We thank Dr. Frank Wolschendorf for access to the Cytation 3 automated imaging plate reader. This work was funded in part by NIH grant R01-AI104499 to OK. Parts of the work were performed in the UAB CFAR facilities and by the UAB CFAR Flow Cytometry Core/Joint UAB Flow Cytometry Core, which are funded by NIH/NIAID P30 AI027767 and by NIH 5P30 AR048311.

참고 :이 6206는 avirulent 변형 17, 18 결핵균의 MC 바와 같이,이 프로토콜의 모든 작품은 바이오 안전성 레벨 2 시설 (BSL-2)에서 수행 할 수 있습니다. M. 결핵 엠씨 2 표현 녹색 형광 단백질 1. 문화 조건 6206 (MTB -GFP) 참고 : M. 결핵 H37Rv는 영양 요구 균주에게 6206 엠씨 (2) 유도 (Δ panCD, Δ leuCD) 플라스미드 pMN437을 발현하는 GFP가이 프로토콜 16에서 사용되는 형질 전환. 비 GFP 연구자의 변형 쉽게 접근 가능하도록 야생형 균주를 발현과 MTB -GFP의 피로를 대체 할 수있다. 그러나, GFP 발현은 식균 작용과 MTB / 대식 세포의 응집체 형성을 육안으로 확인 가능하게하는 것이 바람직하다. 경도의 경우g의 장기 저장은, MTB -GFP 20 % 글리세롤로 보충 (단계 1.1에서 설명) 전체 7H9 미디어에서 -80 ° C에서 동결했다. <ol><li> 10 % 미들 OADC, 0.02 %의 틸록 사폴, 24 μg의 / mL의 D-판토텐산, 50 μg의 / ML의 L-류신 50 μg의 / mL의 하이 그로 마이신 B를 함께 7H9 매체를 보완하여 완전한 MTB -GFP 미디어 (7H9-C)를 준비 </li><li> MTB -GFP의 한 유리 병을 해동하고, 30 mL의 사각형 바닥이 PETG 플라스크에 7H9-C 10 mL를 추가 할 수 있습니다. </li><li> 회전 진탕 (50 회전)에 37 ° C에서 품어. 광학 밀도 측정 (OD 600) 며칠마다 OD 600 1.0 (약 5-8일)에 도달 할 때까지 가져 가라. </li><li> OD를 0.2 ~ 600 사이에 유지하기 위해 희석 통로 배양. </li><li> 감염의 경우, 대수 성장 단계 (OD 0.6-1 600) 내에서 MTB -GFP의 확립 된 문화를 사용합니다. 큰 덩어리의 문화를 방지하기 위해 수조에 MTB 문화 플라스크를 초음파 처리 (130 W 3 × 5의 펄스) ONC전자 또는 일주일에 두 번 </li></ol> 2. 문화 조건 THP-1 세포 <ol><li> 10 % 열 - 불 활성화 소 태아 혈청, 2 mM L- 글루타민 및 10 mM의 HEPES과 RPMI 1640 배지를 보충함으로써 완전한 THP-1 세포 배지 (RPMI-C)를 준비한다. </li><li> 5 % CO 2의 가습 된 분위기 하에서 37 ℃에서 T-75 플라스크에서 세포를 부화. </li><li> 혈구를 사용하여 세포를 계산하거나 매일 유동 세포 계측법 0.1 mL를 당 만 0.6 사이의 밀도 THP-1 세포를 유지한다. </li></ol> 3. 감염 프로토콜 MTB / 대 식세포 집계 구조를 생성합니다 <ol><li> THP-1 세포의 준비 (96 웰 플레이트 당) <ol><li> 5 분 250 XG에 원심 분리기 7 × 10 6 THP-1 세포. 7 mL의 RPMI-C에 재현 탁. </li></ol></li><li> MTB -GFP 준비 <ol><li> 원심 분리기 2.8 × 10 8 MTB -GFP 스윙 - 양동이 원심 분리기에서 3,200 XG에 5 분에서 유OD 600 1.0 = 3 × 10 8 박테리아 / mL로의 변환을 노래. </li><li> 단계 3.2.1에서와 같이 RPMI-C와 원심 분리기로 한 번 씻으십시오. </li><li> 10 초 7 mL의 RPMI-C에 재현 탁하고 소용돌이. </li></ol></li><li> 감염 참고 : 템플릿 그림 1을 참조하십시오. <ol><li> 배지의 증발을 방지하기 위해 물 림 로우 A 및 H 및 칼럼 1, 12 멸균 물 200 μL를 첨가하여 96- 웰 플레이트를 준비한다. </li><li> 배경 제어 (빈)에 대한 2 열 (G2에 B2) 200 μL RPMI-C를 추가합니다. </li><li> 감염, THP-1 세포 현탁액 (3.1 단계)에 MTB -GFP 서스펜션 (단계 3.2)을 추가하고 피펫으로 잘 섞는다. 최종 THP-1 세포 밀도는 용액 당 5 × 105이고, 감염에 대응하는 다수의 40이다. </li><li> 25 mL의 저수지에 THP-1 / MTB -GFP 서스펜션을 따르십시오. </li><li> (G11를 통해 B3) THP-1 나머지 96 우물에 / MTB -GFP 서스펜션의 200 μL 날기 추가GA 멀티 채널 피펫. 참고 : 여러 96 웰 플레이트로 작업하는 경우, 정기적으로 더 혼합물을 각 웰에 첨가되어 있는지 확인하기 위해 탱크에 남아있는 THP-1 / MTB 서스펜션을 재현 탁. </li><li> 7-10일 5 % CO 2, 37 ℃에서 인큐베이션. </li><li> 천천히 (100)을 제거하여 2 일마다 미디어를 변경 μL 100 μL 멀티 채널 피펫을 사용하여 RPMI-C를 미리 예열 추가 잘 부드럽게 각각의 상단에서 미디어를 보냈다. 우물을 재현 탁하고 우물의 바닥에있는 MTB / 대 식세포 집계를 방해하지 마십시오. </li><li> 시각 MTB / 대식 세포 응집체의 크기의 노트를 가지고, 매일 형광 현미경 (4-10X 목적)에 의해 우물을 검사합니다. 하루에 7-10으로 MTB / 대 식세포 집계가 충분히 커야한다 약효 시험 (제 4 절) 동안 진행 (참조 그림 2 참조). </li><li> 원하는 경우, 자동 세포 이미징 시스템을 이용하여 96 웰 캡처 이미지 끼워밝은 필드와 GFP 필터 MTB / 대 식세포 골재 형성을 문서로 설정합니다. 주 : 사용자가 자동화 된 이미징 시스템에 대한 액세스 권한이없는 경우, 대표적인 웰 GFP 이미지 및 시야 기능을 가진 임의의 적당한 현미경을 사용하여 측정 할 수있다. </li></ol></li></ol> 4. 성장 억제 분석은 MTB / 대 식세포 집계에서 파생 의약품 효능에 대해 MTB 평가하기 <ol><li> 약물 준비 (두 약물에 대한 세중의 조건) 참고 : 템플릿도 1a를 참조하십시오. <ol><li> 별도의 96 웰 플레이트에서 G10까지 B2에 7H9-C 미디어의 125 μL를 추가합니다. </li><li> 단계 4.2.4의 희석을 고려하여 7H9-C의 1 mL에 이중 가장 원하는 최종 농도에서 두 약물을 준비합니다. </li><li> 세중 트리트먼트 각각 우물에 B11-C11-D11와 E11-F11-G11 각 약물의 250 μL를 추가합니다. </li><li> 멀티 채널 피펫을 사용하여 직렬 DILUTE 시험 약물 B10-G10에 B11-G11에서 125 μL를 이동하여 두 가지. 각 단계에서 5 회 피펫 팅하여 섞는다. </li><li> 플레이트에서 (오른쪽에서 왼쪽) 열에 열에서 125 μL를 이동하고, 열 4 후 중지를 계속합니다. </li><li> 4 열을 혼합 한 후, 폐기물 용기에 125 μL를 폐기합니다. 열 2와 3은 배경 (빈)과 긍정적 인 성장 컨트롤로 사용할 수 있도록 어떤 약물을 포함 할 수 없습니다. , 또는 테스트 약물 라이브러리도 1b에 템플릿을 따르 참고. 각각의 96 웰 플레이트는 단일 농도에서 58 약물을 수용 할 수있다. 이 단계 4.2.4 절반에 희석되기 때문에 이중 원하는 최종 농도를 사용하여이 약물 플레이트를 준비합니다. </li></ol></li><li> 약물 치료 : <ol><li> MTB를 포함하는 96 웰 플레이트는 대 식세포 (MTB / 대 식세포 집계) -infected 검색합니다. </li><li> 조심 멀티 채널 피펫과 관련된 모든 웰 (G11 내지 B2)을 디 캔트.두 단계의 방법이 수행 첫번째 플레이트 기울임없이 150 μL를 제거하고 플레이트를 기울 웰의 저부 에지에 피펫 팁을 삽입하여 나머지 미디어 (~ 50 μL)을 제거한다. 대 식세포 MTB / 집계가 잘 바닥에 부착 한, 어떤 자료가 손실 될 수 없습니다. </li><li> 조심스럽게 MTB / 대식 세포 집합체를 포함하는 판의 모든 관련 우물 (G11를 통해 B2)에 7H9-C 미디어의 100 μL를 추가합니다. </li><li> 멀티 채널 피펫을 사용하여 감염 판의 대응 웰에 96 웰 플레이트 (단계 4.1)을 함유하는 약물의 100 μL 옮긴다. </li><li> 밀폐 된 가방에 넣어 37 ℃에서 3 일간 배양한다. </li></ol></li></ol> 레사 주린 미세 적정 분석을 사용하여 정량 약물 효능의 (5) <ol><li> 0.8 mg의 최종 농도 원액 레사 주린 준비 / ㎖가 H 2 O.은 멸균 0.22 ㎛의 공극 크기의 PVDF 막 필터를 통해.</li><li> 1 : 1 비율의 2 레사 주린 원액, H 2 O 및 트윈 80 (H 2 O 20 % 용액)을 혼합하여 용액을 준비 작업 레사 주린. 최종 농도가 0.4 ㎎ / ㎖ 레사 주린의 5 % 트윈 80이다. </li><li> 플레이트 판독기 설치 530 nm의 여기 및 590 nm의 발광 37 ° C에서 24 시간 동안 매 30 분에서 형광을 판독 할 수있는 프로그램을 사용. 37 ° C까지 사전 따뜻한 플레이트 리더. </li><li> 멀티 채널 피펫을 사용하여 약물 치료 플레이트 (단계 4.11)의 모든 관련 우물 (G11를 통해 B2)에 레사 주린 작업 용액 20 μL를 추가합니다. </li><li> 플레이트 리더에 접시를 놓고 단계 5.3에서 설정 프로그램을 시작합니다. 주 : 분석 플레이트의 대량의 처리를 용이하게하기 위해, 37 ° C의 배양기에서 분석을 개발하고 단일 형광 24 시간마다 판독을 수행하기에 충분하다. 이것은 또한 운동 기능 배양 플레이트 리더에 대한 액세스 권한이없는 사용자들에 적용 가능하다. </li></ol>

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M., Ramakrishnan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+granuloma+in+expansion+and+dissemination+of+early+tuberculous+infection.">The role of the granuloma in expansion and dissemination of early tuberculous infection.</a> Cell. 136 (1), 37-49 (2009).</li><li>Collins, L., Franzblau, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microplate+alamar+blue+assay+versus+BACTEC+460+system+for+high-throughput+screening+of+compounds+against+Mycobacterium+tuberculosis+and+Mycobacterium+avium.">Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium.</a> Antimicrob Agents Chemother. 41 (5), 1004-1009 (1997).</li><li>Snewin, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+immunity+to+mycobacterial+infection+with+luciferase+reporter+constructs.">Assessment of immunity to mycobacterial infection with luciferase reporter constructs.</a> Infect Immun. 67 (9), 4586-4593 (1999).</li></ol>

The authors have nothing to disclose.

여기서는 상세히 약효 시험에 적합한 다른 MTB 감염 모델을 설명 하였다. 이 모델은 계정 초기 결핵 약물 개발 과정에서 더 고려를 부여해야 두 가지 주요 요인을 취 약물 침투와 감염시 MTB의 대사 변화에 생리 학적으로 관련 장벽의 존재. 우리는 이전에 우리 감염 모델의 이점을 도시 처리량 호환성 16 감염을 스케일링 가능성을 제시하고 있지만, 여기에서 우리는 96 ...

<html> 결핵 (TB)는 40 년 1 항 결핵 화학 요법의 유용성에도 불구하고 심각한 세계 보건 위협 남아있다. 이 비 준수 (2) 환자에 이르게 여러 약물 조합을 사용하여 6 개월의 긴 치료 기간에 대한 요구에 부분적으로 기인한다. 임상 적으로 승인 된 약물의 개발에 성공 3 사실상 존재이다 최근 약제 내성 결핵의 출현은 상기 전계의 문제를 배합했다. 사실, 철저한 항 결핵 약물 개발에도 불구하고, 단 하나의 약물은 FDA가 지난 40 년 4 임상 사용이 승인되었습니다. 따라서, 항 결핵 약물의 새로운 세대는 시급히이 문제를 해결하기 위해 필요하다. 결핵 신약 개발의 핵심 문제는 임상에서 효능을 시험 관내 활성을 가진 화합물에서 성공적으로 전송의 부족이다= &quot;외부 참조&quot;&gt; 5, 6, 7. 처음에, 목표 기반의 접근 방식은 전체 박테리아 세포로 번역하는 데 실패 방지 MTB 약 5,을 선별하기 위해 사용되었다. MTB 세포가 사용되는 경우에도, 그것은 종종 정확하게 생체 8,9 약물 효능을 예측할 수없는 배양액 성장 배양 물을 사용하여 수행된다. 이러한 문제가 인식되었고 MTB 잠복 또는 MTB 대 식세포를 함유하는 약물 스크리닝 분석은 성공적으로 8, 10, 11, 12을 확립하고있다. 그러나, 이들 고급 분석법 약물 비 혈관 폐 병변 발생 관통 장벽 충분한 고려를하고,하지 감염 부위의 괴사 병소이다. 과연심지어 첫번째 줄 TB 약물 리팜피신위한 차선 투여 인해 생체 조직 및 뇌척수액 (CSF)의 불충분으로 문제시 된 세포 MTB (8, 9)에 대해 효능을 감소뿐만 아니라 13 14 15 침투. 의심 할 여지없이 TB 신약 개발 노력을 향상시킬 것이다 초기 리드 개발 과정에서 계정에 이러한 매개 변수를 걸릴 것 같은, 새로운 모델과 분석으로. 이러한 요구를 해결하기 위해, 우리는 최근 MTB 약물 효능 검사 (16)에 대한 저렴하고 신속하고 BSL-2 호환 대안 감염 모델을 설립했다. 생리 학적으로 관련 세포 침투 장벽을 효과적으로 요약 및 대 식세포 계대를 생성 밀도가 생산 큰 식세포 집계 구조 내에서 MTB 포장이 감염 모델, <em&gt; 잠재 MTB를 닮은 변경된 생리 학적 상태와 MTB. 이 감염 모델로부터 유도 MTB 다른 세포 내 감염 모델과 일치하는 결과를 생성 약효를 평가하는 레사 주린 마이크로 타이 세이 (REMA)와 함께 혈중 농도에 비해 높은 CSF 농도를 달성하기 위해 공통 TB 약물의보고 능력을 잘 상관 관계 16. 여기서는 상세히 REMA를 사용 약제 감수성 테스트 식세포 계대 MTB 적합한 생산하는 MTB / 대식 세포 집합체 구조의 생성을 설명한다. 특히,이 감염 시스템을 후보 항 결핵 약물의 스크리닝과의 호환성을 위해 96- 웰 포맷으로 구성 될 수있는 방법을 보여준다.

<table><tbody><tr><td>7H9</td><td>BD Difco</td><td>271310</td><td>Follow manufacturer&#039;s recommendations</td></tr><tr><td>Middlebrook OADC</td><td>BD Biosciences</td><td>212351</td><td></td></tr><tr><td>Tyloxapol</td><td>Sigma</td><td>T8761</td><td>Prepare 20% stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>D-Pantothenic acid hemicalcium salt</td><td>Sigma</td><td>P5710</td><td>Prepare 24 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>L-leucine</td><td>MP Biomedicals</td><td>194694</td><td>Prepare 50 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>Hygromycin B</td><td>EMD Millipore</td><td>400051</td><td>Prepare 200 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Nalgene Square PETG media bottle</td><td>Thermo Fisher</td><td>2019-0030</td><td></td></tr><tr><td>RPMI 1640 media</td><td>Hyclone</td><td>SH30027.01</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S12450H</td><td></td></tr><tr><td>L-glutamine</td><td>Corning</td><td>MT25005CI</td><td></td></tr><tr><td>HEPES</td><td>Hyclone</td><td>SH30237.01</td><td></td></tr><tr><td>Cytation 3 plate reader</td><td>Biotek</td><td></td><td>Interchangable with any fluorescent plate reader and microscope</td></tr><tr><td>Gen5 Software</td><td>Biotek</td><td></td><td>Recording and analysis of rezasurin coversion</td></tr><tr><td>Rifampicin&amp;nbsp;</td><td>Fisher Scientific</td><td>BP2679250</td><td>Prepare 10 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Moxifloxacin Hydrochloride</td><td>Acros Organics</td><td>457960010</td><td>Prepare 10 mg/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Resazurin Sodium Salt</td><td>Sigma</td><td>R7017</td><td>Prepare 800 &amp;mu;g/mL stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr><tr><td>Tween-80</td><td>Fisher Scientific</td><td>T164500</td><td>Prepare 20% stock solution in H&lt;sub&gt;2&lt;/sub&gt;O; filter sterilize</td></tr></tbody></table>

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.

96 웰 플레이트 형식으로이 감염 모델을 적응의 견고성을 확인하기 위해, 우리는 여기에 그림에 주어진 템플릿에 따라 약물 우리의 96 웰에서 파생 된 MTB의 감수성 감염 리팜피신에 모델 (RIF) 및 목시 플록 사신 (MOXI를) 적응을 조사 (1A). 우리는이 분석의 핵심 MTB / 대식 세포 집합체 구조의 생성을 확실하게함으로써, 스루풋의 호환성 <s...

Watch this Scientific Journal Video about A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis at JoVE.com

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 ...

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Research

JoVE Journal

Immunology and Infection

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

7.8K 조회수.  University of Alabama at Birmingham. 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. 

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

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

의 성장 결핵은 Mycobacterium Biofilms

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

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 ...

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.

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

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

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 ...

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. ...

Fluorescence Assays for the Study of Mycobacterium tuberculosis Interaction with the Immune Receptor SLAMF1

The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic strains and genetically engineered cells. Here, an alternative method to detect biochemical interaction between the macrophage microbial sensor SLAMF1 and Mycobacterium tuberculosis is described. Two technical approaches employing flow cytometry and fluorescence microscopy were developed. Total cell protein extracts from human macrophages were generated, then incubated with whole cells of M. tuberculosis (WCMtb)&#160;or M. tuberculosis antigens (Mtb Ags) overnight at 4 &#176;C and finally cross-linked using formaldehyde/glycine/ethylene glycol bis (succinimidyl succinate) treatment. SLAMF1 interaction with WCMtb by flow cytometry was detected with a PE-specific anti-SLAMF1 antibody. The existence of interaction by fluorescence microscopy was performed by attaching Rhodamine-PE stained Mtb Ags to poly-D-lysine coated slides, which were incubated with the total protein extract from monocyte-derived macrophages. After cross-linking treatment, SLAMF1 was visualized using primary (anti-SLAMF1) and secondary (Alexa Fluor 488) antibodies. The assays provided a strong biochemical tool to measure pathogen-immunoreceptor interactions, overcoming the difficulties associated with transgenic cell lines and protein gene expression modulation experiments.

This study provides a protocol for evaluating the interaction of Mycobacterium tuberculosis with the SLAMF1 microbial sensor. The assays were conducted on human monocyte-derived macrophages using flow cytometry and fluorescence microscopy. The described tools are relevant for studying interactions between pathogens and immunoreceptors.

Mycobacterium tuberculosis Immune Receptor SLAMF1과의 상호 작용 연구를 위한 형광 분석

The evaluation of direct interaction between pathogens and immune receptors usually involves sophisticated techniques or implies the use of transgenic ...

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 ...

높은 처리량을 지원 분석은 대식 세포 계대에 대한 약물 효능을 평가하기

요약

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의 성장 결핵은 Mycobacterium Biofilms

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

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

세포 내 타겟팅 억제제의 효능 및 세포 독성 검사를위한 시스템

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

Mycobacterium tuberculosis Immune Receptor SLAMF1과의 상호 작용 연구를 위한 형광 분석

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