Small RNAs of Bartonella bacilliformis; the agent of Carrion's disease in humans

杆状巴尔通体的小RNA；

基本信息

批准号：
9227738
负责人：
Michael F Minnick
金额：
$ 22.7万
依托单位：
UNIVERSITY OF MONTANA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-12-01 至 2018-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9227738
关键词：
Acute Address Altitude Andean Area Arthropod Vectors Arthropods Bacteremia Bacteria Bartonella Bartonella Infections Bartonella bacilliformis Biological Process Biology Bite Blood Cells Chemistry Childhood Chronic Colombia Complex Complication Contracts Cues Disease Disease Outbreaks EMSA Ecuador Endothelial Cells Environment Erythrocytes Etiology Exposure to Fatality rate Fever Genes Genetic Transcription Goals Gram-Negative Bacteria Growth Hematocrit procedure Hemolytic Anemia Human Icterus Incidence Infection Insect Vectors Intervention Left Lesion Life Light Lutzomyia genus Mediating Messenger RNA Metabolic Pathway Metabolism Midgut Modeling Molecular Myalgia Natural History Organism Oroya Fever Parasites Pathogenesis Pathogenicity Persons Peru Phagocytosis Phase Play Population Populations at Risk Post-Transcriptional Regulation Prevalence Proteins Reactive Oxygen Species Regulation Reporting Research Risk Role Sand Flies Skin Small RNA South America Surface Symptoms Syndrome Temperature Transcript Translations Untranslated RNA Vascular Endothelial Cell Verruga Peruana Virulence Virulent antimicrobial axenic culture base biological adaptation to stress climate change cost differential expression environmental change extracellular genetic manipulation genetic strain high risk mutant neglected tropical diseases overexpression pathogen pathogenic bacteria response secondary infection skin lesion transcriptome transcriptome sequencing transcriptomics tumor vector

项目摘要

Bartonella bacilliformis (Bb) is a highly virulent, sand fly-borne, Gram-negative bacterium that causes Carrión’s disease in humans. Carrión’s disease is emerging in endemic regions of the Andes and expanding into historically non-endemic areas of Ecuador, Colombia and Peru. The at-risk population is currently ~1.7 million people in a 56,000 square-mile area. Incidence rates of 12.7/100 person years have been reported in endemic regions. Bb infections can be life-threatening, with fatality rates of 40-88%, if left untreated. Pediatric populations are especially at high-risk. In non-endemic regions, the disease often manifests as an acute illness with an ~80% reduction in erythrocyte hematocrit (Oroya fever), whereas in endemic regions, angiomatous skin lesions (verruga peruana) and chronic bacteremia prevail, effectively creating a human reservoir. Little is known about Bb’s molecular pathogenesis and relationship with its phlebotomine sand fly vector, Lutzomyia verrucarum. We hypothesize that small, noncoding RNAs (sRNAs) play key roles in optimizing Bb’s ability to survive and replicate in human cells and sand flies via transcriptional and post-transcriptional regulation. Bb’s natural history offers an exceptional model to examine regulatory roles of sRNAs, as the pathogen is frequently subjected to dramatic shifts in temperature, pH, and disparate chemistries of the extracellular and intracellular niches of the sand fly vector and human host, respectively. Our long-term goal is to elucidate how Bb regulates the adaptive responses necessary for infection, with overarching objectives of: a) generating information that can be applied towards the eventual eradication of Carrión's disease and b) providing a model of sRNA-mediated regulation of survival genes (e.g., stress response, metabolism, virulence, etc.) in Bartonella and other arthropod-borne, intracellular pathogenic bacteria. The hypothesis will be addressed by three specific aims. In aim 1, we will characterize Bb’s baseline transcriptome (sRNAs and mRNAs) during axenic growth using RNA-Seq. Differential transcription as a function of relevant environmental cues will also be explored. Second, we will compare transcriptomes of Bb grown axenically to those infecting human vascular endothelial cells and erythrocytes. These results will allow us to identify infection-specific transcripts produced in the intracellular niche. In aim 2, we will characterize the transcriptome expressed during infection of sand flies. From these results, we can identify infection-specific RNAs produced during extracellular growth in the insect vector. In aim 3, we will analyze functions of the two dominant, infection-specific sRNAs identified above; one produced in host cells and a second in sand flies. First, we will generate respective sRNA mutant and overexpression strains by genetic manipulation. Second, strains will be analyzed for growth and survival during infection, and their transcriptomes analyzed to identify potential targets. The mRNA targets of the dominant sRNAs will be verified by EMSA and the results used to formulate the respective sRNA-mediated regulatory networks. These results will define the functions for two dominant, “infection-specific” sRNAs of Bb.

杆状巴尔通体 (Bb) 是一种高毒力、白蛉传播的革兰氏阴性细菌，可引起腐肉病腐肉病正在安第斯山脉流行地区出现，并蔓延至人类。厄瓜多尔、哥伦比亚和秘鲁历史上的非流行地区目前的高危人群约为 170 万。据报告，56,000 平方英里地区的发病率为 12.7/100 人年。如果不及时治疗，Bb 感染可能会危及生命，死亡率为 40-88%。在非流行地区，该病通常表现为急性疾病。红细胞比容降低约 80%（奥罗亚热），而在流行地区，血管瘤性皮肤病变（秘鲁疣）和慢性菌血症盛行，有效地创造了人体储存库。了解 Bb 的分子发病机制以及与其白蛉载体 Lutzomyia 的关系我们发现小型非编码 RNA (sRNA) 在优化 Bb 的能力方面发挥着关键作用。通过转录和转录后调节 Bb 在人类细胞和沙蝇中生存和复制。自然史提供了一个特殊的模型来检查 sRNA 的调节作用，因为病原体经常被经历温度、pH 值以及细胞外和细胞内不同化学成分的巨大变化我们的长期目标是分别阐明白蛉载体和人类宿主的生态位。调节感染所需的适应性反应，总体目标是：可用于最终根除腐肉病的信息；b) 提供模型巴尔通体中 sRNA 介导的生存基因调节（例如应激反应、代谢、毒力等）和其他节肢动物传播的细胞内致病细菌将由三个来解决。在目标 1 中，我们将描述无菌过程中 Bb 的基线转录组（sRNA 和 mRNA）。使用 RNA-Seq 进行差异转录作为相关环境线索的函数也将是其次，我们将比较无菌生长的 Bb 转录组与感染人类血管的转录组。这些结果将使我们能够识别产生的感染特异性转录本。在目标 2 中，我们将描述沙子感染过程中表达的转录组。从这些结果中，我们可以识别果蝇细胞外生长过程中产生的感染特异性 RNA。在目标 3 中，我们将分析已识别的两种主要的感染特异性 sRNA 的功能。上面；一个在宿主细胞中产生，另一个在沙蝇中产生。首先，我们将生成各自的 sRNA 突变体。其次，将分析菌株的生长和存活。感染期间，并对它们的转录组进行分析，以确定潜在的目标 mRNA 目标。 EMSA 将验证 sRNA 的主导地位，并将结果用于制定相应的 sRNA 介导的这些结果将定义 Bb 的两个主要“感染特异性”sRNA 的功能。