Role of surface proteins in sand fly colonization by Bartonella bacilliformis

表面蛋白在杆状巴尔通体定植白蛉中的作用

• 批准号: 8303852
• 负责人: Michael F Minnick
• 金额: $ 21.23万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8303852
• 关键词: Acute; Address; Adult; Age; Altitude; Americas; Anorexia; Area; Arthralgia; Bacillary Angiomatosis; Bacteremia; Bacteria; Bartonella Infections; Bartonella bacilliformis; Basic Science; Bite; Blood; Bone Pain; Child; Childhood; Chloramphenicol; Chronic; Chronic Disease; Cicatrix; Ciprofloxacin; Colombia; Complement; Confocal Microscopy; Country; Data; Death Rate; Disease; Disease Outbreaks; Ecuador; Epithelium; Erythrocytes; Fever; Flagellin; Fostering; Foundations; Frequencies; Genes; Goals; Gram-Negative Bacteria; Habitats; Headache; Health; Heart murmur; Hemangioma of skin; Hematocrit procedure; Hemolytic Anemia; Hepatomegaly; Histoplasmosis; Human; Icterus; Imaging Techniques; Immigration; Immune response; Immune system; Immunocompromised Host; Incidence; Individual; Infection; Ingestion; Insect Vectors; Insecta; Intervention; Left; Lesion; Life; Location; Lutzomyia genus; Lymphatic Diseases; Malaise; Malaria; Membrane Proteins; Midgut; Molecular; Nature; Neurologic; Oroya Fever; Pallor; Pathogenesis; Patients; Persons; Peru; Phase; Play; Pneumocystis; Population; Populations at Risk; Pregnant Women; Protein C; Public Health; Relative (related person); Reporting; Research; Respiratory Tract Infections; Rifampin; Risk; Role; Salivary Glands; Salmonella infections; Sand Flies; Shigella Infections; Solid; South America; South American; Streptomycin; Syndrome; Techniques; Time; Tissues; Toxoplasmosis; Tropical Disease; Verruga Peruana; Virulence; Virulence Factors; Virulent; Visit; antimicrobial; climate change; design; heme-binding protein; migration; mutant; neglect; novel; pathogen; prevent; protein B; restoration; secondary infection; skin lesion; transmission process; unborn child; vector

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.

描述（由申请人提供）：杆状巴尔通体 (Bb) 是一种高毒力、白蛉传播的革兰氏阴性细菌，可引起人类巴尔通体病（卡林氏病），该病在流行地区（安第斯山脉高地）重新出现并正在扩大。厄瓜多尔、哥伦比亚和秘鲁的非流行地区（即海拔较低和栖息地更加多样化）的高危人群。据估计，在美国 56,000 平方英里的地区，Bb 感染的发病率为 12.7 人/100 人，可危及生命，死亡率为 40-88%。如果不及时治疗，在非流行地区，儿童群体尤其面临着 10% 的死亡风险。红细胞比容降低约 80%（奥罗亚热），而在流行地区，血管瘤性皮肤病变（秘鲁疣）和慢性菌血症盛行，形成了 Bb 的人类储存库，但人们对 Bb 的分子发病机制、毒力决定因素及其关联知之甚少。为了解决信息缺乏的问题，我们建议检查 Bb 与 L 的关系。 verrucarum，通过实时成像技术，我们还建议检查三种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 Bb 定植中的作用。我们认为，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，血腔和最终唾液腺被感染，并且由此产生的感染是终生的并且不会跨卵巢传播。最后，我们假设 Bb 突变体缺乏上述毒力。为了解决这些假设，我们提出以下目标：在目标 1 中，我们将分析白蛉的定殖和迁移能力。沙蝇中的低传代 GFP+ Bb 菌株的第一个目标是产生包含稳定的、表达 GFP 的插入物的低传代 Bb 菌株，第二个目标是追踪 GFP+ Bb 菌株在沙子中的解剖位置。随着时间的推移，通过共聚焦显微镜，我们将在低传代 Bb 菌株中产生 hbpC、ialB 和 flaA 突变体。其 hbpC、ialB 和 flaA 毒力决定簇已通过新技术诱变的 Bb 菌株第二个目标是通过等位基因恢复创建相应的互补菌株。在目标 3 中，我们将比较野生型 (WT)、突变型和互补型 Bb。第一个目标是比较这些 Bb 菌株在白蛉中定殖和持续存在的能力，第二个目标是比较这些菌株在白蛉中定殖和持续的能力。目标 3 的结果将用于解决这些基因的分子科赫假设，因为它与白蛉载体有关。该研究的结果预计将为最终开发设计的控制策略提供坚实的基础。打断沙蝇-Bb的关联。 公共健康相关性：杆状巴尔通体是沙蝇传播的人类巴尔通体病（卡林氏病）的细菌病原体；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。拟议的研究将分析巴尔通体。芽孢杆菌在白蛉载体中的定殖和迁移，并将比较缺乏以下三种细菌之一的野生型和突变型细菌菌株的这些活性捕获的与毒力有关的决定因素，包括鞭毛蛋白 (FlaA)、血红素结合蛋白 C (HbpC) 和入侵相关基因座 B (IalB) 蛋白，此外还解决了细菌发病机制及其与昆虫关联的信息匮乏问题。研究结果将为制定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。