Molecular epidemiologic identification of pneumococcal tissue specific genes

描述(申请人提供)：肺炎链球菌居住在人类呼吸道，当它离开鼻咽区并入侵正常无菌部位时会引起疾病。无症状携带者远比疾病常见，但这些细菌是幼儿中耳炎、肺炎、脑膜炎和菌血症的重要原因。尽管我们对这些细菌的流行病学和发病机制的了解取得了进展，但使肺炎球菌菌株易于携带与疾病的确切因素尚不清楚。肺炎沙门氏菌的致病倾向不同，特定菌株的遗传背景可能会影响其成功入侵不同组织部位的能力。我们提出了一种跨学科的分子和流行病学方法来增加对肺炎球菌携带者和临床分离株之间的差异的理解。我们的总体目标是：1)确定5岁儿童肺炎链球菌临床和携带性分离株的分子流行病学；2)确定与导致5岁儿童临床疾病的肺炎链球菌菌株相关的组织特异性毒力因子，并评估它们在大量临床和携带性菌株中的分布；以及3)评估棕鼠中耳炎模型中候选中耳炎特异性毒力基因。具体地说，我们将使用多位点序列分型来确定肺炎链球菌克隆与携带者和特定临床疾病的相关性，使用基因组差减来确定组织特异性毒力的单个遗传决定因素，并使用大量临床相关肺炎球菌分离株的斑点杂交筛选来评估这些假定的毒力决定因素在种群水平的相对重要性。这些分离株来自健康儿童和患有菌血症、肺炎、脑膜炎和中耳炎的儿童。将创建中耳炎相关基因中的肺炎链球菌缺失突变体，并在实验性的栗鼠中耳炎模型中测试它们的毒力。更好地了解影响肺炎球菌克隆毒力潜力的遗传因素，对于我们开发有效方法控制、预防和治疗这些重要感染至关重要。

DESCRIPTION (provided by applicant): Streptococcus pneumoniae inhabits the human respiratory tract and causes disease when it leaves the nasopharyngeal region and invades normally sterile sites. Asymptomatic carriage is far more common than disease, yet these bacteria are important causes of otitis media, pneumonia, meningitis, and bacteremia in young children. Despite advances in our understanding of the epidemiology and pathogenesis of these bacteria, the precise factors that predispose a pneumococcal strain for carriage versus disease are not known. S. pneumoniae strains differ in their propensity to cause disease, and the genetic background of a particular strain is likely to influence its ability to successfully invade different tissue sites. We propose an interdisciplinary molecular and epidemiologic approach to increase understanding of the differences between pneumococcal carriage and clinical isolates. Our overall aims are 1) to determine the molecular epidemiology of S. pneumoniae clinical and carriage isolates from children <5 years of age; 2) to identify tissue specific virulence factors associated with S. pneumoniae strains that cause clinical disease in children <5 years of age and evaluate their distribution among a large collection of clinical and carriage strains; and 3) to evaluate candidate otitis media specific virulence genes in the chinchilla model of otitis media. Specifically, we will determine the association of S. pneumoniae clones with carriage and specific clinical diseases using multilocus sequence typing, identify individual genetic determinants of tissue specific virulence using genomic subtraction, and evaluate the relative importance of these putative virulence determinants at the population level using a dot blot hybridization screen of a large, clinically relevant collection of pneumococcal isolates. These isolates are from healthy children and children with bacteremia, pneumonia, meningitis and otitis media. S. pnueumoniae deletion mutants in otitis media associated genes will be created and their virulence tested in an experimental chinchilla model of otitis media. A greater understanding of the genetic factors that influence the virulence potential of pneumococcal clones is critical to our ability to develop effective methods to control, prevent, and treat these important infections.