450 microliters of RPMI 1640 and use it to resuspend the bacterial pellet at a density of one times 10 to the eighth bacteria per 500 microliters of medium. Allow the mixture to incubate at 37 degrees Celsius for 30 minutes.Next, using a hemocytometer and an inverted microscope, determine the THP1 cell culture density. Then, in sterile centrifuge tubes, pellet the cells at 100 times G in 37 degrees Celsius for 10 minutes. Aspirate the supernatant and resuspend the cells in RPMI in complete medium at a density of one million cells per milliliter.Add PMA to a final concentration of 40 nanograms per milliliter. This is differentiation mix. Combined opsonized M.tuberculosis with THP1 differentiation mix at an MOI of 10 to one.And aliquot the final mix at 100 microliters per well in a 96 well white flat bottom plate. Then allow the differentiation and infection to incubate at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide overnight. The following day, use 100 microliters of RPMI to wash the wells twice.Then add compounds diluted to the desired concentrations and RPMI in complete medium. Incubate the plates for three days. When using multichannel pipettes, remember to practice slow and steady plunger action and always land the pipette tips in the same spot inside each well to minimize disturbance to the THP1 cells.Following the incubation, aspirate the medium from the wells and add 50 microliters of luciferase assay reagent to each well. Then use transparent adhesive plate sealers to seal the plates. Incubate the plates at 22 degrees Celsius for five minutes, then use a luminometer to obtain a readout at one second per well.To differentiate THP1 cells, after preparing differentiation mix as demonstrated earlier in this video, dispense 100 microliters of the differentiation mix into each well of a clear, 96 well plate of cells. Incubate the cells at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide overnight. The following day, aspirate the medium from the wells and use RPMI 1640 to wash them twice.Add 100 microliters of compounds diluted in RPMI incomplete to the wells. Then, incubate the cells for three days. Dissolve 0.5 grams of MTT in 100 milliliters of PBS to make a stock solution of five milligrams per milliliter.Then filter sterilize the solution. Two and a half hours before the end of the three day incubation period, add 25 microliters of MTT solution to each well of cells and complete the incubation period. Prepare 50%NN dimethylformamide or DMF by mixing 250 milliliters of DMF with 250 milliliters of deionized water.Prepare MTT extraction buffer as follows. Lay 100 grams of SDS in a 500 milliliter bottle and add 300 milliliters of 50%DMF. Apply low heat to allow the SDS to dissolve.Add 10 milliliters of pure acetic acid and 12.5 milliliters of one molar hydrochloric acid. Then, use 50%DMF to fill up the bottle to the 500 milliliter mark. At the end of the treatment period, add 100 microliters of extraction buffer to each well.Incubate the mixture at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide overnight. The following day, read the absorbance at 570 nanometers. To carry out a resazurin assay, grow M.tuberculosis in 7H9 ADST to midlock phase.Dilute the culture with 7H9 ADST to an OD600 of 0.01. Use 7H9 ADST to dilute the compounds to two times the testing concentrations. And aliquot 100 microliters of each diluted compound into each well of a clear 96 well plate.Transfer 100 microliters of the diluted bacterial suspension into each well. Incubate the plates at 37 degrees Celsius in a humidified incubator for five days. Dissolve 10 milligrams of resazurin in 100 milliliters of deionized water.And filter sterilize the solution. Add 30 microliters of resazurin solution to the wells. Then incubate the cells for 48 hours.Bacterial growth is indicated by a color conversion from blue to pink. Carry out quantitative analysis according to the text protocol. This scatterplot shows the raw luminescence data measured in relative luminescence units for the treatment of various concentrations of rifampicin on M.tuberculosis and THP1 cells.In this plot, the percent reduction and luminescence in treated wells compared to untreated wells is shown. Rifampicin is capable of reducing 99.9%of the relative light units, or RLUs from intracellular M.tuberculosis at a concentration of 0.1 micrograms per milliliter. Similar to previously published results determined from CFU.In this figure, the cytotoxicity caused by increasing concentrations of G1-1H used in the MTT assay is illustrated. Concentrations of 10 and three micromolar are clearly below the IC 50 cutoff. This figure shows the effects of various concentrations of the antibiotic apramycin on resazurin conversion by M.tuberculosis.The in-broth MIC90 was between 2.5 and five micrograms per milliliter. This is slightly higher than the previously published value of 1.5 micrograms per milliliter. But it is still well within the acceptable range.As demonstrated here, evaporation becomes significant for all experiments with prolonged incubation times. So care must be taken to keep incubators well humidified to avoid inconsistencies. Once mastered, this technique can provide reliable intracellular data in as little as four days.While attempting this procedure it is important to remember to be gentle with THP1 cells, and be consistent with pipetting if attempting without automation. Following this procedure, other methods like high content screening can be used to determine if hit compounds are bacteriostatic or bactericidal. After its development, this protocol paved the way for researchers in the field of tuberculosis drug discovery to rapidly assess compound efficacy against MTB inside human macrophages.Don't forget that working with microbacterium tuberculosis is dangerous. Use precautions and proper equipment when handling this microorganism.

Research

JoVE Journal

Immunology and Infection

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

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

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

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

Growth of Mycobacterium tuberculosis Biofilms

Growth of Mycobacterium tuberculosis Biofilms

의 성장 결핵은 Mycobacterium Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

자연의 전송에 결핵 말라리아 동시 감염을 연구하는 실험 모델 결핵균와 변형체 berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA 바이러스의 넓은 스펙트럼 화학 억제를위한 높은 처리량 검사

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

다중 약물 내성을 보여<em&gt; 결핵균</em&gt; 증폭 마이크로 어레이

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

HIV 역전사 및 인테그라 제 억제제의 빠른 검사

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

에 대한 기능성 연구를위한 3D 인간의 폐 조직 모델<em&gt; 결핵균</em&gt; 감염

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

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

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

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 ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

18FDG PET/CT 영상 공부 결핵균 감염 및 비 인간 영장류에서 치료 도구로 서의 분석

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

기자 효소 형광와 쥐에서 이미징 결핵균

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...