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。

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
<ol><li>
通过共轭 (图 1) 将转转换成钩端螺旋体许可证.
	<ol>
<li>将致病性钩端螺旋体的指数相培养量与 107细胞相对应, 注入10毫升的 Ellinghausen----约翰逊-哈里斯介质 (EMJH)18,19。孵育在30° c 与 150 rpm 晃动, 直到密度达到 2-8 x 108细胞/毫升。 注意: 致病钩的加倍时间是12到 24 h, 这取决于菌株。</li>
		<li>接种50µL 的供体大肠杆菌菌株β216320携带动员转质粒 (pCjTKS2)16到5毫升 Luria 肉汤 (LB) 补充0.3 毫米 26-diaminopimelicacid (磷酸二铵), 50 µg/毫升卡那霉素 (公里) 和50µg/毫升的霉 (Spc) 和地方过夜在一个37° c 孵化器在 255 rpm。</li>
		<li>将60µL 的大肠杆菌细胞接种到3毫升的 EMJH, 辅以0.3 毫米的磷酸二铵 (EMJH + 磷酸二铵)。孵育在37° c 在 255 rpm 鼓动为 3-4 h 由 OD600nm≈0.3。</li>
		<li>要装配过滤单元 (图 1), 将底座放在125毫升侧臂锥形烧瓶上, 将醋酸纤维素过滤器 (孔径 0.1 mm; 直径 25 mm) 放置在底座上, 然后将漏斗夹在底座上。将过滤装置连接到真空系统。</li>
		<li>添加5毫升的钩端螺旋体。培养和0.5 毫升的大肠杆菌文化进入漏斗。通过过滤器将液体真空。</li>
		<li>将过滤器与细菌表面上的 EMJH + DAPplate。在30° c 的条件下, 在夜间孵化。</li>
		<li>将过滤器放入15毫升管中, 其中含有1毫升的 EMJH 和漩涡, 十年代将细菌释放到介质中。使用 10-15 1 毫米无菌玻璃珠或无菌一次性吊具, 将悬浮液的200µL 分布在 5 EMJH platescontaining 50 µg/毫升上。用膜把盘子包起来, 在30° c 的时候把它们倒挂在3至4周, 直到菌落可见。</li>
		<li>转移殖民地单独地入3毫升 EMJH 包含50µg/毫升公里 (EMJH + 公里) 在30° c 之下在 150 rpm 鼓动7到10天直到文化到达密度≈ 108/ml。 注: 培养物可贮存在-80 ° c 或液氮 (含4% 甘油)。</li>
	</ol>
</li>
	<li>
通过嵌套 PCR 识别转插入站点 (图 2)
	<ol>
<li>溶解50µL 的每转突变体 PCR 管由孵育在95° c 为 15 min。 注意: dna 可以被纯化, 使用 dna 提取试剂盒。</li>
		<li>根据表 1, 用 Deg1 和 Tnk1 底漆 (表 3) 准备 PCR 混合。转移23.7 µL 的混合物到每个 PCR 管, 并增加1.3 µL 的裂解细胞。运行程序:95 5 分钟;40周期:95 ° c 为十五年代, 40 ° c 为 1 min, 72 ° c 为 2 min;72° c 为 10 min。</li>
		<li>根据表 2, 使 PCR 与标记和 TnkN1 底漆 (表 3) 混合。将24.2 µL 的混合物转移到每个 pcr 管中, 并添加0.8 µL pcr #1 反应。运行程序:95 ° c 为5分钟;35周期:95 ° c 为十五年代, 55 ° c 为三十年代, 72 ° c 为 2 min;72° c 为 10 min。</li>
		<li>在10-15 伏/cm (图 2B) 的1% 琼脂糖凝胶上运行3µL 的 PCR 产品。</li>
		<li>用 pcr 纯化试剂盒提纯阳性样品的 pcr 产物。洗 dna 与试剂盒所允许的最低体积, 以最大限度地提高 dna 的浓度恢复。</li>
		<li>使用 TnkN1 底漆 (表 3) 发送用于桑格测序的纯化 PCR 产品。</li>
		<li>通过 BLASTN 分析 (http://blast.ncbi.nlm.nih.gov/) 或使用 SpiroScope 数据库 (http://www.genoscope.cns.fr/agc/mage)21, 通过将结果序列与父级应变的基因组序列进行比较来识别插入点。</li>
		<li>用引物退火法对侧翼宿主序列进行转的插入点确认。 注意: 转通过≈ 2 kb 增加了野生类型序列的大小。</li>
	</ol>
</li>
</ol>2. 动物实验 (图 3)
<ol><li>
钩端螺旋体变种的区域性
	<ol>
<li>分别生长在10毫升 EMJH + 公里在30° c 在 150 rpm 鼓动到密度 107-108钩/毫升的转突变体。</li>
		<li>用暗场显微镜和 Petroff-豪塞尔计数器或米勒23所描述的钩计数。</li>
		<li>将 EMJH 中的每个区域性稀释为相同的密度, 例如 106单元格/mL。</li>
		<li>要装配输入池, 将稀释的区域性混合在一起。 注意: 在输入池中加入控件, 添加已知的适应缺陷的变种, 如loa2217、24和不变的适应性的变种, 如ligB17,25,分别.突变体的池可以在-80 ° c 或液氮中储存4% 的甘油。</li>
	</ol>
</li>
	<li>
挑战 注: 这里描述的方法由退伍军人事务大洛杉矶机构动物保护和使用委员会 (协议 #09018-14) 批准。<ol>
<li>注射腹腔1毫升的输入池的每一个动物与胰岛素注射器 U-100 与 26G x ½ "针。 注意: 在试验中, 8 只动物被挑战1毫升的输入池, 即 106细菌总数17。这种感染可以在安乐死前4天进行。</li>
		<li>收集10毫升的输入池, 并旋转20分钟在 3220 x g. 小心清除上清, 而不干扰颗粒。储存细胞颗粒在80˚C 直到使用 (步骤 3.1.3)。</li>
		<li>每天监视动物, 直到它们在预定终点终止。每天称仓鼠, 寻找终点标准: 食欲不振、步态或呼吸困难、虚脱、皱毛或 > 10% 的最大重量损失。</li>
	</ol>
</li>
	<li>
体外实验
	<ol>
<li>在挑战日, 接种3瓶25毫升的 EMJH + 公里与5毫升的输入池。增长文化在30° c 在 150 rpm 鼓动之下。</li>
		<li>使用节2.1.2 的方法之一计算钩的每日计数。当密度达到≈ 1 x 108/毫升, 旋转下来的每一个暂停20分钟, 在 3220 x g。</li>
		<li>将细胞小球储存在-80 ° c 直到使用。</li>
	</ol>
</li>
	<li>
组织的收获和储存
	<ol>
<li>用异氟醚吸入安乐, 其次是双侧胸切口26。</li>
		<li>立即收集1至2毫升的血液心脏穿刺与3毫升注射器和 25G x 5/8 "针。将血液转移到含有 EDTA 的试管中。混合由反演, 5 到6次。</li>
		<li>收集一个肾脏和⅓到½腹正中叶的肝 cryotubes。</li>
		<li>在-80 ° c 贮存组织直至使用。</li>
	</ol>
</li>
</ol>3. 构建用于高通量排序的基因组库 (图 4)
<ol><li>
DNA 提取
	<ol>
<li>从血液中提取 DNA<ol>
<li>将100µL 的血液从 EDTA 管转移到离心管。</li>
			<li>使用 dna 提取试剂盒净化 dna。按照制造商的说明进行操作。</li>
		</ol>
</li>
		<li>从组织中提取 DNA<ol>
<li>使用手术刀和剪刀, 骰子每个器官50至80毫克的小块 (1 毫米 x 1 毫米), 并转移到一个无菌的螺旋帽干管。用精确的天平测量组织的重量。</li>
			<li>在试管中加入500µL 的无菌 PBS。</li>
			<li>质样品, 使用干扰1分钟, 在5运动每秒。</li>
			<li>使用以下等式计算与25毫克组织相对应的体积: <img alt="Equation 1" src="/files/ftp_upload/56442/56442eq1.jpg">
</li>
			<li>将计算出的体积转移到 DNA 提取试剂盒中。按照制造商的指示进行 DNA 纯化。</li>
		</ol>
</li>
		<li>从输入池和体外培养中提取 DNA。<ol>
<li>常温解冻细菌颗粒5-10 分钟。</li>
			<li>按照随试剂盒的指示进行 DNA 提取。</li>
		</ol>
</li>
		<li>将 DNA 储存在-80 ° c 直到使用。</li>
	</ol>
</li>
	<li>
剪切 DNA (图 5)
	<ol>
<li>转移50µL 的提取 DNA 到1.5 毫升离心管。</li>
		<li>把管子放入 sonicator 杯垫的架子上, 装满冷水 (4 ° c)。</li>
		<li>运行 sonicator 3 分钟, 在80% 强度与十年代脉冲和 5 s 关闭脉冲。 注意: 戴上耳罩或耳塞以保护听觉。</li>
		<li>在2% 琼脂糖凝胶上运行2.5 µL 的剪切 dna, 以确认大部分的 dna 是 < 600 bp 的大小。</li>
	</ol>
</li>
	<li>
加法 C 尾巴
	<ol>
<li>用小体积分光光度计测量 DNA 浓度。</li>
		<li>使用以下等式计算与 500 ng DNA 对应的体积: <img alt="Equation 2" src="/files/ftp_upload/56442/56442eq2.jpg">
</li>
		<li>准备尾矿反应 (表 4)。在37° c 下孵育1小时;在75° c 下钝化20分钟。 注: 对于需要调整到大于14.5 µL 的体积的样品, 将反应的最终体积增加到40µL, 并相应地扩大剩余的元件。</li>
		<li>用 PCR 纯化试剂盒清洗样品。用12µL 洗脱缓冲液洗 DNA。</li>
	</ol>
</li>
	<li>
巢式 PCR
	<ol>
<li>PCR #1<ol>
<li>根据表 3和5准备 PCR 混合。转移22µL 的图书馆混合到每个 PCR 管和添加3µL 纯化 DNA。与控制组合进行类似的操作。 注: 底漆 TnkN317特定于转, 而底漆 olj3766是特定于 C 尾的。控制组合缺乏 TnkN3 底漆, 专门针对转。</li>
			<li>运行以下程序:95 ° c 为2分钟;24周期:95 ° c 为三十年代, 60 ° c 为三十年代, 72 ° c 为 2 min;72° c 为 2 min。</li>
		</ol>
</li>
		<li>PCR #2。<ol>
<li>根据表 3和6准备第二个 PCR 混合。将49µL 的文库混合到每个 pcr 管中, 并添加1µL 的 pcr #1 文库反应。将控制组合的24.5 µL 转移到每个 pcr 管上, 并添加0.5 µL pcr #1 控制反应。 注: 底漆 pMargent2 特定于转, 而 IP 引物6包含六基对条码和由下一代测序平台识别的特定序列。</li>
			<li>运行以下程序:95 ° c 为2分钟;18周期:95 ° c 为三十年代, 60 ° c 为三十年代, 72 ° c 为 2 min;72° c 为 2 min。</li>
		</ol>
</li>
		<li>在2% 琼脂糖凝胶上运行3µL。图书馆应该显示一个涂片以信号的多数在200到 600 bp (图 6) 和没有放大为控制反应。</li>
	</ol>
</li>
	<li>
PCR 产物的纯化。 注意: 按照制造商的说明, 用 PCR 纯化试剂盒清洁基因组库。用30µL 洗脱缓冲液洗 DNA。</li>
	<li>用荧光测量 DNA 浓度。平均2到3读。</li>
	<li>使用下面的等式计算每个库的容量, 相当于15或 20 ng: <img alt="Equation 3" src="/files/ftp_upload/56442/56442eq3.jpg">
</li>
	<li>
根据以前的计算将所有库混合在一起.用以下网站的公式确定 DNA 的摩尔浓度: http://www.molbiol.edu.ru/eng/scripts/01_07.html 注意: DNA 浓度和体积要求取决于测序平台。</li>
</ol>4. 高通量测序和数据分析
<ol><li>序
	<ol>
<li>序列库作为 64 bp 单读取使用自定义测序底漆 pMargent3 和标准的商业测序底漆。测序平台将为您提供 FastQ 的文件和所有的顺序读取。</li>
	</ol></li>
	<li>分析与银河软件
	<ol><li>下载基因组序列文件<ol>
<li>在 SpiroScope 数据库主页 (参见步骤1.2.7 为链接), 选择用于实验的有机体并且点击 "装载入基因组浏览器"。</li>
			<li>在工具栏 (位于主页顶部附近), 选择 "搜索/导出 > 下载数据", 在 "序列 (fasta)" 行中, 单击 "基因组" 下载序列。</li>
			<li>打开文件与记事本 (PC) 或 "(Mac) 和重命名的染色体 (例如" 上帝 ")。保持 fasta 格式。</li>
			<li>按照相同的步骤下载染色体 II. 序列通过复制和粘贴将两个染色体序列组合成一个 .txt 文件。 注意: 还可以从 NCBI 网站 (https://www.ncbi.nlm.nih.gov) 下载染色体序列, 并将其合并为单个 .txt 文件。</li>
		</ol></li>
		<li>将文件上载到 Galaxy 服务器 注: Galaxy 是一个开源的网络平台, 用于管理数据密集型的生物信息学工作流27,28,29,30 , 可以在 https://usegalaxy.org/访问。<ol>
<li>在 "工具" 菜单中, 选择 "从计算机获取数据 > 上传文件"。将由测序平台生成的. fastq 文件拖放到窗口中, 然后单击 "开始"。</li>
			<li>按照相同的步骤上载钩端螺旋体基因组序列. txt 文件。</li>
		</ol></li>
		<li>新郎阅读<ol>
<li>从 "工具" 菜单中选择 "NGS: QC 和操作 > FASTQ 美容师"。</li>
			<li>在 "文件到新郎" 旁边, 选择 "步骤4.2.2 中上载的库"。在输入 FASTQ 质量分数类型中, 选择适当的测序系统。对于高级选项, 请保留 "隐藏高级 Office 选定"。</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>删除序列工件<ol>
<li>选择 "NGS: QC 和操作 > 删除序列工件"。在要筛选的库旁边, 选择在步骤4.2.3 中生成的整理文件。单击 "执行"。</li>
		</ol></li>
		<li>删除 C 尾序列 注意: 重复这些步骤一两次, 以确保所有的 C 尾被删除。<ol>
<li>选择 "NGS: QC 和操作 > 剪辑适配器序列"。</li>
			<li>选择或输入以下内容: 库到剪辑: 选择在步骤4.2.4 中生成的文件。 最小序列长度:15 来源: 输入自定义序列 输入自定义剪切序列: CCCCCCC 输入非零值以保持适配器序列和跟随它的 x 基地: 0 丢弃具有未知 (N) 基的序列: 是 输出选项: 输出剪切和 non-clipped 序列</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>删除适配器序列<ol>
<li>选择 "NGS: QC 和操作 > 剪辑适配器序列"。</li>
			<li>选择或输入以下内容:</li>
			<li>库到剪辑: 选择在步骤4.2.5 中生成的文件。</li>
			<li>最小序列长度:15</li>
			<li>来源: 输入自定义序列</li>
			<li>输入自定义剪切序列: CGTATGCCGTCTTCTGCTTG</li>
			<li>输入非零值以保持适配器序列和跟随它的 x 基地: 0</li>
			<li>丢弃具有未知 (N) 基的序列: 是</li>
			<li>输出选项: 输出剪切和 non-clipped 序列</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>基于其质量的筛选器读取<ol>
<li>选择 "NGS: QC 和操作 > 按质量过滤"。 注意: 此工具根据质量分数选择读取。</li>
			<li>选择以下内容: 要筛选的库: 选择在步骤4.2.6 中生成的文件。 质量截止值:20 必须有质量等于/大于截止值的序列的基数百分比:95</li>
			<li>单击 "执行"。 注: 与这些设置, 读短于20核苷酸或以质量比分20或较少为95% 的周期被摒弃。调整你的实验的严谨设置。</li>
		</ol></li>
		<li>映射读取32<ol>
<li>选择 "NGS: 映射 > Bowtie2"32。</li>
			<li>在主窗口的字段中选择以下内容: 这是单个的还是配对的库: 单 FASTQ 文件: 从步骤4.2.7 中选择过滤质量的库。 写入齐 (以 fastq 格式) 读取到单独的文件 (s): 否 写对齐 (以 fastq 格式) 读取到单独的文件: 否 您是否会从历史记录中选择一个参考基因组或使用内置索引？: 使用历史记录和生成索引中的基因组 选择参考基因组: 选择在步骤4.2.2 中上传的钩端螺旋体 genome.txt 文件。 设置读取组信息: 不设置 选择分析模式: 1: 仅限默认设置 是否要使用预设？: 否, 仅使用默认值 将 bowtie2 映射统计信息保存到历史记录中: 否 作业资源参数: 使用默认作业资源参数</li>
			<li>单击 "执行" 以将读取与基因组对齐。</li>
		</ol></li>
		<li>转换文件<ol>
<li>选择 "NGS: SAMtools > BAM-to-SAM 转换 BAM 为 SAM"。</li>
			<li>选择以下内容: 要转换的 BAM 文件: 从步骤4.2.8 中选择映射库。 标题选项: 在 SAM 输出中包含页眉 (-h)</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>转换文件<ol>
<li>选择 "NGS: SAMtools > 转换 SAM 为间隔"。</li>
			<li>选择以下内容: 选择要转换的数据集: 选择在步骤4.2.9 中生成的 SAM 映射库文件。 全部打印: 是</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>排序读取<ol>
<li>选择 "按升序或降序对数据进行筛选和排序 > 排序"。</li>
			<li>选择以下内容: 排序数据集: 选择在步骤4.2.10 中生成的间隔文件。 上一栏: 2 具有风味: 数字顺序 所有内容: 升序排列</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>选择读取匹配的染色体 I<ol>
<li>选择 "筛选并排序 > 选择与表达式匹配的行"。</li>
			<li>选择以下内容: 选择行: 选择在步骤4.2.11 中生成的排序读取的文件。 : 匹配 模式: 输入在步骤4.2.1.3 中确定的 I 染色体的名称 (例如, "上帝")。</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>选择读取匹配的染色体 II 继续下面的步骤4.2.12。</li>
		<li>组读取根据插入点的染色体 I<ol>
<li>选择 "联接、减去和分组 > 按列对数据进行分组, 并对其他列执行聚合操作"。</li>
			<li>选择以下内容: 选择数据: 从步骤4.2.12 中选择结果文件。 按列分组: 2 分组时忽略大小写: 否 忽略以下列字符开头的行: Ø 操作 > + 插入操作 类型: 计数 上一栏: 2 舍入结果为最接近的整数: 否</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>根据第二号染色体的插入部位进行组读 继续下面的步骤4.2.14。</li>
		<li>在 I 染色体上排序插入点<ol>
<li>选择 "按升序或降序对数据进行筛选和排序 > 排序"。</li>
			<li>选择以下内容: 排序数据集: 从步骤4.2.14 中选择文件。 上一栏: 1 具有风味: 数字顺序 所有内容: 升序排列</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>在第二号染色体上排序插入点 继续下面的步骤4.2.16。</li>
	</ol></li>
	<li>统计分析
	<ol>
<li>通过从步骤4.2.16 复制和粘贴两列, 将 Galaxy 数据传输到电子表格文件。和4.2.17。到 Excel 文件中。 注意: 第一列是转插入站点的核苷酸坐标, 第二列是每个插入站点上的读取数。</li>
		<li>使用表中提供的核苷酸坐标来识别窝藏转的基因。 注: 例如, 在 i 染色体的核苷酸 #30718 中插入的转位于LIC10024基因中, 它在染色体 i 中跨越核苷酸29263-31539。</li>
		<li>计算每个组织中的每个突变体的相对频率 (F), 然后在下面的等式中输入池: <img alt="Equation 4" src="/files/ftp_upload/56442/56442eq4.jpg">
</li>
		<li>使用下面的等式计算每个突变体和组织的输出/输入比率 (R): <img alt="Equation 5" src="/files/ftp_upload/56442/56442eq5.jpg">
</li>
		<li>魏氏签名测试<ol>
<li>通过将每种动物组织中的中位比率设置为 1.0, 使所有的输出/输入比率正常化。1.0 的比率是中性的, > 1.0 是不利的, < 1.0 是有利的33。</li>
			<li>使用魏氏秩测试与 P 值 < 0.05 进行比较, 将输出/输入比率与 1.0 (中性适应) 进行对比。</li>
		</ol>
</li>
	</ol></li>
</ol>

<ol>
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作者没有什么可透露的。

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, 可以通过光学密度读数来监测培养密度。要使用的动物数量的确定部分取决于输入池的大小, 并且必须通过功率分析来确定。对于 large-scale 实验, 我们建议在与生物进行协商时进行功率分析。
瓶颈会影响来自输出池的随机突变体的恢复。虽然在我们的试验研究17 (图 7) 中没有发现严重的瓶颈, 但 large-scale 实验可能会受到突变体随机损失的影响。通过增加输入池中每个突变体的单元数来增加挑战剂量, 将使35瓶颈的可能性降到最低。新发布的 Galaxy 工具洋红色提供了必要的工具来评估瓶颈和适用性31。
为替代感染途径、其他钩端螺旋体菌株或其他啮齿动物模型计划的 Tn-Seq 试验需要额外的考虑。如果在被检查的每个组织的感染过程中, 寄主物种的感染动力学不为人知或没有连续的指数, 则应从组织的培养中获取 DNA, 而不是直接从组织中获得。这一额外的步骤将减少高估突变体的适应性由于检测 DNA 从死螺旋体。如果从培养组织中获取输出池, 则应培养输入池以解决体外生长的影响。我们还建议用少量突变体进行试验, 以确定瓶颈是否会引起关注, 并帮助进行 large-scale 实验的功率分析。
根据 Tn-Seq 确定的改变体质的确认将需要对动物模型中的个体突变体进行测试。目前, 每个突变体必须在 Tn-Seq 之前单独测序, 以确定在感兴趣的基因中携带插入的变种。然而, 如果致病性的钩端螺旋体中的等位基因替换变得容易, 个体突变体的测序将不再是必要的。另外, 转录激活剂类似的效应已经成功地用于减少L. 螺旋体36中的lig基因表达式。该技术可用于下调节突变体中被 Tn-Seq 鉴定的改变的基因。
在解释 Tn-Seq 数据时, 细菌之间的相互作用应该被考虑在内。从理论上讲, 降低体质可能是由于突变体之间的竞争, 而不是转插入的直接影响。此外, 在一个池中的突变体的不改变在体内的适应性可能是由于变种人之间的合作。例如, 一个突变体不能产生一个必要的因素可以补充 intercellularly 由生产的因素由其他变种人在池中。我们观察到, 一些突变体的适应度有所提高, 这可能是由于不再合成蛋白质的新陈代谢负担减少了, 而这对生长是不必要的, 如沙门氏菌 enterica37所示。
该协议可用于确定在压力条件下的新陈代谢或生存所涉及的基因在体外 38,39。例如, 在不同的条件下 (如高氯化钠浓度、有限的铁和酸性 pH 值) 中生长的钩端螺旋体) 可以识别出负责酸性生存或抗应力的基因。这些体外实验也可以用腐生菌株进行, 如L. biflexa应变 Patoc I, 因为这种 Tn-Seq 方法可以应用于所有测序的钩端螺旋体菌株。

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。

<table>
	<tbody>
		<tr>
			<td>Kanamycin sulfate from Streptomyces kanamyceticus</td>
			<td>Sigma-Aldrich</td>
			<td>K4000</td>
			<td></td>
		</tr>
		<tr>
			<td>2,6-diaminopimelic acid</td>
			<td>Sigma-Aldrich</td>
			<td>D1377</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectinomycin dihydrochloride pentahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S4014</td>
			<td></td>
		</tr>
		<tr>
			<td>Axio Lab A1 microscope with a darkfieldcondenser</td>
			<td>Zeiss</td>
			<td>490950-001-000</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy blood and tissue kit</td>
			<td>Qiagen</td>
			<td>69504/69506</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute PCR Purification</td>
			<td>Qiagen</td>
			<td>28004/28006</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104/28106</td>
			<td></td>
		</tr>
		<tr>
			<td>Model 505 Sonic Dismembrator</td>
			<td>Fisher Scientific</td>
			<td>FB-505</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5&#34; Cup horn</td>
			<td>Fisher Scientific</td>
			<td>FB-4625</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead Ruptor 24</td>
			<td>Omni International</td>
			<td>19-010</td>
			<td>Step 3.1.2.4</td>
		</tr>
		<tr>
			<td>Terminal deoxynucleotidyl transferase</td>
			<td>Promega</td>
			<td>M828C</td>
			<td></td>
		</tr>
		<tr>
			<td>Master mix Phusion</td>
			<td>Thermo Scientific</td>
			<td>F531</td>
			<td>Preparation of genomic libraries, step 3.4.</td>
		</tr>
		<tr>
			<td>DreamTaq Master Mix</td>
			<td>Thermo Scientific</td>
			<td>K9011/K9012</td>
			<td>Identification of the transposon insertion site, step 1.2.</td>
		</tr>
		<tr>
			<td>dCTP</td>
			<td>Thermo Scientific</td>
			<td>R0151</td>
			<td></td>
		</tr>
		<tr>
			<td>ddCTP</td>
			<td>Affymetrix/ USBProducts</td>
			<td>77112</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal cycler</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 fluorometer</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td>step 3.6.</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS assay kit</td>
			<td>Invitrogen</td>
			<td>Q32851/Q32854</td>
			<td>step 3.6.</td>
		</tr>
		<tr>
			<td>Qubit assay tubes</td>
			<td>life technologies</td>
			<td>Q32856</td>
			<td>step 3.6.</td>
		</tr>
		<tr>
			<td>PBS pH 7.2 (1X)</td>
			<td>Gibco</td>
			<td>20012-027 20012-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable scalpel No10</td>
			<td>Feather</td>
			<td>2975#10</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic K2 EDTA 2 ml tubes</td>
			<td>BD vacutainer</td>
			<td>367841</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe U-100 with 26G x &#189;&#8221; needle</td>
			<td>BD vacutainer</td>
			<td>329652</td>
			<td>IP challenge, step 2.2.1.</td>
		</tr>
		<tr>
			<td>3 mL Luer-Lok tip syringe</td>
			<td>BD vacutainer</td>
			<td>309657</td>
			<td>Cardiac puncture, step 2.4.2.</td>
		</tr>
		<tr>
			<td>25G X 5/8&#8221; needle</td>
			<td>BD vacutainer</td>
			<td>305901</td>
			<td>Cardiac puncture, step 2.4.2.</td>
		</tr>
		<tr>
			<td>25 mm fritted glass base with stopper</td>
			<td>EMD Millipore</td>
			<td>XX1002502</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>25 mm aluminum spring clamp</td>
			<td>EMD Millipore</td>
			<td>XX1002503</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>15 ml borosilcate glass funnel</td>
			<td>EMD Millipore</td>
			<td>XX1002514</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>125 ml side-arm Erlenmeyer flask</td>
			<td>EMD Millipore</td>
			<td>XX1002505</td>
			<td>Filtration unit system, step 1.1.7.</td>
		</tr>
		<tr>
			<td>Acetate-cellulose filter VVPP (pore size 0.1 mm; diameter 25 mm)</td>
			<td>EMD Millipore</td>
			<td>VVLP02500</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-throughput parallel sequencing to measure fitness of leptospira interrogans transposon insertion mutants during golden syrian hamster infection

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。

Watch this Scientific Journal Video about High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection at JoVE.c

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

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。

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

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

high-throughput-parallel-sequencing-to-measure-fitness-leptospira

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

8.8K 次观看.  Veterans Affairs Greater Los Angeles Healthcare System. 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。

视频: High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

科研

免疫与感染

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.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

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 Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

高通量基因定位在<em&gt;布氏锥虫</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

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.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

复制的有序, 非冗余图书馆的铜绿假单胞菌应变 PA14 座子插入突变体

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

哺乳动物细胞血浆膜检测效率的高通量测量

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. NETs have been demonstrated to serve as an important host defense mechanism that traps and kills microorganisms. On the other hand, they have been implicated in diverse systemic autoimmune diseases. NETs are immunogenic and toxic structures that contain a pool of relevant autoantigens including anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) and systemic lupus erythematosus (SLE). Different forms of NETs can be induced depending on the stimulus. The amount of NETs can be quantified using different techniques including measuring DNA release in supernatants, measuring DNA-complexed with NET-molecules like myeloperoxidase (MPO) or neutrophil elastase (NE), measuring the presence of citrullinated histones by fluorescence microscopy, or flow cytometric detection of NET-components which all have different features regarding their specificity, sensitivity, objectivity, and quantity. Here is a protocol to quantify ex vivo NET formation in a highly-sensitive, high-throughput manner by using three-dimensional immunofluorescence confocal microscopy. This protocol can be applied to address various research questions about NET formation and degradation in health and disease.

This protocol describes a highly sensitive and high throughput neutrophil extracellular trap (NET) assay for the semi-automated quantification of ex vivo NET formation by immunofluorescence three-dimensional confocal microscopy. This protocol can be used to evaluate NET formation and degradation after different stimuli and can be used to study potential NET-targeted therapies.

一种评估和定量中性粒细胞外细胞陷阱形成的高通量检测

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

在血清型 1 型肺炎链球菌 菌株 519/43 中构建突变体

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...