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

杆状巴氏杆菌(Bartonella bacilliformis，BB)是一种由沙蝇传播的高毒力革兰氏阴性细菌，可引起卡里翁氏病。 人类的疾病。卡里翁病正在安第斯山脉的流行地区出现，并蔓延到 厄瓜多尔、哥伦比亚和秘鲁历史上的非流行区。高危人群目前约为170万人 人们聚集在56,000平方英里的区域内。报告的发病率为12.7/100人年。 地区。BB感染可能危及生命，如果不治疗，死亡率可达40%-88%。儿科 人群尤其处于高危状态。在非流行地区，该病通常表现为急性疾病。 红细胞压积下降约80%(奥罗亚热)，而在流行地区，血管瘤 皮肤损害(Peruana)和慢性菌血症盛行，有效地造成了人类的蓄水池。小才是 已知的BB的分子发病机制及其与其血吸虫沙蝇载体Lutzmyia的关系 寻常疣。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控在人类细胞和沙蝇中生存和复制。BB‘s 自然病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 易受温度、pH以及细胞外和细胞内不同化学成分的影响 分别为沙蝇媒介和人类宿主的生态位。我们的长期目标是阐明BB是如何 调节感染所需的适应性反应，首要目标是：a)产生 可应用于最终根除卡里翁氏病的信息和b)提供一个模式 SRNA介导的生存基因调控(如应激反应、新陈代谢、毒力等)在巴尔通氏杆菌 以及其他节肢动物传播的细胞内致病菌。这一假说将由三个方面来阐述 明确的目标。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.作为相关环境线索的函数的差异转录也将是 探索过了。第二，我们将比较轴向生长的BB和感染人类血管的BB的转录 内皮细胞和红细胞。这些结果将使我们能够识别产生的感染特异性转录本 在细胞内的壁龛里。在目标2中，我们将对沙子感染过程中表达的转录组进行鉴定。 苍蝇。从这些结果中，我们可以确定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，我们将分析已确定的两个主要的、感染特异的sRNA的功能。 上图；一种在宿主细胞中产生，另一种在沙蝇中产生。首先，我们将产生各自的sRNA突变体 以及通过基因操作过量表达菌株。其次，将分析菌株的生长和存活情况。 在感染期间，并分析它们的转录本以确定潜在的靶标。基因的信使核糖核酸靶点 EMSA将验证主要的sRNA，并使用结果来制定各自的sRNA介导的 监管网络。这些结果将确定BB的两个主要的“感染特异性”sRNAs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.