在参与宿主因素的高通量筛选基于显微镜的检测方法<em>布鲁氏菌</em> Hela细胞感染
Summary
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.
Abstract
布鲁氏菌的物种是感染的动物作为天然宿主兼细胞内病原体。传播给人类最常通过与受感染的动物的直接接触或通过污染的食物摄入引起的，并可能导致严重的慢性感染。
布鲁氏菌可以侵入的专业和非专业的吞噬细胞和内质网（ER）衍生空泡内复制。 布鲁氏菌对进入宿主细胞，溶酶体降解的回避和复制在ER形腔室所需的宿主因素仍是未知。在这里，我们描述两个测定以鉴定参与在HeLa细胞布鲁氏条目和复制宿主因素。该协议描述了使用RNA干扰的同时，可应用替代的筛选方法。该测定法基于检测荧光标记的细菌使用自动化在荧光标记的宿主细胞宽视野显微镜。荧光图像用CellProfiler标准化图像分析管道，允许单细胞为基础的感染得分进行分析。
在端点检测，细胞内的复制感染两天后进行测量。这使得细菌交通到增殖的细菌进入。 布鲁氏菌已成功建立了一种细胞内的利基将因此已在宿主细胞内增殖强烈围绕后12小时开始他们的复制利基。因为细胞内的细菌会的数量大大超过个别的细胞外或细胞内非复制性的细菌，应变组成型表达绿色荧光蛋白可以使用。然后强GFP信号是用来识别被感染的细胞。
与此相反，对于条目测定是必不可少的细胞内和细胞外的细菌区分开来。这里，被用于四环素诱导的GFP的菌株的编码。感应与由庆大霉素胞菌的同时失活的GFP使细胞内和细胞基于所述GFP信号外细菌之间的分化，与仅细胞内细菌能够表达GFP。这允许鲁棒检测单胞菌的启动细胞内增殖之前。
Introduction
布鲁氏菌物种是属于类α-变形菌的革兰氏阴性，兼性细胞内病原体。它们引起流产和不孕在其自然宿主如牛，山羊，绵羊或导致在流行地区严重的经济损失。布鲁氏菌病是最重要的人畜共患病引起全球每年超过五十万新的人类感染1之一。传输给人类最常用的是与受感染动物的直接接触或通过污染的食物摄入引起的，如未经高温消毒的牛奶。发热疾病的症状是非特异性的，这会导致困难布鲁氏菌病诊断。如果不治疗，患者可发展为慢性感染更严重的症状，如关节炎，心内膜炎，和神经病2。
细胞水平上的布鲁氏菌能够侵入吞噬细胞和非吞噬细胞和细胞内内复制车厢被称为布鲁氏菌含泡（BCV）。细菌的内化外消旋，卢，和Cdc42 3直接激活需要肌动蛋白骨架重排。真核宿主细胞内部，沿BCV内吞途径贩卖，尽管与溶酶体的相互作用，细菌设法避免降解4。由囊泡ATP酶的BCV酸化时需要引起细菌IV型分泌系统（T4SS）5的表达。据认为，由T4SS分泌细菌效应是必不可少的布鲁氏菌 ，以确立其复制利基，由于T4SS 6的缺失或囊泡ATP酶引线抑制在建立细胞小生7的缺陷。细菌中，直到他们贩卖的阶段不复制 达成ER衍生的液泡车厢8。一旦细胞内增殖时，BCV在CL发现与ER标志物如钙联接蛋白和葡萄糖-6-磷酸酶6 OSE关联。
由布鲁氏菌进入细胞，避免溶酶体降解，最后复制在ER-状隔间的分子机制仍是未知。参与感染的不同步骤宿主因素主要被确定的目标的方法或小规模小干扰RNA（siRNA）在果蝇细胞9执行屏幕。这些都揭示个人的宿主因素的布鲁氏菌感染过程中的贡献，但我们仍远未整个过程有全面的了解。
这里，协议，允许使用与自动宽视场荧光显微镜和自动图像分析相结合的大型RNA干扰（RNAi）筛选人类宿主因子的鉴定呈现。 HeLa细胞反向siRNA转染作为describ进行ED早期10,11稍作修改。端点测定覆盖除了出口和感染邻近细胞的布鲁氏菌细胞内的生命周期的很大一部分。为了进一步表征在端点测定法鉴定的命中，则使用一个修改协议，以确定所涉及的感染的早期步骤的因素。
组成型表达GFP一个流产布鲁氏菌菌株用于细菌的地方允许感染细胞两天端点检测。在此期间，细菌进入细胞，投放到ER衍生的复制利基，并在周围核空间复制。高水平的GFP信号然后可用于可靠地检测含复制的细菌的单个细胞。
为了研究在高通量检测布鲁项，它是能够细胞内外细菌区分重要。该方法在这里circumv介绍经济需求测试的细胞内和细胞外的细菌差速器抗体染色。它是基于一个布氏杆菌菌株表达结合的四环素诱导的GFP与DsRed的组成型表达。组成型DsRed的标志物的存在允许在样品中存在的所有的细菌的鉴定。 GFP表达是由庆大霉素加入无毒四环素类似物无水四环素（ATC）与细胞外细菌的灭活同时的（GM）诱导。而细胞不透抗生素了Gm杀死胞外细菌，的aTc可以进入宿主细胞并在细胞内细菌选择性诱导GFP表达。此双报告允许使用宽视场显微镜从胞菌（仅DsRed的信号）单胞菌（GFP和DsRed的信号）的健壮的分离。为了通过细胞内布鲁氏菌达到可检测的GFP表达，我们已经发现，通过的aTc 4小时的诱导导致RELI能信号。类似的诱导方案在胞内细菌选择性地表达GFP先前已用于研究细胞内的志贺氏菌 12。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Tryptic Soy Agar (TSA) | BD | 236950 | |
| Tryptic Soy Broth (TSB) | Fluka | 22092 | |
| Kanamycin sulfate | Sigma-Aldrich | 60615 | |
| Skim milk | |||
| 250 ml screw cap bottle | Corning | 8396 | |
| DMEM | Sigma-Aldrich | D5796 | |
| Fetal Calf Serum (FCS) | Gibco | 10270 | Heat-inactivated |
| Trypsin-EDTA (0.5%) | Gibco | 15400-054 | 10x stock solution, dilute 1:10 in PBS |
| Scepte 2.0 Cell Counter | Merck Milipore | PHCC20060 | Alternative cell counting devices can be used. |
| Greiner CELLSTAR 384-well plate | Sigma-Aldrich | M2062 | |
| Peelable aluminum foil | Costar | 6570 | |
| Reagent dispenser: "Multidrop 384 Reagent Dispenser" | Thermo Scientific | 5840150 | Alternative reagent dispenser can be used. |
| Transfection reagent: "Lipofectamine RNAiMAX | Invitrogen | 13778-150 | |
| Automated plate washer: "Plate washer ELx50-16" | BioTek | ELX5016 | 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. |
| Gentamicin | Sigma-Aldrich | G1397 | |
| Anhydrotetracycline hydrochloride | Sigma-Aldrich | 37919 | 100 ug/ml solution in 100% ethanol is kept at -20°C protected from light in aluminum-foil |
| PBS | Gibco | 20012 | |
| Paraformaldehyde | Sigma-Aldrich | P6148 | Dissolve in 0.2 M HEPES buffer, pH 7.4. Store at -20°C and thaw freshly the day before use. Caution, PFA is toxic by inhalation, in contact with skin and if swallowed. |
| HEPES | Sigma-Aldrich | H3375 | |
| Triton X-100 | Sigma-Aldrich | T9284 | |
| DAPI | Roche | 10236276001 | |
| Scrambled siRNA | Dharmacon | D-001810-10 | |
| Kif11 siRNA | Dharmacon | L-003317-00 | |
| ARPC3 siRNA | Dharmacon | L-005284-00 | |
| Brucella abortus 2308 | |||
| pJC43 (apHT::GFP) | Celli et al.12 | ||
| pAC042.08 (apht::dsRed, tetO::tetR-GFP) | Construction: pJC44 4 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. | ||
| Primer prAC090 | Sigma-Aldrich | TTTTTGAATTCTGGCAATTCCGACGTCTAAGAAACC | |
| Primer prAC092 | Sigma-Aldrich | TTTTTGTCGACTTTGTCCTACTCAGGAGAGCGTTC | |
| HeLa CCL-2 | ATCC | CCL-2 | |
| ImageXpress Micro | Molecular Devices | IXM IMAGING MSCOPE | 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. |
| High-Speed Laser Auto-Focus | Molecular Devices | 1-2300-1037 | |
| CFI Super Fluor 10x objective | Nikon | MRF00100 | N.A 0.50, W.D 1.20mm, DIC Prism: 10x, Spring loaded |
| Photometrics CoolSNAP HQ Monochrome CCD Camera | Molecular Devices | 1-2300-1060 | 1392 x 1040 imaging pixels, 6.45 x 6.45-µm pixels, 12 bits digitization |
| MetaXpress software | Molecular Devices | 9500-0100 | |
| LUI-Spectra-X-7 | Lumencor | SPECTRA X V-XXX-YZ | Light engine. The following light sources are used: violet (DAPI), cyan (GFP), green/yellow (RFP) |
| Single Band Exciter for DAPI | Semrock | FF01-377/50-25 | |
| Single Band Emitter for DAPI | Semrock | FF02-447/60-25 | |
| Single Band Dichroic for DAPI | Semrock | FF409-Di03-25×36 | |
| Single Band Exciter for GFP | Semrock | FF02-472/30-25 | |
| Single Band Emitter for GFP | Semrock | FF01-520/35-25 | |
| Single Band Dichroic for GFP | Semrock | FF495-Di03-25×36 | |
| Single Band Exciter for RFP | Semrock | FF01-562/40-25 | |
| Single Band Emitter for RFP | Semrock | FF01-624/40-25 | |
| Single Band Dichroic for RFP | Semrock | FF593-Di03-25×36 |
References
- Pappas, G., Papadimitriou, P., Akritidis, N., Christou, L., Tsianos, E. V. The new global map of human brucellosis. Lancet Infect Dis. 6, 91-99 (2006).
- Atluri, V. L., Xavier, M. N., de Jong, M. F., den Hartigh, A. B., Tsolis, R. E. Interactions of the human pathogenic Brucella species with their hosts. Annu Rev Microbiol. 65, 523-541 (2011).
- Guzman-Verri, C., et al. GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes: direct activation of Cdc42. J Biol Chem. 276, 44435-44443 (2001).
- Starr, T., Ng, T. W., Wehrly, T. D., Knodler, L. A., Celli, J. Brucella intracellular replication requires trafficking through the late endosomal/lysosomal compartment. Traffic. 9, 678-694 (2008).
- Boschiroli, M. L., et al. The Brucella suis virB operon is induced intracellularly in macrophages. Proc Natl Acad Sci U S A. 99, 1544-1549 (2002).
- Celli, J., et al. Brucella evades macrophage killing via VirB-dependent sustained interactions with the endoplasmic reticulum. J Exp Med. 198, 545-556 (2003).
- Porte, F., Liautard, J. P., Kohler, S. Early acidification of phagosomes containing Brucella suis is essential for intracellular survival in murine macrophages. Infect Immun. 67, 4041-4047 (1999).
- Anderson, T. D., Cheville, N. F. Ultrastructural morphometric analysis of Brucella abortus-infected trophoblasts in experimental placentitis. Bacterial replication occurs in rough endoplasmic reticulum. Am J Pathol. 124, 226-237 (1986).
- Qin, Q. M., et al. RNAi screen of endoplasmic reticulum-associated host factors reveals a role for IRE1alpha in supporting Brucella replication. PLoS Pathog. 4, e1000110 (2008).
- Ramo, P., et al. Simultaneous analysis of large-scale RNAi screens for pathogen entry. BMC genomics. 15, 1162 (2014).
- Kuhbacher, A., Gouin, E., Cossart, P., Pizarro-Cerda, J. Imaging InlC secretion to investigate cellular infection by the bacterial pathogen Listeria monocytogenes. J Vis Exp. , e51043 (2013).
- Kentner, D., et al. Shigella reroutes host cell central metabolism to obtain high-flux nutrient supply for vigorous intracellular growth. Proc Natl Acad Sci U S A. 111, 9929-9934 (2014).
- Celli, J., Salcedo, S. P., Gorvel, J. P. Brucella coopts the small GTPase Sar1 for intracellular replication. Proc Natl Acad Sci U S A. 102, 1673-1678 (2005).
- von Bargen, K., Gorvel, J. P., Salcedo, S. P. Internal affairs: investigating the Brucella intracellular lifestyle. FEMS microbiology reviews. 36, 533-562 (2012).
- Kamentsky, L., et al. Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software. Bioinformatics. 27, 1179-1180 (2011).
