Genome-wide assessment of Group B Streptococcus fitness and virulence

项目摘要/摘要 无乳链球菌是新生儿最常见的感染原因 美国的发病率和死亡率是全球死产和感染的主要原因 新生儿期。虽然无症状的生殖器直肠GBS定植在健康成年人中很常见，但阴道 妊娠晚期定植是新生儿感染的主要危险因素。为此，改进了 对促进阴道定植和从定植向定植转变的细菌基因的理解 侵犯性的生活方式可能会为新的预防策略开辟道路。我们的团队构建了高度的 饱和的GBS转座子突变文库，在全基因组评估中发挥了重要作用 在各种实验环境中，基因对细菌适合性的贡献。最近，我们开发了一种高- 使用这些复杂、混合的文库来生成全面的、有序的突变体的吞吐量策略 非必需GBS基因库(其中约有1700个)。在目标1中，我们建议使用 我们开发的技术和技术是为了组装完整的、索引的突变体文库 两株代表从感染患者中恢复的常见包膜亚型的GBS菌株(Ia和Ia血清型 v)。这些索引库将免费与广大研究社区共享，将代表一种 发病机制研究的重大进展，允许研究单个突变体或精选的文库亚库 广泛的实验设计。在目标2中，我们将在一系列研究中使用索引库，以充分 GBS双组分信号通路在小鼠阴道定植中的作用 模特。将评估包含所有19个GBS双组件系统淘汰点的库子池 在阴道定植模型中具有显著适合性缺陷的成分突变。顶尖候选人将是 用目标等基因敲除菌株进行验证，这也将通过rna-seq来鉴定 下游基因受每个候选双组分系统调控。最后，索引的文库敲除 每个候选的双组分系统调节剂都将被评估为阴道内主要的健身贡献者 殖民模式。这些目标将为研究GBS的发病机制建立新的、灵活的和强大的工具。 它们将允许对双组件系统及其受监管的系统进行公正和全面的评估 基因网络是预防新生儿感染的潜在目标。

Project Summary/Abstract Streptococcus agalactiae (group B Streptococcus; GBS) is the most common infectious cause of neonatal morbidity and mortality in the United States and a major global contributor to stillbirth and infection in the newborn period. While asymptomatic genitorectal GBS colonization is common among healthy adults, vaginal colonization in late pregnancy is a major risk factor for neonatal infection. For this reason, improved understanding of bacterial genes that promote vaginal colonization and the transition from a colonizing to an invasive lifestyle may open avenues to new preventative strategies. Our group has constructed highly saturated GBS transposon mutant libraries, which have been instrumental in genome-wide assessments of gene contributions to bacterial fitness in a variety of experimental settings. Recently, we developed a high- throughput strategy for using these complex, intermixed libraries to generate comprehensive, ordered mutant libraries of nonessential GBS genes (of which there are approximately 1,700). In Aim 1, we propose to use techniques and technologies we have developed in order to assemble complete, indexed mutant libraries of two GBS strains, representing common capsular subtypes recovered from infected patients (serotypes Ia and V). These indexed libraries—which will be freely shared with the research community at large—will represent a major advance in pathogenesis research, allowing study of single mutants or curated library sub-pools in a wide range of experimental designs. In Aim 2, we will use the indexed libraries in a series of studies to fully characterize the contribution of GBS two-component signaling pathways to vaginal colonization in a mouse model. Library sub-pools containing knockouts of all 19 GBS two-component systems will be assessed for constituent mutants with significant fitness defects in the vaginal colonization model. Top candidates will be validated with targeted isogenic knockout strains, which will also be characterized via RNA-seq to identify downstream genes regulated by each candidate two-component system. Finally, indexed library knockouts of each candidate two-component system regulon will be assessed for major fitness contributors in the vaginal colonization model. These aims will establish new, flexible, and powerful tools for studying GBS pathogenesis. They will permit unbiased and comprehensive assessment of two-component systems and their regulated genetic networks as potential targets for preventing neonatal infection.