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: 10216957
• 负责人: Lamar S Thomas
• 金额: $ 3.12万
• 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 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10216957
• 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; Structure; 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.

项目总结 新生儿中的无乳链球菌(B群链球菌，GBS)感染通常是致命的，与 母体GBS阴道定植。预防性产中抗生素的使用，虽然对早发性疾病有效， 有自己的陷阱，对迟发性新生儿GBS疾病没有影响。因此，更长期的战略是 控制GBS感染所需的。这项建议关注的是一种新的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蛋白质疫苗候选者的资格。SAK_1753以前是 与阴道相比，被确定为GBS A909转录组中上调最高的基因(~3000倍) 液体培养中的定植到生长。SAK_1753由两个组件系统SAERS直接调节，没有 已知的链球菌外的同源物或预测的蛋白质结构域，使其功能成为一个谜。这种蛋白质是保守的 在所有已测序的GBS菌株中，它由多个重复结构域组成，重复数不同于 菌株。我们将对SAK_1753的调控和定位进行研究，并确定其在阴道定植中的作用。我们有 在野生型(WT)GBSA909中创建了基因敲除(KO；ΔSAK_1753：：SPEC)和过度表达(OE；PRECA-SAK_1753)菌株 背景资料。与人阴道上皮细胞(VK2)的初步黏附试验表明，OE株是 能与上皮细胞结合的能力明显高于WT，KO结合明显低于WT。SAK_1753也显示出结合 对1型胶原和纤维蛋白原具有特异性。此外，SAK_1753 KO株的阴道分泌物显著减少。 在小鼠模型中的殖民。初步免疫荧光显微镜显示，SAK_1753存在于细胞表面， 这表明该蛋白可能具有粘附性。在这项提案中，我们将进一步描述SAK_1753的作用。GBS 将创建包含SAK_1753截断的菌株，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与VK2细胞和体内小鼠的黏附试验中进行测试 殖民主义。进一步确定SAK_(1753)的细胞定位 将使用显微镜。这项研究的成功将有助于减少GBS粘附性的策略 阴道组织，从而减少与GBS相关的新生儿疾病和死亡。