Brucella melitensis bacteria cause persistent, intracellular infections in small ruminants as well as in humans, leading to significant morbidity and economic loss worldwide. The majority of experiments on the transcriptional responses of Brucella to conditions inside the host have been performed following invasion of cultured mammalian cells, and do not address gene expression patterns during long-term infection. Here, we examine the application of the previously developed coincidence cloning methodology to recover and characterize B. melitensis RNA from the supramammary lymph node of experimentally-infected goats. Using coincidence cloning, we successfully recovered Brucella RNA from supramammary lymph nodes of B. melitensis-infected goats at both short-term (4 weeks) and long-term (38 weeks) infection time points. Amplified nucleic acid levels were sufficient for analysis of Brucella gene expression patterns by RNA-sequencing, providing evidence of metabolic activity in both the short-term and the long-term samples. We developed a workflow for the use of sequence polymorphism analysis to confirm recovery of the inoculated strain in the recovered reads, and utilized clustering analysis to demonstrate a distinct transcriptional profile present in samples recovered in long-term infection. In this first look at B. melitensis gene expression patterns in vivo, the subset of Brucella genes that was highly upregulated in long-term as compared to short-term infection included genes linked to roles in murine infection, such as genes involved in proline utilization and signal transduction. Finally, we demonstrated the challenges of qPCR validation of samples with very low ratios of pathogen:host RNA, as is the case during in vivo brucellosis, and alternatively characterized intermediate products of the coincidence cloning reaction. Overall, this study provides the first example of recovery plus characterization of B. melitensis RNA from in vivo lymph node infection, and demonstrates that the coincidence cloning technique is a useful tool for characterizing in vivo transcriptional changes in Brucella species. Genes upregulated in long-term infection in this data set, including many genes not previously demonstrated to be virulence factors in mice or macrophage experiments, are candidates of future interest for potential roles in Brucella persistence in natural host systems.

中文翻译：

---

从山羊组织中克隆布鲁氏菌RNA的巧合恢复：推进布鲁氏菌病中病原体基因表达的体内分析。

布鲁氏菌细菌在小反刍动物和人类中引起持续的细胞内感染，从而导致全世界范围内严重的发病率和经济损失。布鲁氏菌对宿主内部条件的转录反应的大多数实验是在入侵培养的哺乳动物细胞后进行的，并且不能解决长期感染过程中的基因表达模式。在这里，我们检查了先前开发的巧合克隆方法在从受感染山羊的超淋巴结中回收和鉴定B. melitensis RNA的应用。使用巧合克隆，我们在短期（4周）和长期（38周）感染时间点成功地从感染了B. melitensis的山羊的超细菌淋巴结中回收了布鲁氏菌RNA。扩增的核酸水平足以通过RNA测序分析布鲁氏菌基因表达模式，为短期和长期样品的代谢活性提供了证据。我们开发了一种工作流程，用于使用序列多态性分析来确认已恢复的读数中接种菌株的恢复，并利用聚类分析来证明长期感染中恢复的样品中存在独特的转录谱。在首次观察体内的B. melitensis基因表达模式时，与短期感染相比，长期高度上调的布鲁氏菌基因子集包括与鼠类感染相关的基因，例如脯氨酸利用相关基因和信号转导。最后，我们证明了病原体：宿主RNA比例极低的样品进行qPCR验证所面临的挑战（如体内布鲁氏菌病期间的情况），以及特征在于重合克隆反应的中间产物。总的来说，这项研究提供了从体内淋巴结感染中恢复和鉴定meliensis B. RNA的第一个实例，并证明了同时克隆技术是表征布鲁氏菌属物种体内转录变化的有用工具。在该数据集中长期感染中上调的基因，包括许多先前未在小鼠或巨噬细胞实验中证明为毒力因子的基因，对于布鲁氏菌在天然宿主系统中的持久性潜在作用可能是未来感兴趣的候选者。如在体内布鲁氏菌病期间一样，并且其特征是同时克隆反应的中间产物。总的来说，这项研究提供了从体内淋巴结感染中恢复和鉴定meliensis B. RNA的第一个实例，并证明了同时克隆技术是表征布鲁氏菌属物种体内转录变化的有用工具。在该数据集中长期感染中上调的基因，包括许多先前未在小鼠或巨噬细胞实验中证明为毒力因子的基因，对于布鲁氏菌在天然宿主系统中的持久性潜在作用可能是未来感兴趣的候选者。如在体内布鲁氏菌病期间一样，并且其特征是同时克隆反应的中间产物。总的来说，这项研究提供了从体内淋巴结感染中恢复和鉴定meliensis B. RNA的第一个实例，并证明了同时克隆技术是表征布鲁氏菌属物种体内转录变化的有用工具。在该数据集中长期感染中上调的基因，包括许多以前未在小鼠或巨噬细胞实验中证明为致病因子的基因，对于布鲁氏菌在天然宿主系统中的持久性潜在作用可能是未来感兴趣的候选者。这项研究提供了从体内淋巴结感染中恢复和鉴定B. melitensis RNA的第一个实例，并证明了同时克隆技术是表征布鲁氏菌属物种体内转录变化的有用工具。在该数据集中长期感染中上调的基因，包括许多先前未在小鼠或巨噬细胞实验中证明为毒力因子的基因，对于布鲁氏菌在天然宿主系统中的持久性潜在作用可能是未来感兴趣的候选者。这项研究提供了从体内淋巴结感染中恢复和鉴定B. melitensis RNA的第一个实例，并证明了同时克隆技术是表征布鲁氏菌属物种体内转录变化的有用工具。在该数据集中长期感染中上调的基因，包括许多先前未在小鼠或巨噬细胞实验中证明为毒力因子的基因，对于布鲁氏菌在天然宿主系统中的持久性潜在作用可能是未来感兴趣的候选者。