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

杆状巴尔通体（Bb）是一种高毒性、沙蝇传播的革兰氏阴性细菌，可引起Carrión's 人类的疾病。卡里翁病在安第斯山脉的流行地区出现，并扩大到 厄瓜多尔、哥伦比亚和秘鲁历史上非流行地区。目前的高危人群约为170万 56,000平方英里区域内的人据报道，在地方性流行病中， 地区如果不及时治疗，Bb感染可能危及生命，死亡率为40- 88%。儿科 人口尤其处于高风险之中。在非流行地区，该病常表现为急性病 红细胞压积降低约80%（奥罗亚热），而在流行地区，血管瘤性 皮肤病变（秘鲁疣）和慢性菌血症普遍存在，有效地形成了人类宿主。之甚少 了解Bb分子致病机制及其与白蛉媒介Lutzomyia的关系 疣状。我们假设小的非编码RNA（sRNA）在优化Bb的能力方面发挥关键作用， 通过转录和转录后调节在人类细胞和白蛉中存活和复制。Bb的 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 受到温度、pH值以及细胞外和细胞内不同化学物质的剧烈变化的影响， 白蛉病媒和人类宿主的生态位。我们的长期目标是阐明BB 调节感染所必需的适应性反应，总体目标是： 可以应用于最终根除Carrión病的信息和B）提供模型 sRNA介导的存活基因调节（例如，应激反应、代谢、毒力等）巴尔通体 以及其他节肢动物传播的细胞内致病细菌。该假设将由三个解决 具体目标。在目标1中，我们将描述Bb在无菌过程中的基线转录组（sRNAs和mRNAs）， 使用RNA-Seq.差异转录作为相关环境线索的功能也将被 探讨了其次，我们将比较无菌生长的Bb的转录组与感染人类血管的Bb的转录组。 内皮细胞和红细胞。这些结果将使我们能够确定感染特异性转录本产生 在细胞内的小生境。在目标2中，我们将描述在感染沙子过程中表达的转录组 苍蝇从这些结果中，我们可以鉴定出在细胞外生长过程中产生的感染特异性RNA。 昆虫媒介。在目标3中，我们将分析鉴定的两种主要的感染特异性sRNAs的功能 一种是在宿主细胞中产生，另一种是在白蛉中产生。首先，我们将产生各自的sRNA突变体， 和通过遗传操作的过表达菌株。其次，将分析菌株的生长和存活 在感染过程中，和他们的转录组分析，以确定潜在的目标。mRNA的靶点 将通过EMSA验证显性sRNA，并将结果用于配制相应的sRNA介导的 监管网络。这些结果将定义Bb的两个主要的“感染特异性”sRNA的功能。

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.