Role of surface proteins in sand fly colonization by Bartonella bacilliformis

描述（由申请人提供）：Bartonella bacilliformis （Bb）是一种高毒力的，由沙蝇传播的革兰氏阴性细菌，可导致人类的巴尔通体病（Carri¿n病）。巴尔通体病正在流行地区（安第斯山脉高地区）重新出现，并扩大到厄瓜多尔、哥伦比亚和秘鲁的非流行地区（即海拔较低和生境更多样化的地区）。据估计，在南美洲56,000平方英里的区域内，有170万人处于危险之中。在流行地区报告的发病率为12.7 / 100人年。Bb感染可危及生命，如果不及时治疗，死亡率为40-88%，使用抗菌素治疗后死亡率为10%。儿科人群尤其处于危险之中。在非流行地区，该病表现为急性疾病，红细胞红细胞压积减少约80%（奥罗亚热），而在流行地区，血管瘤性皮肤病变（秘鲁斑疹）和慢性菌血症普遍存在，形成人类乙型肝炎病毒库。关于Bb的分子发病机制、毒力决定因素及其与沙蝇载体疣状Lutzomyia verrucarum的关系知之甚少。为了解决这种信息的缺乏，我们建议通过实时成像技术来检查Bb与疣状乳杆菌的关系。我们还建议研究三个假定的毒力决定因素[即鞭毛蛋白（FlaA），血红素结合蛋白C （HbpC）和入侵相关基因座B （IalB）蛋白]在疣状乳杆菌的Bb定植中的作用。我们假设Bb最初在摄入被污染的血粉后感染昆虫的中肠上皮。由于Bb的高度侵袭性，从感染的焦点开始，血液和最终的唾液腺受到感染，并且由此产生的感染是终身的，不会经卵巢传播。最后，我们假设缺乏上述毒力因子的Bb突变体在沙蝇中的定殖、复制和/或传播能力将受到损害。为了解决这些假设，我们提出了流动目标：在目标1中，我们将分析低传代GFP+ Bb菌株在沙蝇中的定殖和迁移。第一个目标是产生低传代的Bb菌株，该菌株含有稳定的、表达gfp的插入物。第二个目标是通过共聚焦显微镜跟踪GFP+ Bb菌株在沙蝇中的解剖位置。在Aim 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毒力决定因素已被一种新技术诱变。第二个目标是通过等位基因修复创造相应的互补菌株。在目标3中，我们将比较野生型（WT）、突变型和补充型Bb菌株在沙蝇中的定殖和持续存在能力。第一个目标是比较这些Bb菌株在沙蝇上定居的相对能力。第二个目标是比较这些菌株在沙蝇体内复制和存活的能力。Aim 3的结果将用于解决这些基因的分子Koch假设，因为它与沙蝇载体有关。该研究结果有望为最终开发旨在中断沙蝇- bb关联的控制策略提供坚实的基础。

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.