Genetic Requirement for Pneumococcal Ear Infection

BackgroundEar infection or otitis media (OM) accounts for most bacterial respiratory infections in children in both developed and developing nations. Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis are the major OM pathogens. However, little is known about the genetic basis of bacterial OM largely due to practical difficulties in conducting research in ear infection models and genetically manipulating clinical isolates. Here, we report the first genome-scale in vivo screen for bacterial genes required for ear infection in a chinchilla model by signature tagged mutagenesis (STM), a high throughput mutant screen technique.Methodology/Principal FindingsSTM strains were constructed with a multi-drug resistant OM isolate ST556 (serotype 19F) and screened in a chinchilla OM model. Out of 5,280 mutants tested, 248 mutants were substantially underrepresented in the mutant pools recovered from the middle ear fluids of the infected chinchillas, indicating the impaired ability to survive and replicate in the middle ears due to genetic disruptions in the chromosome of strain ST556. Further DNA sequencing analysis mapped the mutations to 169 pneumococcal genes. Surprisingly, only 52 of these genes were required for pneumococcal nasopharyngeal colonization in a murine model. This infection site-specific gene requirement was verified by targeted mutagenesis in the selected genes.Conclusions/SignificanceThese findings suggest that there are a subset of pneumococcal genes required for ear infection and that these may be distinct from those required for nasal colonization. Our data thus provide comprehensive gene targets for mechanistic understanding of pneumococcal ear infection. Finally, this study has also developed a model for future genome-scale search for virulence determinants in other pathogens associated with ear infections.背景 中耳炎（otitis media, OM）是发达国家与发展中国家儿童细菌性呼吸道感染的首要病因。肺炎链球菌（Streptococcus pneumoniae）、未分型流感嗜血杆菌（nontypeable Haemophilus influenzae）以及卡他莫拉菌（Moraxella catarrhalis）是引发OM的主要致病菌。然而，由于中耳炎模型构建以及临床分离株的遗传操作存在实际难题，目前学界对细菌性OM的遗传基础仍知之甚少。本研究首次借助标签突变诱变（signature tagged mutagenesis, STM）这一高通量突变筛选技术，在毛丝鼠OM模型中开展了致病菌中耳感染必需基因的全基因组体内筛选。 方法与主要结果 本研究以耐多药OM临床分离株ST556（血清型19F）构建STM突变菌株，并在毛丝鼠OM模型中完成筛选。在共计测试的5280株突变株中，从感染宿主中耳液回收的突变株库内，248株突变体的丰度显著降低，提示这些突变体因ST556染色体上的基因破坏，在中耳内的存活与增殖能力受损。进一步的DNA测序分析将这些突变位点定位至169个肺炎链球菌基因。令人意外的是，其中仅52个基因是肺炎链球菌在小鼠模型鼻咽定植所必需的。这种感染位点特异性的基因需求，通过所选基因的定向诱变得到了验证。 结论与意义 本研究结果表明，存在一类肺炎链球菌中耳感染所特需的基因，且这类基因与鼻咽定植所需的基因存在显著差异。本研究的数据为解析肺炎链球菌中耳炎的致病机制提供了全面的基因靶点。此外，本研究还为后续针对其他与OM相关致病菌的毒力决定因子开展全基因组规模筛选，建立了可复用的研究模型。