GENETICS OF H. AEGYPTIUS BRAZILIAN PURPURIC FEVER CLONE

巴西紫癜热（BPF）是一种致命疾病，由一种独特的侵入性克隆流感嗜血杆菌生物群埃及伊蚊（埃及伊蚊）引起，这种细菌通常是非侵入性的，可引起轻度结膜炎。埃及伊蚊BPF克隆不同寻常的入侵特性，使该病原体成为研究从一种古老的细菌病原体中产生一种表达新的毒力决定因子的入侵衍生物的理想模型。此外，利用天然宿主-靶细胞和存在的相互作用，细菌基因转移和细菌毒力基因表达调控的组织培养模型的可用性。此外，对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知之甚少。因此，本研究项目的长期目标是阐明BPF克隆入侵和破坏内皮宿主细胞的遗传和分子机制。在本提案中，我们通过几种方法来实现这一总体目标，将细菌遗传学中使用的经典方法与旨在分离存在于毒性菌株中的独特基因并检查差异基因表达的技术相结合。第一个特异性目标是分离和鉴定独特的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。第二个特异性目标是利用northern和western blotting结合逆转录酶-聚合酶链反应，在转录和翻译水平上分析独特的BPF基因的表达。第三个特定目的检查独特的BPF基因在入侵和破坏人类内皮细胞使用等基因突变的作用。这些突变体将通过定点插入诱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。这些提出的研究解决了埃及伊蚊BPF克隆引起的血管破坏的发病机制的一个重要的和很大程度上未被探索的方面。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.