This work was supported by grants 51RT 0_126008 and 51RTP0_151029 for the Research and Technology Development (RTD) project InfectX and TargetInfectX, respectively, in the frame of SystemsX.ch, the Swiss Initiative for Systems Biology. We acknowledge grant 310030B_149886 from the Swiss National Science Foundation (SNSF). Work of S.H.L and A.C. was supported by the International PhD Program "Fellowships for Excellence" of the Biozentrum. Simone Muntwiler is acknowledged for technical assistance. We would like to thank Dirk Bumann for providing pNF106 and Jean Celli for pJC43 and pJC44.

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The authors declare no conflicts of interest.

Bacterial pathogens have evolved numerous strategies to manipulate eukaryotic host cells to their benefit. Pathogens causing acute infections often show rapid proliferation which is accompanied by significant alarming of the immune system and loss of viability of infected cells. In contrast, Brucella and other pathogens that cause chronic infections manage to establish long-lasting interactions within host cells. Therefore, bacteria need to fine tune host cell functions to their benefit without disrupting cellul...

브루셀라 종은 α-프로 테오 박테리아의 클래스에 속하는 그람 음성, 통성 세포 내 병원균이다. 그들은 이러한 풍토 지역에서 심각한 경제적 손실을 초래 소, 염소, 또는 양 등 자연 호스트에서 낙태와 불임의 원인. 브루셀라 병은 전 세계적으로 매년 50 만 새로운 인간 감염 1 위에 일으키는 가장 중요한 인수 공통 질병 중 하나입니다. 인간에 ​​전송 가장 일반적으로 이러한 저온 살균하지 않은 우유로 감염된 동물과 직접 접촉하거나 오염 된 음식의 섭취에 의해 발생합니다. 열성 질환의 증상은 브루셀라 병의 진단에 어려움이 발생하는 불특정입니다. 처리되지 않은 경우, 환자는 관절염, 심내막염, 및 신경 병증이 같은 더 심각한 증상으로 만성 감염을 개발할 수 있습니다. 세포 수준에서 브루셀라 식세포는 비 식세포에 침입 할 수 있으며, 세포 내에서 복제브루셀라 함유 공포 (BCV)라고 함. 박테리아의 내면화는 라 세미,의 Rho와 Cdc42 3의 직접적인 활성화에 의해 액틴 세포 골격의 재 배열이 필요합니다. 진핵 숙주 세포 내부에서 BCV는 세포 내 이입 경로를 따라 트래 피킹과 리소좀과의 상호 작용에도 불구하고, 세균 분해 4 않도록 관리한다. 소낭 ATPase에 의해 BCV의 산성화가 박테리아 타입 IV 분비 시스템 (T4SS) (5)의 발현을 유도 할 필요가있다. 브루셀라는 복제 성 틈새 시장을 확립하기위한 T4SS 분비 세균 이펙터는 세포 내 틈새 시장 (7)의 설립의 결함에 T4SS 6의 삭제 또는 기공을 ATP 아제 리드의 억제 때문에, 필수적인 것으로 생각된다. 박테리아는 그들이 때까지 인신 매매의 단계에서 복제되지 않는다 응급실에서 파생 된 액포 실 (8)에 도달한다. 세포 증식이 발생되면, BCV는 CL에서 발견이러한 calnexin 및 글루코스 -6- 인산 (6)와 같은 ER 마커 OSE 협회. 브루셀라는 ER-같은 구획, 세포를 입력 리소좀 저하를 방지하고, 마지막으로 복제하는 분자 메커니즘은 크게 알려지지 않은 남아있다. 감염의 여러 단계에 관여하는 호스트 요소는 주로 대상으로 접근 또는 초파리 세포 (9)에서 수행되는 작은 규모의 작은 간섭 RNA (siRNA를) 스크린에 의해 확인되었다. 다음은 브루셀라 감염시 개별 호스트 요인의 기여에 광명을 비춰했지만 우리는 전체 과정의 포괄적 인 이해에서 멀리 아직도이다. 여기서, 자동 폭 필드 형광 현미경 및 자동화 된 이미지 분석과 함께 대규모 RNA 간섭 (RNAi의) 검사를 사용하여 인간 숙주 인자의 식별을 허용 프로토콜이 제시된다. HeLa 세포의 뒷면의 siRNA로 형질 describ 수행에드 이전 10, 11 약간의 수정과. 엔드 포인트 분석은 상기 출구와 인접 셀의 감염을 제외한 브루셀라 세포 수명의 많은 부분을 포함한다. 상기 엔드 분석에서 확인 된 히트를 특성화하기 위해, 감염의 초기 단계에 관여하는 인자들을 확인하는 변형 프로토콜이 사용된다. 구조적 GFP를 발현하는 브루셀라 아 보르 투스 균주 박테리아 이틀 동안 세포를 감염시킬 수있는 엔드 포인트 분석에 사용된다. 이 기간 동안, 박테리아는 ER-파생 된 복제 성 틈새 시장 세포, 트래픽을 입력하고 요정 핵 공간에 복제합니다. GFP 신호의 하이 레벨은 안정적으로 복제 박테리아를 포함 개별 셀을 검출 할 수있다. 고 처리량 분석에 브루셀라 항목을 연구하기 위해, 세포 내 및 세포 외 박테리아를 구별 할 수있는 것이 중요하다. 이 방법은 여기 circumv 제시세포 내 및 세포 외 박테리아의 엔트 차 항체 염색. 그것은을 DsRed의 구성 성 발현과 함께 테트라 사이클린 - 유도 GFP를 발현하는 브루셀라 변형에 기초한다. 구성 적을 DsRed 마커의 존재는 샘플에 존재하는 모든 균을 식별 할 수있다. GFP 발현 젠타 의해 동시에 세포 외 박테리아의 불 활성화와 함께 무독성 테트라 사이클린 유사체 anhydrotetracycline (ATC)의 첨가 (GM)에 의해 유도된다. 셀 배리어 성 항생제 GM은 세포 외 박테리아를 죽이고 있지만, ATC는 숙주 세포를 선택하고 박테리아 세포 내에서 선택적으로 GFP 발현을 유도 할 수있다. 이 듀얼 기자는 넓은 필드 현미경을 사용하여 세포 외 박테리아 (만을 DsRed 신호)에서 단일 세포 내 세균 (GFP 및을 DsRed 신호)의 강력한 분리 할 수​​ 있습니다. 세포 브루셀라 의해 검출 GFP 발현에 도달하기 위해 우리는 ATC에 의해 유도 4 시간이 RELI 초래한다는 발견수 신호. 선택적으로 세포 내 세균에 GFP를 표현하는 유사 유도 방식은 세포 내 시겔 (12)을 연구하기 위해 이전에 사용되었습니다.

<table><tbody><tr><td>Tryptic Soy Agar (TSA)</td><td>BD</td><td>236950</td><td></td></tr><tr><td>Tryptic Soy Broth (TSB)</td><td>Fluka</td><td>22092</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Sigma-Aldrich</td><td>60615</td><td></td></tr><tr><td>Skim milk</td><td></td><td></td><td></td></tr><tr><td>250 ml screw cap bottle</td><td>Corning</td><td>8396</td><td></td></tr><tr><td>DMEM</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>Fetal Calf Serum (FCS)</td><td>Gibco</td><td>10270</td><td>Heat-inactivated</td></tr><tr><td>Trypsin-EDTA (0.5%)</td><td>Gibco</td><td>15400-054</td><td>10x stock solution, dilute 1:10 in PBS</td></tr><tr><td>Scepte 2.0 Cell Counter</td><td>Merck Milipore</td><td>PHCC20060</td><td>Alternative cell counting devices can be used</td></tr><tr><td>Greiner CELLSTAR 384-well plate</td><td>Sigma-Aldrich</td><td>M2062</td><td></td></tr><tr><td>Peelable aluminum foil</td><td>Costar</td><td>6570</td><td></td></tr><tr><td>Reagent dispenser: &quot;Multidrop 384 Reagent Dispenser&quot;</td><td>Thermo Scientific</td><td>5840150</td><td>Alternative reagent dispenser can be used.</td></tr><tr><td>Transfection reagent: &quot;Lipofectamine RNAiMAX</td><td>Invitrogen</td><td>13778-150</td><td></td></tr><tr><td>Automated plate washer: &quot;Plate washer ELx50-16&quot;</td><td>BioTek</td><td>ELX5016</td><td>This plate washer contains a 16-channel manifold suitable for 384-well plates. It fits into a biosafety cabinet and has a lid covering the plate during washing which reduces the risk of aerosol production. Alternative plate washers with similar features could be used.</td></tr><tr><td>Gentamicin</td><td>Sigma-Aldrich</td><td>G1397</td><td></td></tr><tr><td>Anhydrotetracycline hydrochloride</td><td>Sigma-Aldrich</td><td>37919</td><td>100 &amp;#956;g/ml solution in 100% ethanol is kept at -20 &amp;deg;C protected from light in aluminum-foil</td></tr><tr><td>PBS</td><td>Gibco</td><td>20012</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>Dissolve in 0.2 M HEPES buffer, pH 7.4. Store at -20 &amp;deg;C and thaw freshly the day before use. Caution, PFA is toxic by inhalation, in contact with skin and if swallowed.</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>DAPI</td><td>Roche</td><td>10236276001</td><td></td></tr><tr><td>Scrambled siRNA</td><td>Dharmacon</td><td>D-001810-10</td><td></td></tr><tr><td>Kif11 siRNA</td><td>Dharmacon</td><td>L-003317-00</td><td></td></tr><tr><td>ARPC3 siRNA</td><td>Dharmacon</td><td>L-005284-00</td><td></td></tr><tr><td>&lt;em&gt;Brucella abortus 2308&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>pJC43 (&lt;em&gt;apHT::GFP&lt;/em&gt;)</td><td></td><td></td><td>Celli &lt;em&gt;et al.&lt;/em&gt;&lt;sup&gt;12&lt;/sup&gt;</td></tr><tr><td>pAC042.08 (&lt;em&gt;apht::dsRed, tetO::tetR-GFP&lt;/em&gt;)</td><td></td><td></td><td>Construction: pJC44 &lt;sup&gt;4&lt;/sup&gt; was digested with EcoRI followed by generation of blunt ends with Klenov enzyme and subsequent digestion with SalI. TetR-GFP was amplified from pNF106 using primer prAC090 and prAC092. Following digestion with SalI, the TetR-GFP product was ligated to the digested pJC44 vector.</td></tr><tr><td>Primer prAC090</td><td>Sigma-Aldrich</td><td></td><td>TTT TTG AAT TCT GGC AAT TCC GAC GTC TAA GAA ACC</td></tr><tr><td>Primer prAC092</td><td>Sigma-Aldrich</td><td></td><td>TTT TTG TCG ACT TTG TCC TAC TCA GGA GAG CGT TC</td></tr><tr><td>HeLa CCL-2</td><td>ATCC</td><td>CCL-2</td><td></td></tr><tr><td>ImageXpress Micro</td><td>Molecular Devices&amp;nbsp;</td><td>IXM IMAGING MSCOPE</td><td>Automated cellular imaging microscope equipped with a precision motorized Z-stage. Alternative systems for automated microscopy and alternative components for hard- and software specified below can be employed.</td></tr><tr><td>High-Speed Laser Auto-Focus</td><td>Molecular Devices&amp;nbsp;</td><td>1-2300-1037</td><td></td></tr><tr><td>CFI Super Fluor 10X objective</td><td>Nikon&amp;nbsp;</td><td>MRF00100</td><td>N.A 0.50, W.D 1.20 mm, DIC Prism: 10X, Spring loaded</td></tr><tr><td>Photometrics CoolSNAP HQ Monochrome CCD Camera</td><td>Molecular Devices&amp;nbsp;</td><td>1-2300-1060</td><td>1,392 x 1,040 imaging pixels, 6.45 x 6.45 &amp;micro;m pixels, 12 bits digitization</td></tr><tr><td>MetaXpress software</td><td>Molecular Devices&amp;nbsp;</td><td>9500-0100</td><td></td></tr><tr><td>LUI-Spectra-X-7</td><td>Lumencor</td><td>SPECTRA X V-XXX-YZ</td><td>Light engine. The following light sources are used: violet (DAPI), cyan (GFP), green/yellow (RFP)</td></tr><tr><td>Single Band Exciter for DAPI</td><td>Semrock</td><td>FF01-377/50-25</td><td></td></tr><tr><td>Single Band Emitter for DAPI</td><td>Semrock</td><td>FF02-447/60-25</td><td></td></tr><tr><td>Single Band Dichroic for DAPI</td><td>Semrock</td><td>FF409-Di03-25x36</td><td></td></tr><tr><td>Single Band Exciter for GFP</td><td>Semrock</td><td>FF02-472/30-25</td><td></td></tr><tr><td>Single Band Emitter for GFP</td><td>Semrock</td><td>FF01-520/35-25</td><td></td></tr><tr><td>Single Band Dichroic for GFP</td><td>Semrock</td><td>FF495-Di03-25x36</td><td></td></tr><tr><td>Single Band Exciter for RFP</td><td>Semrock</td><td>FF01-562/40-25</td><td></td></tr><tr><td>Single Band Emitter for RFP</td><td>Semrock</td><td>FF01-624/40-25</td><td></td></tr><tr><td>Single Band Dichroic for RFP</td><td>Semrock</td><td>FF593-Di03-25x36</td><td></td></tr></tbody></table>

microscopy-based assays for high-throughput screening of host factors involved in brucella infection of hela cells

브루셀라 종은 자연 호스트로 동물을 감염 통성 세포 내 병원균이다. 인간에게 전송은 가장 일반적으로 감염된 동물과 직접 접촉하거나 오염 된 음식의 섭취에 의해 발생되고 심한 만성 감염으로 이어질 수 있습니다. 브루셀라 전문 및 비 전문 식세포의 세포 침입과 소포체 (ER) 유래 액포 내에서 복제 할 수 있습니다. 응급실과 같은 구획 브루셀라 숙주 세포에 진입, 리소좀 분해 회피 및 복제에 필요한 숙주 요인은 거의 알려져 남아있다. 여기에서 우리는 HeLa 세포에서 브루셀라 항목 및 복제에 관련된 호스트 요인을 파악하기 위해이 분석에 대해 설명합니다. 다른 검사 방법을 적용 할 수 있지만 프로토콜은 RNA 간섭의 사용을 설명한다. 분석법은 형광 표지 된 세균의 검출에 사용 자동 형광 표지 된 숙주 세포에 기초와이드 필드 현미경. 형광 이미지는 단일 셀 기반 감염 득점을 허용 CellProfiler에서 표준 이미지 분석 파이프 라인을 이용하여 분석한다. 엔드 분석에서, 세포 내 복제 이틀 감염 후 측정한다. 이 확산 성공적으로 이렇게 강하게 숙주 세포 내에서 증식 할 것이다 세포 내 틈새를 설립 세균 항목입니다. 브루셀라 후 12 시간의 주위에 시작되는 자신의 복제 성 틈새 트래픽에 박테리아를 할 수 있습니다. 세포 내 박테리아가 크게 개별 세포 또는 세포 비 - 복제 성 박테리아를 능가하기 때문에, 구조적으로 GFP를 발현하는 균주를 사용할 수있다. 강한 GFP 신호는 감염된 세포를 식별하기 위해 사용된다. 대조적으로, 엔트리 분석을 위해 세포 내 및 세포 외 박테리아를 구별하는 것이 필수적이다. 여기에서, 테트라 사이클린 - 유도 GFP에 대한 변형 부호화가 사용된다. 유도겐타 마이신에 의한 세포 외 박테리아의 불 활성화와 동시에 GFP의 세포 내 박테리아 GFP를 발현 할 수있는 함께 세포 및 GFP 신호에 기초하여 박테리아 세포의 분화를 가능하게한다. 세포 내 증식이 시작되기 전에이 단일 세포 내 박테리아의 강력한 탐지 할 수 있습니다.

도 1a는 자동으로 엔드 분석에서 감염된 세포를 식별하는 데 사용되는 이미지 분석의 일례를 나타낸다. DAPI 염색 HeLa 세포의 핵이 확인되었고, 25 픽셀 핵 연장하여 핵 주위 8 화소의 폭 조갑 핵 및 보로 노이 셀 바디를 산출 하였다. 박테리아가 주로 조갑 핵 공간 확산 때문에, 셀이 영역에서 강도 GFP 감염 및 비감염 세포를 구별 할 수있는 가장 강력한 측정이다....

Watch this Scientific Journal Video about Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells at JoVE.com

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

에 참여 호스트 요인의 높은 처리량 검사에 대한 현미경 기반 분석 실험<em&gt; 브루셀라</em&gt; HeLa 세포의 감염

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

microscopy-based-assays-for-high-throughput-screening-host-factors

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JoVE Journal

Immunology and Infection

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

8.2K 조회수.  University of Basel. Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

비디오: Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

세포에 붙어 Antileishmanial 검사에 대한 기생충 구조 및 변화 분석<em&gt; Leishmania donovani</emTHP1 인간 급성 백혈병 Monocytic 셀 라인에서&gt; Amastigotes

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

연구

면역학 및 감염병학

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

액포 파열의 단일 세포 측정은 세포 내 병원균에 의해 발생

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

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 ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

세대와의 멀티 표현형 높은 콘텐츠 상영<em&gt; Coxiella burnetii의</em&gt; 트랜스포존 돌연변이

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

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 Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or cellular processes. With the advancement of biotechnologies and the high translational potential of small molecules, a number of innovative approaches in drug discovery have evolved, which explains the resurgent interest in the use of HTS. The oncology field is currently the most active research area for drug screening, with no major breakthrough made for the identification of new immunomodulatory compounds targeting transplantation-related complications or autoimmune ailments. Here, we present a novel in vitro murine fluorescent-based lymphocyte assay easily adapted for the identification of new immunomodulatory compounds. This assay uses T or B cells derived from a transgenic mouse, in which the Nur77 promoter drives GFP expression upon T- or B-cell receptor stimulation. As the GFP intensity reflects the activation/transcriptional activity of the target cell, our assay defines a novel tool to study the effect of given compound(s) on cellular/biological responses. For instance, a primary screening was performed using 4,398 compounds in the absence of a &#34;target hypothesis&#34;, which led to the identification of 160 potential hits displaying immunomodulatory activities. Thus, the use of this assay is suitable for drug discovery programs exploring large chemical libraries prior to further in vitro/in vivo validation studies.

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

작은 분자의 높은 처리량 스크리닝 적합한 형광 계 분석 림프구

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

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 ...

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 ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate hosts, respectively. While pathogenic symptoms of leishmaniasis can vary widely, from benign cutaneous lesions to highly fatal visceral disease forms depending on the infective species, all Leishmania species reside inside host macrophages during the vertebrate stage of their lifecycle. Leishmania infectivity is therefore directly related to its ability to invade, survive and replicate within parasitophorous vacuoles (PVs) inside macrophages. Thus, assessing the parasite&#39;s ability to replicate intracellularly serves as a dependable method for determining virulence. Studying leishmaniasis development using animal models is time-consuming, tedious and often difficult, particularly with the pathogenically important visceral forms. We describe here a methodology to follow the intracellular development of Leishmania in bone marrow-derived macrophages (BMMs). Intracellular parasite numbers are determined at 24 h intervals for 72 - 96 h following infection. This method allows for a reliable determination of the effects of various genetic factors on Leishmania virulence. As an example, we show how a single allele deletion of the Leishmania Mitochondrial Iron Transporter gene (LMIT1) impairs the ability of the Leishmania amazonensis mutant strain LMIT1/&#916;Lmit1 to grow inside BMMs, reflecting a drastic reduction in virulence compared to wild-type. This assay also allows precise control of experimental conditions, which can be individually manipulated to analyze the influence of various factors (nutrients, reactive oxygen species, etc.) on the host-pathogen interaction. Therefore, the appropriate execution and quantification of BMM infection studies provide a non-invasive, rapid, economical, safe and reliable alternative to conventional animal model studies.

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

All pathogenic Leishmania species reside and replicate inside macrophages of their vertebrate hosts. Here, we present a protocol to infect murine bone marrow-derived macrophages in culture with Leishmania, followed by precise quantification of intracellular growth kinetics. This method is useful for studying individual factors influencing host-pathogen interaction and Leishmania virulence.

대 식 세포 안의 세포내 성장의 정량화는 빠르고 Leishmania 기생충의 독성을 평가 하기 위한 신뢰할 수 있는 방법

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

Obtainment of Macrophages from Human Monocytes to Assess Leishmania braziliensis Infection Rate and Innate Host Immune Response

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These cells are specialized in microorganism recognition and phagocytosis, but also activate other immune cells and present antigens, as well as promote inflammation and tissue repair. Here, we describe a protocol to obtain mononuclear cells from peripheral blood (PBMC) of healthy donors to separate monocytes that then differentiate into macrophages. These cells can then be infected in vitro at different Lb concentrations to evaluate the ability to control infection, as well as evaluate host cell immune response, which can be measured by several methods. PBMCs were first isolated by centrifuging with Ficoll-Hypaque gradient and then plated to allow monocytes to adhere to culture plates; non-adherent cells were removed by washing. Next, adherent cells were cultured with macrophage-colony stimulating factor (M-CSF) for 7 days to induce macrophage differentiation. We suggest plating 2 x 106&#160;cells per well on 24-well plates in order to obtain 2 x 105&#160;macrophages. Fully differentiated macrophages can then be infected with Lb for 4 or 24 hours. This protocol results in a significant percentage of infected cells, which can be assessed by optical or fluorescence microscopy. In addition to infection index, parasite load can be measured by counting the numbers of parasites inside each cell. Further molecular and functional assays can also be performed in culture supernatants or within the macrophages themselves, which allows this protocol to be applied in a variety of contexts and also adapted to other intracellular parasite species.

<meta charset="utf8"></head><body>Leishmania braziliensis 감염률 및 선천성 숙주 면역 반응을 평가하기 위해 인간 단핵구에서 대식세포 획득

This protocol describes the process for obtaining human macrophages from monocytes for infection with Leishmania braziliensis. It also allows researchers to evaluate infection rate and parasite viability, ROS production by fluorescence microscopy, and the production of inflammatory mediators in culture supernatants to investigate macrophage response to infection.

Leishmania braziliensis 감염률 및 선천성 숙주 면역 반응을 평가하기 위해 인간 단핵구에서 대식세포 획득

Macrophages are multifunctional cells essential to the immune system function, and the primary host cell in Leishmania braziliensis (Lb) infection. These ...

에 참여 호스트 요인의 높은 처리량 검사에 대한 현미경 기반 분석 실험

요약

더 많은 비디오 탐색

세포에 붙어 Antileishmanial 검사에 대한 기생충 구조 및 변화 분석

액포 파열의 단일 세포 측정은 세포 내 병원균에 의해 발생

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

세대와의 멀티 표현형 높은 콘텐츠 상영

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

작은 분자의 높은 처리량 스크리닝 적합한 형광 계 분석 림프구

세포 내 타겟팅 억제제의 효능 및 세포 독성 검사를위한 시스템

높은 처리량을 지원 분석은 대식 세포 계대에 대한 약물 효능을 평가하기

대 식 세포 안의 세포내 성장의 정량화는 빠르고 Leishmania 기생충의 독성을 평가 하기 위한 신뢰할 수 있는 방법

Leishmania braziliensis 감염률 및 선천성 숙주 면역 반응을 평가하기 위해 인간 단핵구에서 대식세포 획득