The Characterization of Streptococcus agalactiae Novel Protein, Sak_1753, in the Colonization of the Vaginal Tract

项目摘要 新生儿无乳链球菌（B组链球菌，GBS）感染通常是致命的，与 母体GBS阴道定植。预防性分娩期抗生素的使用虽然对早发性疾病有效， 它们有自己的缺陷，对晚发性新生儿GBS疾病没有影响。因此，一个更长期的战略是 控制GBS感染所必需的。这项提案的重点是一种新的GBS蛋白，Sak_1753，我们的目标是确定 其在GBS阴道定殖中的作用及其作为基于蛋白质的候选疫苗的资格。sak_1753以前是 在GBS A909转录组中被鉴定为最高度上调的基因（~3000倍）， 在液体培养中定殖至生长。Sak_1753由双组分系统SaeRS直接调节， 已知的同源物以外的链球菌或预测的蛋白质结构域，留下它的功能是一个谜。蛋白质是保守的 在所有测序的GBS菌株中，它由多个重复结构域组成， 菌株我们将表征Sak_1753的调节和定位，并确定其在阴道定植中的作用。我们有 在野生型（WT）GBS A909中创建敲除（KO; Δsak_1753：：Spec）和过表达（OE; PreA-sak_1753）菌株 背景用人阴道上皮细胞（VK 2）进行的初步粘附测定证明OE菌株是 Sak_1753能够比WT显著更多地结合上皮细胞，并且KO结合显著更少。 对胶原蛋白1和纤维蛋白原的特异性。此外，sak_1753 KO菌株表现出阴道分泌物的显著减少， 在小鼠模型中的定殖。最初的免疫荧光显微术表明在细胞表面发现了Sak_1753， 表明该蛋白可能具有粘附作用。在本提案中，我们将进一步描述Sak_1753的作用。GBS 将创建包含Sak_1753截短的菌株，以鉴定重复结构域在宿主中的作用 附件.表达截短蛋白的菌株将在VK 2细胞和体内鼠粘附试验中进行检测。 殖民化为了进一步确定Sak_1753的细胞定位，采用免疫印迹技术和免疫荧光技术， 将使用显微镜。这项研究的成功将有助于减少GBS粘附的策略， 阴道组织，从而减少GBS相关的新生儿疾病和死亡。

PROJECT SUMMARY Streptococcus agalactiae (Group B Strep, GBS) infections in neonates are often fatal and are strongly associated with maternal GBS vaginal colonization. The use of preventive intrapartum antibiotics, while effective against early onset disease, have their own pitfalls and have no effect on late onset neonatal GBS diseases. As such, a more long-term strategy is required to control infections by GBS. This proposal focuses on a novel GBS protein, Sak_1753, and we aim to determine its role in GBS vaginal colonization and its eligibility as a protein-based vaccine candidate. sak_1753 was previously identified as the most highly upregulated gene (~3000 fold) in the GBS A909 transcriptome when comparing vaginal colonization to growth in liquid culture. Sak_1753 is regulated directly by a two-component system SaeRS and has no known homologs outside streptococci or predicted protein domains, leaving its function a mystery. The protein is conserved among all sequenced strains of GBS and is made up of multiple repeat domains with the number of repeats differing between strains. We will characterize Sak_1753 regulation and localization and determine its role in vaginal colonization. We have created knockout (KO; Δsak_1753::Spec) and over-expression (OE; PrecA-sak_1753) strains in a wildtype (WT) GBS A909 background. Preliminary adherence assays with human vaginal epithelial cells (VK2) demonstrated that the OE strains were able to bind to epithelial cells significantly more than WT and the KO bound significantly less. Sak_1753 also showed binding specificity to collagen 1 and fibrinogen. In addition, the sak_1753 KO strain exhibited significant decreased vaginal colonization in a murine model. Initial immunofluorescence microscopy indicates that Sak_1753 is found on the cell surface, indicating a possible adhesive role of this protein. In this proposal we will further characterize the role of Sak_1753. GBS strains will be created to contain truncations of Sak_1753 in an effort to identify the role of the repeated domains in host attachment. Strains expressing truncated proteins will be tested in adhesion assays with VK2 cells and in vivo murine colonization. To further ascertain the cellular localization of Sak_1753 western blot techniques and immunofluorescence microscopy will be used. The success of this research will contribute to the strategies for reduction of GBS adhesion to vaginal tissue and hence a reduction in GBS-related neonatal diseases and deaths.