Genome-wide assessment of Group B Streptococcus fitness and virulence

项目概要/摘要 无乳链球菌（B组链球菌; GBS）是新生儿最常见的感染性病原 发病率和死亡率在美国和一个主要的全球贡献死产和感染， 新生儿时期虽然无症状的生殖器直肠GBS定植在健康成人中很常见，但阴道GBS定植在健康成人中很常见。 妊娠后期的定植是新生儿感染的主要危险因素。因此，改进 了解促进阴道定植的细菌基因以及从定植到定植的转变， 侵入性生活方式可能为新的预防策略开辟道路。我们的团队建立了高度的 饱和的GBS转座子突变体库，这在全基因组评估 在各种实验环境中基因对细菌适应性的贡献。最近，我们开发了一个高- 使用这些复杂的混合文库来产生全面的有序突变体的通量策略 非必需GBS基因文库（其中约有1，700个）。在目标1中，我们建议使用 技术和技术，我们已经开发，以组装完整的，索引的突变库， 两种GBS菌株，代表从感染患者中回收的常见荚膜亚型（血清型Ia和 （五）。这些索引库将免费与广大研究社区共享， 发病机制研究的重大进展，允许在一个特定的环境中研究单个突变体或策展的文库子库。 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， 表征GBS双组分信号通路对小鼠阴道定植的贡献 模型将评估含有所有19个GBS双组分系统敲除的文库子池， 在阴道定殖模型中具有显著适应性缺陷的组成突变体。最佳候选人将是 用靶向同基因敲除菌株进行验证，这些菌株也将通过RNA-seq进行表征，以鉴定 每个候选双组分系统调控的下游基因。最后，索引库敲除 将评估每个候选的双组分系统调节子在阴道中的主要适合度贡献者 殖民模式这些目标将建立新的，灵活的，强大的工具，研究GBS的发病机制。 它们将允许对双组分体系及其受监管的 遗传网络作为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.