NTHi Type IV pili: expression and vaccine potential

NTHi IV 型菌毛：表达和疫苗潜力

• 批准号: 7036418
• 负责人: ROBERT S. MUNSON
• 金额: $ 45.19万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-12-16 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7036418
• 关键词: Haemophilus influenzae; bacteria infection mechanism; bacterial cytopathogenic effect; bacterial genetics; bacterial proteins; bacterial vaccines; chinchilla; disease /disorder model; epitope mapping; gene expression profiling; gene mutation; genetic regulatory element; immunoprecipitation; laboratory rabbit; molecular assembly /self assembly; otitis media; peptide chemical synthesis; phenotype; pilin; polymerase chain reaction; protein biosynthesis; protein localization; protein structure function; synthetic antigens; transfection /expression vector; vaccine development

DESCRIPTION (provided by applicant): Nontypeable Haemophilus influenzas (NTHi) is a predominant etiologic agent of otitis media (OM) in chil- dren as well as a major cause of pneumonia and other respiratory tract diseases in adults. Although much has been learned about several outer membrane proteins and the lipooligosaccharide produced by this or- ganism, overall we have a poor understanding of the pathogenesis of Haemop/7/7i;s-induced infection. Type IV pili (Tfp) play a significant role in the pathogenesis of disease caused by numerous Gram-negative pathogens and some Tfp-based immunogens induce protective activity against these organisms. Although a cryptic gene cluster that might encode a Tfp in Haemophilus has long been recognized, expression of Tfp has never been demonstrated by this organism. In fact, in a review entitled DNA uptake during Bacterial Transformation, published in Nature Reviews Microbiology in March 2004, Chen and Dubnau state "Other competent organisms, such as H. influenzae or the Gram-positive bacteria B. subtilis and S. pneumoniae, require similar genes for DNA uptake, but do not possess filamentous structures that extend from the cell surface". Further, twitching motility, a non-flagellar-based Tfp-dependent motility has never been observed among the Pasteurellaceae. We recently demonstrated that NTHi produces Tfp under defined environmental conditions and further, that Tfp are responsible for twitching motility in NTHi. We hypothesize that Tfp will play an important role in pathogenesis. We have also determined that H. influenzae has a single copy of the pilA gene (encodes the major pilin subunit) and we have now sequenced this gene in 11 NTHi strains. Unlike the situation for N. gonorrhoeae, the derived amino acid sequence of these genes is highly conserved which is a considerable advantage for our vaccine development efforts that intend to target the H. influenzae Tfp. Experiments are thereby proposed herein to determine how the Tfp are assembled as well as how syn- thesis and assembly of these structures are regulated. Additionally, experiments are proposed design a type IV-pilin based immunogen that will induce protection against infection caused by the majority of nontypeable H. influenzae isolates. These experiments will lead to a better understanding of the pathogenesis of NTHi- induced disease as well as new approaches for the prevention of disease caused by NTHi.

描述（由申请人提供）：不可分型流感嗜血杆菌（NTHi）是儿童中耳炎（OM）的主要病原体，也是成人肺炎和其他呼吸道疾病的主要原因。尽管人们对这种生物体产生的几种外膜蛋白和脂寡糖了解很多，但总的来说，我们对 Haemop/7/7i;s 诱导感染的发病机制了解甚少。 IV 型菌毛 (Tfp) 在多种革兰氏阴性病原体引起的疾病发病机制中发挥着重要作用，一些基于 Tfp 的免疫原可诱导针对这些生物体的保护活性。虽然嗜血杆菌中可能编码 Tfp 的神秘基因簇早已被识别，但这种生物体从未证明 Tfp 的表达。事实上，在 2004 年 3 月发表在《自然评论微生物学》上的一篇题为“细菌转化过程中的 DNA 摄取”的评论中，Chen 和 Dubnau 指出“其他有能力的生物体，例如流感嗜血杆菌或革兰氏阳性菌枯草芽孢杆菌和肺炎链球菌，需要相似的基因来摄取 DNA，但不具有从细胞表面延伸的丝状结构”。此外，在巴斯德氏菌科中从未观察到抽搐​​运动，一种基于非鞭毛的Tfp依赖性运动。我们最近证明 NTHi 在特定的环境条件下产生 Tfp，并且 Tfp 负责 NTHi 的抽搐运动。我们假设 Tfp 在发病机制中发挥重要作用。我们还确定流感嗜血杆菌具有 pilA 基因的单个拷贝（编码主要菌毛蛋白亚基），并且我们现已在 11 个 NTHi 菌株中对该基因进行了测序。与淋病奈瑟菌的情况不同，这些基因的衍生氨基酸序列高度保守，这对于我们针对流感嗜血杆菌Tfp的疫苗开发工作来说是一个相当大的优势。因此本文提出了实验来确定 Tfp 如何组装以及如何调节这些结构的合成和组装。此外，实验建议设计一种基于 IV 型菌毛蛋白的免疫原，该免疫原将诱导针对大多数不可分型流感嗜血杆菌分离株引起的感染的保护。这些实验将有助于更好地了解 NTHi 诱导疾病的发病机制以及预防 NTHi 引起的疾病的新方法。