Role of surface proteins in sand fly colonization by Bartonella bacilliformis

描述(申请人提供)：巴尔通体杆菌(BB)是一种由沙蝇传播的高度毒力的革兰氏阴性细菌，可导致人类巴尔通体病(卡里恩病)。巴氏杆菌病正在流行地区(安第斯高地)重新出现，并向厄瓜多尔、哥伦比亚和秘鲁等非流行地区(即较低海拔和更多样化的栖息地)扩散。据估计，在南美洲5.6万平方英里的地区，高危人口约为170万人。在流行地区报告的发病率为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，红细胞压积下降约80%(Oroya热)，而在流行地区，血管瘤性皮肤损害(Verruga Peruana)和慢性菌血症盛行，造成人类的BB蓄水池。关于BB的分子致病机制、毒力决定因素以及它与白粉病病媒--寻常褐飞虱的关系，人们知之甚少。为了解决这一信息匮乏的问题，我们建议通过活体成像技术来研究BB与疣状乳杆菌的关系。我们还建议研究三个可能的毒力决定因素[即鞭毛蛋白(FLAA)、血红素结合蛋白C(HBPC)和入侵相关位点B(IALB)蛋白]在Bb侵染疣状乳杆菌定植中的作用。我们假设，BB最初是在摄入受污染的血粉后感染昆虫的中肠上皮。由于BB的高度侵袭性，血腔和最终的唾液腺从这个感染灶开始感染，由此产生的感染是终生的，不会通过卵巢传播。最后，我们假设缺乏上述毒力因子的BB突变体在沙蝇体内的定殖、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，我们将分析一种低传代的GFP+BB菌株在沙蝇体内的定植和迁移。第一个目标是产生一个低传代的BB菌株，它包含一个稳定的、表达GFP的插入片段。第二个目标是通过共聚焦显微镜追踪GFP+BB菌株在沙蝇中随时间的解剖位置。在目标2中，我们将在低传代的BB菌株中产生HBPC、IALB和FlaA突变株。第一个目标是产生低传代的BB菌株，其HBPC、IALB和FlaA毒力决定簇已被一种新技术诱变。第二个目标是通过等位基因恢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菌株在沙蝇体内定居和存活的能力。第一个目标是比较这些BB菌株对沙蝇定居的相对能力。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。目的3的结果将用于解决这些基因的分子科赫假设，因为它与沙蝇载体有关。这项研究的结果有望为最终制定旨在中断沙蝇-BB关联的控制策略提供坚实的基础。 与公共卫生相关：巴氏杆菌是一种由沙蝇传播的人类巴尔通体病(卡里氏病)的细菌剂；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。这项拟议的研究将分析杆菌在沙蝇载体中的定植和迁移，并将比较缺乏鞭毛蛋白(FlaA)、血红素结合蛋白C(HBPC)和入侵相关基因B(IALB)蛋白的野生型和突变菌株的这些活性。除了解决有关这种细菌的致病机理及其与昆虫媒介的关联的信息匮乏问题外，这项研究的结果还将为开发破坏沙蝇病原体关联并防止传播给人类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.