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Immunologia e infezioni

应用荧光共振能量转移（FRET）审查效应转运效率由<em>贝氏柯克斯体</em> siRNA沉默中

Published: July 06, 2016
doi:
10.3791/54210
, ,
1Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity,University of Melbourne

Summary

调查细菌病原体和宿主之间的相互作用是生物学研究的一个重要领域。在这里，我们描述了必要的技术测量使用布拉姆基板的siRNA的基因沉默过程中贝氏柯克斯体效应易位。

Abstract

贝氏柯克斯体 ，Q热的病原体，是一种细胞内病原体依赖于IV型点/ ICM分泌系统建立一个复制的利基。效应的队列，通过该系统转移进入宿主细胞来操纵主机进程和允许建立用于复制一个独特溶酶体衍生的液泡的。这里提出的方法涉及的两个成熟的技术相结合：使用的siRNA特异性基因沉默和使用依赖于β内酰胺酶活性的基于FRET的衬底效应易位测定。运用这两种方法，我们可以开始了解宿主因素在细菌分泌系统的功能和效应易位的作用。在此研究中，我们检查RAB5A和Rab7A，无论是内吞贩卖通路的重要调节器的作用。我们表明，沉默在效应translocatio下降或者蛋白质结果的表达氮效率。这些方法可以很容易地修改，以研究其他细胞内外的病原体也利用分泌系统。以这种方式，所涉及的细菌效应易位宿主因子的全局图像可以显现出来。

Introduction

贝氏柯克斯体是一种独特的细胞内病原体引起人畜共患的人类感染Q热。此疾病与延伸从无症状的血清转化到威胁生命的慢性感染，往往表现为心内膜炎年曝光1后临床 ​​表现的广谱相关联。人类感染主要通过与反刍动物感染的主要储存器，特别是奶牛，绵羊和山羊污染气溶胶的吸入发生。虽然感染贝氏在这些动物通常是亚临床型，感染可能引发流产和分娩流体和胎盘中的相当细菌量会污染当地环境1。巨大的负担，这种污染的一个例子可能对公共健康和农业产业化中的Q热疫情发生在荷兰2最近观察。 2007年之间，2010年，Q热4000人感染病例被确诊，这爆发挂山羊养殖场3显著污染。另外， 贝氏是潜在的生物武器，如划分由美国疾病控制和预防中，由于细菌的和必要的低感染剂量的环境稳定性造成严重的发病率和死亡率4。

贝氏存在于两个阶段：第一阶段的生物体，从天然来源分离的，是非常强毒和II期生物体在体内高度衰减。例如，在贝氏柯克斯体九里期生物体几个体外通路，制作了第二阶段的细菌包含一个不可逆转的染色体缺失导致截短的脂多糖（LPS）5。本品系，C. burnetii NMII，是表型上类似的I期的组织培养模型，并提供一个更安全的模式l对于研究人员研究实验室5发病贝氏 。近年来几个突破进展迅速贝氏遗传学领域。最值得注意的是，无菌媒体的发展（酸化柠檬酸半胱氨酸介质- ACCM-2）已经允许贝氏的无细胞生长在液体和固体培养基上6,7。这导致了包括可诱导的基因表达系统，穿梭载体和随机转座子系统8-11可贝氏遗传工具直接改进。最近，两种方法靶向的基因的失活也已经开发铺平用于检查特定毒力基因的候选12的方式。

肺泡巨噬细胞以下内化， 贝氏复制到高号码的膜-结合的隔室中称为含液泡（CCV）的Coxiella-。该CCV需要主机内吞贩运日粗糙的早期和晚期内涵体，直到成熟为溶酶体衍生的细胞器13。在这整个过程中，CCV获得，要么出现瞬时或保持与液泡相关联，包括但不限于，Rab5的，RAB7，CD63和LAMP-1月13日至15日宿主因素。在宿主细胞内的贝氏复制完全依赖于一个全功能点/ ICM型IVB分泌系统（T4SS）8,16,17。此分泌系统是祖先相关的共轭系统多蛋白质结构和跨越细菌和液泡膜提供细菌蛋白，称为效应，到宿主细胞质18。在贝氏 T4SS在功能上非常相似的特点以及IVB型点/ ICM分泌嗜肺军团菌 19,20制度。有趣的是，不会发生T4SS和随后的效应易位激活直到贝氏达到酸性溶酶体衍生细胞器，大约8小时后感染17,21。迄今为止，已有超过130点/ ICM效应已经确定9,17,22-24。许多这些效应的宿主细胞内的贝氏在复制过程中可能发挥重要作用;然而，只有少数的效应已在功能上表征25-29。

在这项研究中，我们利用依赖于通过在宿主细胞的细胞质内的β内酰胺酶的活性的CCF2-AM FRET底物（以下简称为BLAM基板）（ 图1）的裂解的基于荧光易位测定。感兴趣的基因融合到TEM-1上的报道质粒，提供组成型表达β内酰胺酶（BLAM）。的BLAM基板由两种荧光团（香豆素和荧光素），该形成FRET对。在荧光素和在不存在效应易位绿色荧光发射的FRET香豆素结果的激励;然而，如果该布拉M-效应融合蛋白易位到宿主胞质，所得β内酰胺酶活性裂解BLAM基板的β内酰胺环，分离FRET对生产下列激发的蓝色荧光发射。这种易位试验得到了证明作为一种方法，从各种不同的细胞内外的细菌，其中包括C.确定的效应蛋白burnetii，L.肺 ，L. longbeachae， 沙眼衣原体 ，肠致病性大肠杆菌 ， 沙门氏菌和布鲁氏菌 17,30-35。

为了确定对效应贝氏易位主机特定因素的作用下，我们采用了称为RNA干扰基因沉默一套行之有效的方法，特别是小干扰RNA（siRNA）。原本在秀丽隐杆线虫鉴定，RNA干扰是用于先天防卫厅一个保守的内源性细胞过程对病毒NSE以及基因调节36,37。序列特异性的siRNA结合后，mRNA的降解通过RISC发生（RNA诱导的沉默复合物），导致特异性基因沉默或击倒38。在这项研究中，将siRNA用于靶向两个主机的蛋白质，RAB5A和Rab7A，这是内吞途径的重要调节剂。沉默的效应易位RAB5A和Rab7A的影响是用C.确定burnetii pBlaM-CBU0077。 CBU0077被选定为它以前被证明由17 贝氏的点/ ICM分泌系统易位。

同时利用siRNA的基因沉默和这里所描述的fluorescencebased易位检测，我们开始建立的效应蛋白通过在贝氏的易位宿主因素的作用。这种方法可以适用于宽范围的具有类似secretio细胞内和细胞外的细菌N个系统负责效应蛋白的易位。

Protocol

注：所有涉及贝氏柯克斯体 RSA439 NMII的生长或操作过程应在物理遏制二级实验室，并在遵守当地指南的生物安全柜中进行。下面描述的反向转染和易位试验工作流程的示意图示于图2。 1. C.制备burnetii文化表达CBU0077融合到β-内酰胺酶（pBlaM-CBU0077）（第1天） 准备1X ACCM-2 6： 结合表1中列出的组分。调整用6M的NaOH…

Representative Results

对于这项研究中，C.被选定burnetii pBlaM-CBU0077菌株作为CBU0077先前已经证明是贝氏点/ ICM分泌系统17的易位效应。在感染前，的基因组/毫升在七天C.总数burnetii pBlaM-CBU0077培养，使用qPCR列举。 图4展示了循环阈值的一个例子来自标准和样品定量PCR如下预期（CT）的值。如在4.3.3中描述的Ct值（在扩增曲线（ ?…

Discussion

分泌系统，以及细菌效应蛋白这些系统输送到宿主细胞的细胞质中，是许多致病性细菌利用独特复制壁龛内建立的感染的重要毒力的组成部分。许多研究小组的重点是研究细菌效应和宿主蛋白和宿主细胞通路的影响，这些效应都之间的相互作用。非常有限的研究，如果有的话，已研究了宿主蛋白是必要成功细菌效应易位的潜力。

在这项研究中，我们使用了专，细胞内病原体<e…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

This work was supported by National Health and Medical Research Council (NHMRC) grants (Grant ID 1062383 and 1063646) awarded to HJN. EAL is supported by an Australian Postgraduate Award.

Materials

Reagents
Citric acid Sigma C0759 ACCM-2 medium component
Sodium citrate Sigma S4641 ACCM-2 medium component
Potassium phosphate Sigma 60218 ACCM-2 medium component
Magnesium chloride Calbiochem 442611 ACCM-2 medium component
Calcium chloride Sigma C5080 ACCM-2 medium component
Iron sulfate Fisher S93248 ACCM-2 medium component
Sodium chloride Sigma S9625 ACCM-2 medium component
L-cysteine Sigma C6852 ACCM-2 medium component
Bacto-neopeptone BD 211681 ACCM-2 medium component
Casamino acids Fisher BP1424 ACCM-2 medium component
Methyl beta cyclodextrin Sigma C4555 ACCM-2 medium component
RPMI + Glutamax ThermoFisher Scientific 61870-036 ACCM-2 medium component
Chloramphenicol Sigma C0378 For bacterial culture
ON-TARGETplus Non-targeting Control Pool (OTP) Dharmacon D-001810-10-05 Non-targeting control
siGENOME Human PLK1 (5347) siRNA – SMARTpool Dharmacon M-003290-01-005 Causes cell death; measure of transfection efficiency
siGENOME Human RAB5A (5968) siRNA – SMARTpool Dharmacon M-004009-00-0005
siGENOME Human RAB7A (7879) siRNA – SMARTpool Dharmacon M-010388-00-0005
5X siRNA buffer Dharmacon B-002000-UB-100 Use sterile RNase-free water to dilute 5X siRNA buffer to 1X siRNA buffer
DharmaFECT-1 Transfection Reagent Dharmacon T-2001-01 For transfection
Opti-MEM + GlutaMAX ThermoFisher Scientific 51985-034 Reduced-serum medium used for transfection
DMEM + GlutaMAX ThermoFisher Scientific 10567-014 For cell culture and infection
Heat-inactivated fetal calf serum (FCS) Thermo Scientific SH30071.03 Can use alternate equivalent product
DMSO Sigma D8418 For storage of Coxiella strain
PBS For cell culture
0.05% Trypsin + EDTA ThermoFisher Scientific 25300-054 For cell culture
dH2O For dilution of samples and standards for qPCR
Quick-gDNA Mini Prep ZYMO Research D3007 Can use alternate equivalent product to extract gDNA
SensiFAST SYBR No-ROX Kit Bioline BIO-98020 Can use alternate equivalent qPCR master mix product for qPCR reaction
LiveBLAzer FRET-B/G Loading kit with CCF2-AM Invitrogen K1032 For measurement of translocation using fluorescence and generation of the 6X loading solution (contains Solution A, B, C and DMSO)
Sodium hydroxide Merck 106469 Probenicid solution component
Sodium phosphate monobasic Sigma 71505 Probenicid solution component
di-Sodium hydrogen orthophosphate Merck 106586 Probenicid solution component
Probenicid Sigma P8761 Probenicid solution component
DRAQ5 Fluorescent Probe Solution (5 mM) ThermoFisher Scientific 62251 Nuclei stain to determine cell viablity. Use 1:4000 diluted in PBS. 
4% PFA (paraformaldehyde) solution in PBS Sigma P6148 Fixing solution for HeLa 229 cells
25cm2 tissue culture flask with vented cap Corning 430639 For growth of bacterial strain
96 Well Flat Clear bottom Black Polystyrene TC-Treated Microplates, Individually wrapped, with Lid, Sterile Corning 3603
75cm2 tissue culture flask with vented cap Corning 430641 For growth of HeLa 229 cells
175cm2 tissue culture flask with vented cap Corning 431080 For growth of HeLa 229 cells
Haemocytometer For quantification of HeLa 229 cells
Tear-A-Way 96 Well PCR plates 4titude 4ti-0750/TA For qPCR reaction
8-Lid chain, flat Sarstedt 65.989.002 For qPCR reaction
Name Company Catalog Number Comments
Equipment
Bench top microfuge
Bench top vortex
Orbital mixer
Centrifuge Eppendorf 5810 R For pelleting bacterial culture
Nanodrop For gDNA quantification
Mx3005P QPCR machine Aligent Technologies 401456 For quantification of Coxiella genomes in 7 day culture
ClarioSTAR microplate reader BMG LabTech For measurement of fluorescence
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Tags

Keywords: FRETEffector TranslocationSiRNA SilencingCoxiella BurnetiiCellular MicrobiologyHost-pathogen InteractionsChlamydiaSalmonellaE. ColiBeta-lactamaseACCM2HeLa CellsTransfection96-well Plate
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Newton, P., Latomanski, E. A., Newton, H. J. Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing. J. Vis. Exp. (113), e54210, doi:10.3791/54210 (2016).
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