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

无乳链球菌新型蛋白 Sak_1753 在阴道定植中的表征

• 批准号: 10442754
• 负责人: Lamar S Thomas
• 金额: $ 2.87万
• 依托单位: STATE UNIVERSITY OF NY,BINGHAMTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10442754
• 关键词: Achievement; Adherence; Adhesions; Adhesives; Africa; Antibiotics; Bacteria; Bacterial Adhesins; Binding; Binding Proteins; Biological Assay; Birth; Caribbean region; Cell surface; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Child; Collagen; Data; Disease; Eligibility Determination; Epithelial Cells; Exhibits; Extracellular Matrix; Far East; Far-Western Blotting; Fibrinogen; Genes; Genetic Transcription; Goals; Growth; Human; Immunofluorescence Microscopy; Infection; Infection Control; Intestines; Intravenous; Investigation; Knock-out; Liquid substance; Membrane; Meningitis; Mothers; Mucous Membrane; Mus; Neonatal; Onset of illness; Patients; Perinatal; Pharmacotherapy; Play; Population; Pregnant Women; Prevalence; Preventive; Property; Proteins; Protocols documentation; Regulation; Research; Risk; Role; Sepsis; Specificity; Streptococcus; Streptococcus Group B; Surface; System; Techniques; Tertiary Protein Structure; Testing; Tissues; United States; Vagina; Western Blotting; base; commensal microbes; cooking; early onset; host colonization; human microbiota; in vivo; intrapartum; mouse model; mutant; neonatal death; neonate; novel; overexpression; prevent; protein expression; small hairpin RNA; success; tissue culture; transcriptome; transcriptomics; transmission process; urogenital tract; vaccine candidate; vaccine development; vaginal mucosa

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.

项目概要 新生儿无乳链球菌（B 族链球菌，GBS）感染通常是致命的，并且与以下疾病密切相关： 母亲 GBS 阴道定植。使用预防性产时抗生素，在有效对抗早发疾病的同时， 有其自身的缺陷，并且对迟发性新生儿 GBS 疾病没有效果。因此，更长远的战略是 需要控制 GBS 感染。该提案重点关注一种新型 GBS 蛋白 Sak_1753，我们的目标是确定 它在 GBS 阴道定植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 在比较阴道时，被确定为 GBS A909 转录组中最高度上调的基因（约 3000 倍） 定殖到液体培养中的生长。 Sak_1753 由双组分系统 SaeRS 直接调节，并且没有 链球菌或预测蛋白质结构域之外的已知同源物，使其功能成为一个谜。蛋白质被保存 在所有已测序的 GBS 菌株中，由多个重复结构域组成，各重复结构域之间的重复次数不同 菌株。我们将描述 Sak_1753 的调控和定位，并确定其在阴道定植中的作用。我们有 在野生型 (WT) GBS A909 中创建了敲除 (KO; Δsak_1753::Spec) 和过表达 (OE; PrecA-sak_1753) 菌株 背景。对人阴道上皮细胞 (VK2) 的初步粘附测定表明，OE 菌株是 与上皮细胞的结合能力显着高于WT，而KO与上皮细胞的结合显着低于WT。 Sak_1753也显示出结合 对胶原蛋白 1 和纤维蛋白原具有特异性。此外，sak_1753 KO菌株表现出显着降低的阴道 小鼠模型中的定植。初始免疫荧光显微镜表明在细胞表面发现了 Sak_1753， 表明该蛋白质可能具有粘附作用。在本提案中，我们将进一步描述 Sak_1753 的角色。 GBS 将创建包含 Sak_1753 截短的菌株，以努力确定重复结构域在宿主中的作用 依恋。表达截短蛋白的菌株将在 VK2 细胞和体内小鼠的粘附测定中进行测试 殖民化。进一步确定 Sak_1753 蛋白质印迹技术和免疫荧光的细胞定位 将使用显微镜。这项研究的成功将有助于制定减少 GBS 粘附的策略 阴道组织，从而减少与 GBS 相关的新生儿疾病和死亡。