Molecular epidemiologic identification of pneumococcal tissue specific genes

描述（由申请方提供）：肺炎链球菌栖息于人类呼吸道，当其离开鼻咽区域并侵入正常无菌部位时引起疾病。无症状携带远比疾病常见，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有所进展，但使肺炎球菌菌株易于携带与疾病的确切因素尚不清楚。S.肺炎菌株在它们引起疾病的倾向上是不同的，并且特定菌株的遗传背景可能影响其成功侵入不同组织部位的能力。我们提出了一个跨学科的分子和流行病学的方法，以增加对肺炎球菌携带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S。肺炎链球菌临床和携带分离株<5岁的儿童; 2）确定组织特异性毒力因子与S。在<5岁的儿童中引起临床疾病的肺炎菌株，并评估它们在大量临床和携带菌株中的分布;和3）评估中耳炎的灰鼠模型中的候选中耳炎特异性毒力基因。具体地说，我们将确定S的关联。使用多位点序列分型鉴定携带和特定临床疾病的肺炎球菌克隆，使用基因组消减鉴定组织特异性毒力的个体遗传决定因素，并使用大量临床相关肺炎球菌分离株集合的斑点杂交筛选来评估这些推定毒力决定因素在群体水平上的相对重要性。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。S.将在中耳炎相关基因中产生肺炎克雷伯氏菌缺失突变体，并在实验性灰鼠中耳炎模型中测试它们的毒力。更深入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来控制、预防和治疗这些重要感染的能力至关重要。

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.