高通量并行测序在金黄叙利亚仓鼠感染期间测量钩端螺旋体螺旋体转插入突变体的适应性
1Veterans Affairs Greater Los Angeles Healthcare System, 2Departments of Medicine,David Geffen School of Medicine at University of California Los Angeles, 3Departments of Urology,David Geffen School of Medicine at University of California Los Angeles, 4Departments of Microbiology, Immunology, and Molecular Genetics,University of California Los Angeles
Summary
我们在这里描述的技术, 结合转诱变和高通量测序, 以确定和量化转端突变体在组织后的挑战, 仓鼠。该协议可用于筛选动物生存和传播的变种人, 也可用于体外研究。
Abstract
在这份手稿中, 我们描述了一个转测序 (Tn) 技术, 以确定和量化的钩端螺旋体螺旋体突变在健康期间改变了金黄色的叙利亚仓鼠感染。将随机转突变与高通量测序技术结合起来。动物受到转突变体 (输入池) 的挑战, 然后在几天后采集血液和组织, 以确定和量化每个器官 (产出池) 的突变株数。将输出池与输入池进行比较, 以评估每个突变体的体内适用性。这种方法可以在数量有限的动物中筛选出一大批突变体。稍加修改, 本协议可以与任何动物模型的钩端螺旋体病, 水库宿主模型, 如老鼠和急性感染模型, 如仓鼠, 以及体外研究。Tn-Seq 提供了一个强大的工具, 筛选的变种人与在体内和体外健身缺陷。
Introduction
由于可用的遗传工具数量有限, 某些细菌 (如钩端螺旋体) 的毒力基因鉴定很困难。一个常用的方法是创建一个突变体的随机转诱变后, 在每个突变体的插入点的识别和毒性测试的个别转突变动物模型。这种方法费时费钱, 需要大量的动物。
当随机诱变首次为病原体钩端螺旋体螺旋体时, 通过在动物模型1中测试个体突变株来鉴定致病基因。突变体的选择依据的标准, 如他们的潜在作用, 信号或运动或他们预测的外膜或表面位置。由于大多数端基因编码未知函数的假设蛋白质 2, 选择基于这些标准的突变体限制了发现新的端毒力基因的能力。
最近, L. 螺旋体转突变体的池在仓鼠和鼠模型3中被筛选出具有传染性。每只动物都被一个多达10变种人的水池所挑战。从血液和肾脏获得的培养物 PCR 检测出突变体的传染性。pcr 测试是费力的, 因为它需要一个单独的 pcr 反应为每个突变池。由于不同文化中每个突变体的频率没有被量化, 这种方法偏向于识别高度减毒突变体。
我们描述一个转测序 (Tn-Seq) 技术, 作为一种策略, 更有效地筛选毒力基因。Tn-seq 是由转诱变的变种人库的创建, 其次是大规模的并行排序4,5,6。简单地, 转突变体被汇集, 接种入动物, 并且以后从不同的器官恢复了 (输出水池)。从输出池的 DNA 提取和消化限制性酶或被超声。针对转插入点的交界处进行两轮 PCR。此步骤允许添加用于排序的必要适配器。通过高通量测序分析产生的 PCR 产物, 以确定池中每个突变体的转插入点及其相对丰度, 与突变体池的初始组成进行比较。
这种方法的主要优点是能够同时筛选大量的突变体和少量的动物。Tn-Seq 不需要事先知道的转插入点, 这增加了发现新的钩端螺旋体特定基因的机会, 涉及的毒力较少的时间和更高的效率。由于端在组织中的负担是相对较高的啮齿动物模型易受致命感染 (通常 104到 108细菌/g 的组织)7,8,9以及在水库主机10,11, 可以直接分析组织, 而无需培养, 减少由于体外生长而引起的偏见。
在迄今所描述的大多数细菌病原体的 Tn-Seq 研究中, 插入诱变的高频率允许感染的大池, 其中包含了变种, 在每个基因中都有多个紧密间隔的转插入物4 ,12,13,14。还开发了一种细菌, 其诱变频率远低于6。使用钩端螺旋体, 转突变体的库可以通过结合 Slamti et al15所描述的共轭动员质粒引入转的方法生成。然而, 转突变的频率在L. 螺旋体是低的。当在共轭质粒上引入Himar1转时, 报告的 transconjugant 频率仅为每个接收单元的 8.5 x 10-8 , 且其 Lai 应变为L. 螺旋体16 , 很可能是同样贫困与大多数其他菌株的L. 螺旋体。这里描述的协议部分基于为螺旋体螺旋体开发的, 其中转插入诱变的频率也是低的6。
对于我们的试验与协议17, 我们进行了转诱变与L. 螺旋体螺旋体 Manilae 菌株 L495 由于其他组的成功隔离转插入突变体在应变沿其低LD50 (致死剂量) 为毒力1。我们筛选了42变种人的 Tn, 并确定了几个突变候选者的毒力缺陷, 包括两个插入在候选腺苷酸苷基因。对仓鼠的两个突变体进行的单独检测证实它们的毒力不足17。
Protocol
注意:钩端螺旋体病株) 的致病菌株必须在生物安全2级 (BSL-2) 控制程序下处理。必须佩戴适当的个人防护装备 (PPE)。第二类生物安全柜必须用于致病性钩端螺旋体的所有操纵。 1. 创建转变种库15 通过共轭 (图 1) 将转转换成钩端螺旋体许可证. 将致病性钩端螺旋体?…
Representative Results
在L. 螺旋体中创建转突变体库需要一个过滤单元, 如图 1所示。我们从每个交配中恢复了 100-200 transconjugants 在每个突变体中, 通过对随机 pcr 生成的 pcr 产物进行测序, 以转和相邻宿主序列的末端为目标, 转插入点被识别为15 (图 2A)。随机 PCR 结果的一个例子显?…
Discussion
虽然我们的试验结果为仓鼠挑战腹腔与 42 L. 螺旋体突变体提出了17, 我们预计, 更大的突变池可以筛选的 Tn-Seq。由于 transconjugants 的频率较低 (100-200 transconjugants/交配), 因此需要数交配来产生足够数量的突变体进行大规模的 Tn-Seq 实验。在液态文化中维持大量的变种人, 这就带来了必须解决的后勤挑战。文化可以在深96好的板块中孵化。在 420 nm 的分光光度计上, 可以通过光学…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了退伍军人事务优异奖 (D.A.H.) 和国家卫生补助金 R01 AI 034431 (D.A.H.) 的支持。
Materials
| Kanamycin sulfate from Streptomyces kanamyceticus | Sigma-Aldrich | K4000 | |
| 2,6-diaminopimelic acid | Sigma-Aldrich | D1377 | |
| Spectinomycin dihydrochloride pentahydrate | Sigma-Aldrich | S4014 | |
| Axio Lab A1 microscope with a darkfieldcondenser | Zeiss | 490950-001-000 | |
| DNeasy blood and tissue kit | Qiagen | 69504/69506 | |
| MinElute PCR Purification | Qiagen | 28004/28006 | |
| QIAquick PCR purification kit | Qiagen | 28104/28106 | |
| Model 505 Sonic Dismembrator | Fisher Scientific | FB-505 | |
| 2.5" Cup horn | Fisher Scientific | FB-4625 | |
| Bead Ruptor 24 | Omni International | 19-010 | Step 3.1.2.4 |
| Terminal deoxynucleotidyl transferase | Promega | M828C | |
| Master mix Phusion | Thermo Scientific | F531 | Preparation of genomic libraries, step 3.4. |
| DreamTaq Master Mix | Thermo Scientific | K9011/K9012 | Identification of the transposon insertion site, step 1.2. |
| dCTP | Thermo Scientific | R0151 | |
| ddCTP | Affymetrix/ USBProducts | 77112 | |
| T100 Thermal cycler | BioRad | 1861096 | |
| Qubit 2.0 fluorometer | Invitrogen | Q32866 | step 3.6. |
| Qubit dsDNA HS assay kit | Invitrogen | Q32851/Q32854 | step 3.6. |
| Qubit assay tubes | life technologies | Q32856 | step 3.6. |
| PBS pH 7.2 (1X) | Gibco | 20012-027 20012-050 | |
| Disposable scalpel No10 | Feather | 2975#10 | |
| Plastic K2 EDTA 2 ml tubes | BD vacutainer | 367841 | |
| syringe U-100 with 26G x ½” needle | BD vacutainer | 329652 | IP challenge, step 2.2.1. |
| 3 mL Luer-Lok tip syringe | BD vacutainer | 309657 | Cardiac puncture, step 2.4.2. |
| 25G X 5/8” needle | BD vacutainer | 305901 | Cardiac puncture, step 2.4.2. |
| 25 mm fritted glass base with stopper | EMD Millipore | XX1002502 | Filtration unit system, step 1.1.7. |
| 25 mm aluminum spring clamp | EMD Millipore | XX1002503 | Filtration unit system, step 1.1.7. |
| 15 ml borosilcate glass funnel | EMD Millipore | XX1002514 | Filtration unit system, step 1.1.7. |
| 125 ml side-arm Erlenmeyer flask | EMD Millipore | XX1002505 | Filtration unit system, step 1.1.7. |
| Acetate-cellulose filter VVPP (pore size 0.1 mm; diameter 25 mm) | EMD Millipore | VVLP02500 |
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Citar este artigo
Lourdault, K., Matsunaga, J., Evangelista, K. V., Haake, D. A. High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection. J. Vis. Exp. (130), e56442, doi:10.3791/56442 (2017).