Role of surface proteins in sand fly colonization by Bartonella bacilliformis

描述（由申请方提供）：杆状巴尔通体（Bb）是一种高毒力、沙蝇传播的革兰氏阴性细菌，可引起人类巴尔通体病（Carrión病）。巴尔通体病在流行地区（高安第斯山脉）重新出现，并扩展到非流行地区（即，厄瓜多尔、哥伦比亚和秘鲁海拔较低，栖息地更多样化。在南卡罗来纳州56,000平方英里的地区，估计有170万人处于危险之中。美国参考据报告，流行地区的发病率为12.7 / 100人年。Bb感染可能危及生命，如果不治疗，死亡率为40- 88%，使用抗菌剂治疗后死亡率为10%。儿童群体尤其处于危险之中。在非流行性地区，该疾病表现为红细胞压积降低约80%的急性疾病（Oroya热），而在流行性地区，血管瘤性皮肤病变（秘鲁疣）和慢性菌血症占主导地位，形成了Bb的人体储存库。关于Bb的分子发病机制、毒力决定因素及其与白蛉媒介疣鼻虱的关系知之甚少。为了解决这个信息的缺乏，我们建议检查Bb与L的关系。verrucarum，通过实时成像技术。我们还建议研究三个假定的毒力决定因素的作用[即，鞭毛蛋白（FlaA）、血红素结合蛋白C（HbpC）和侵袭相关位点B（Ial B）蛋白]在B B定殖中的作用。疣状。我们假设，Bb最初感染昆虫的中肠上皮细胞后，摄入污染的血粉。由于Bb的高度侵袭性，从该感染病灶开始，血腔和最终唾液腺被感染，并且所导致的感染是终身的并且不经卵巢传播。最后，我们假设缺乏上述毒力因子的Bb突变体在白蛉中定殖、复制和/或传播的能力将受损。为了解决这些假设，我们提出了流动的目标：在目标1中，我们将分析低传代，GFP+ Bb菌株在白蛉中的定殖和迁移。第一个目标是产生含有稳定的GFP表达插入物的低传代Bb菌株。第二个目标是通过共聚焦显微镜观察GFP+ Bb菌株随时间推移在白蛉中的解剖位置。在目标2中，我们将在低传代Bb菌株中产生hbpC、ialB和flaA突变体。第一个目标是产生低传代的Bb菌株，其hbpC、ialB和flaA毒力决定簇已通过新技术诱变。第二个目标是通过等位基因恢复产生相应的互补菌株。在目标3中，我们将比较野生型（WT），突变体和补充Bb菌株的定殖能力，并坚持在白蛉。第一个目标是比较这些Bb菌株定殖白蛉的相对能力。第二个目标是比较这些菌株在白蛉中复制和持续存在的能力。目标3的结果将用于解决这些基因的分子科赫假设，因为它涉及到白蛉载体。该研究的结果预计将提供一个坚实的基础，最终制定控制策略，旨在中断沙蝇Bb协会。 公共卫生相关性：杆状巴尔通体（Bartonella bacilliformis）是一种由沙蝇传播的人类巴尔通体病（Carrión's disease）的细菌病原体;这是一种被忽视的热带疾病，威胁着南美洲56，000平方英里的约170万人。本研究将对B.本发明的目的是研究白蛉载体中的杆状菌定殖和迁移，并将这些活性与野生型和突变型细菌菌株进行比较，所述细菌菌株缺乏假设参与毒力的三种决定因素之一，包括鞭毛蛋白（FlaA）、血红素结合蛋白C（HbpC）和侵袭相关位点B（Ial B）蛋白。除了解决有关细菌致病机理及其与昆虫媒介相关性的信息缺乏外，该研究的结果还将为开发破坏白蛉-病原体关联并防止传播给人类的策略的长期目标奠定基础。

DESCRIPTION (provided by applicant): Bartonella bacilliformis (Bb) is a highly virulent, sand fly-borne, gram-negative bacterium that causes bartonellosis (Carri¿n's disease) in humans. Bartonellosis is reemerging in endemic regions (high Andes) and expanding into non-endemic areas (i.e., lower altitudes and more diverse habitats) of Ecuador, Colombia and Peru. The at-risk population is estimated at 1.7 million people in a 56,000 square-mile area of S. America. 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, and 10% fatalty following treatment with antimicrobials. Pediatric populations are especially at risk. In non-endemic regions, the disease 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, creating a human reservoir of Bb. Little is known about Bb's molecular pathogenesis, virulence determinants and its association with the sand fly vector, Lutzomyia verrucarum. To address this dearth of information, we propose to examine Bb's relationship with L. verrucarum, by live imaging techniques. We also propose to examine the role of three putative virulence determinants [i.e., flagellin (FlaA), heme-binding protein C (HbpC) and the invasion-associated locus B (IalB) protein] in Bb's colonization of L. verrucarum. We hypothesize that Bb initially infects the insect's midgut epithelium upon ingestion of a contaminated blood-meal. From this focus of infection, the hemocoel and eventually the salivary glands become infected, owing to the highly invasive nature of Bb, and that the resulting infection is life-long and not trans-ovarially transmitted. Finally, we hypothesze that Bb mutants which lack the aforementioned virulence factors will be impaired in their ability to colonize, replicate and/or spread in the sand fly. To address these hypotheses, we propose the flowing aims: In Aim 1, we will analyze colonization and migration of a low-passage, GFP+ Bb strain in sand flies. The first goal is to generate a low-passage Bb strain that contains a stable, GFP-expressing insert. The second goal is to follow the anatomical locations of the GFP+ Bb strain in the sand fly over time, by confocal microscopy. In Aim 2, we will generate hbpC, ialB and flaA mutants in low-passage Bb strains. The first goal is to generate low-passage, Bb strains whose hbpC, ialB and flaA virulence determinants have been mutagenized by a novel technique. The second goal is to create corresponding, complemented strains by allelic restoration. In Aim 3, we will compare wild-type (WT), mutant and complemented Bb strains for the ability to colonize and persist in sand flies. The first goal is to compare the reltive ability of these Bb strains to colonize sand flies. The second goal is to compare the ability of these strains to replicate and persist in the sand fly. Aim 3 results will be used to address molecular Koch's postulates for these genes, as it relates to the sand fly vector. The results of the study are expected to provide a solid foundation for eventually developing control strategies designed to interrupt the sand fly-Bb association. PUBLIC HEALTH RELEVANCE: Bartonella bacilliformis is the sand fly-borne, bacterial agent of bartonellosis (Carri¿n's disease) in humans; a neglected tropical disease that threatens ~1.7 million people in 56,000 square miles of South America. The proposed research will analyze B. bacilliformis colonization and migration in the sand fly vector and will compare these activities i wild-type and mutant strains of the bacterium that lack one of three determinants hypothesized to be involved in virulence, including flagellin (FlaA), heme-binding protein C (HbpC) and invasion-associated locus B (IalB) proteins. In addition to addressing a dearth of information on the bacterium's pathogenesis and association with its insect vector, results of the study will establish a foundation for the long- range goal of developing strategies that disrupt the sand fly-pathogen association and prevent transmission to humans.