GENETICS OF H. AEGYPTIUS BRAZILIAN PURPURIC FEVER CLONE

巴西紫热病(BPF)是一种致命疾病，由埃及埃及嗜血杆菌(H.aegyptius)的一种独特的入侵克隆引起，这种细菌通常是非侵袭性的，会导致轻微的结膜炎。埃及伊蚊BPF克隆的不同寻常的侵袭属性使该病原体成为研究从旧细菌病原体中出现表达新毒力决定因素的入侵衍生物的理想模型。此外，利用天然宿主-靶细胞的组织培养模型的可用性和存在的相互作用、细菌基因转移和细菌毒力基因表达的调控。此外，关于BPF毒力因子的分子和遗传性质以及由该病原体引起的暴发性紫癜的发病机制还知之甚少。因此，本研究的长期目标是阐明BPF克隆侵袭和破坏内皮细胞宿主细胞的遗传和分子机制。在这项建议中，我们通过几种方法来解决这一总体目标，将细菌遗传学中使用的经典方法与旨在分离毒力菌株中存在的独特基因并检查差异基因表达的技术相结合。第一个具体目标涉及分离和鉴定独特的BPF基因，其中一些可能与毒力有关。这将通过使用创新的减法基因组杂交技术构建基因库来实现。第二个特定目的是利用Northern和Western印迹结合逆转录聚合酶链式反应在转录和翻译水平上分析独特的BPF基因的表达。第三个特定目的是利用同基因突变体检测独特的BPF基因在人类内皮细胞入侵和破坏中的作用。这些突变体将通过定点插入突变和等位基因交换产生，将在HMEC-1组织培养模型中进行测试，该模型模拟埃及伊蚊入侵菌株在感染过程中产生的血管破坏。这些拟议的研究涉及埃及伊蚊BPF克隆引起的血管破坏的一个重要且在很大程度上尚未被探索的致病机制。此外，这些研究将有助于更好地理解其他血管破坏性传染病的性质。

Brazilian purpuric fever (BPF) is a fatal disease caused by a unique invasive clone of Haemophilus influenzae biogroup aegyptius (H. aegyptius), a bacterial species that is normally non-invasive and causes mild cases of conjunctivitis. The unusual invasive attributes of the BPF clone of H. aegyptius make this pathogen an ideal model to study the emergence of an invasive derivative, which expresses new virulence determinants, from an old bacterial pathogen. Furthermore, the availability of a tissue culture model using the natural host-target cells and the existence interactions, bacterial gene transfer, and the regulation of the expression of bacterial virulence genes. In addition, little is known about the molecular and genetic nature of the BPF virulence factors and the pathogenesis of the purpura fulminans caused by this pathogen. Thus, the long-term goal of this research project is the elucidation of the genetic and molecular mechanisms involved in the invasion and destruction of the endothelial host-cells by the BPF clone. In this proposal, we address this overall goal through several approaches, combining classical methods used in bacterial genetics with techniques designed to isolate unique genes present in virulent strains and examine differential gene expression. The first Specific Aim involves the isolation and characterization of unique BPF genes, some of which may be involved in virulence. This will be achieved by constructing a gene library using the innovative subtraction genomic hybridization technique. The second Specific Aim is focused on the expression analysis of the unique BPF genes at the transcriptional and translational levels using northern and western blotting together with reverse transcriptase- polymerase chain reaction. The third Specific Aim examines the role of unique BPF genes in the invasion and destruction of human endothelial cells using isogenic mutants. These mutants, which will be generated by site-directed insertion mutagenesis and allelic exchange, will be tested in the HMEC-1 tissue culture model that mimics the vascular destruction produced during the infection process caused by the invasive strains of H. aegyptius. These proposed studies address an important and largely unexplored aspect of the pathogenesis of the vascular destruction caused by the BPF clone of H. aegyptius. Furthermore, these studies will lead to a better comprehension of the nature of other vascular destructive infectious diseases.