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

基本信息

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
项目类别：
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

项目摘要

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：产妇 GBS 阴道定植使用预防性产时抗生素，同时可有效预防早发型疾病，有其自身的缺陷，并且对迟发性新生儿 GBS 疾病没有影响。因此，更长期的策略是。该提案重点关注一种新型 GBS 蛋白 Sak_1753，我们的目标是确定其在 GBS 阴道定植中的作用及其作为基于蛋白质的候选疫苗 sak_1753 的资格此前已被确定。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） Sak_1753 的定殖到液体培养中的生长直接由双组分系统 SaeRS 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。在所有已测序的 GBS 菌株中，由多个重复域组成，重复次数不同我们将表征 Sak_1753 的调节和定位并确定其在阴道定植中的作用。在野生型 (WT) GBS A909 中创建 (KO; Δsak_1753::Spec) 和敲除过度表达 (OE; PrecA-sak_1753) 菌株人阴道上皮细胞 (VK2) 的初步粘附试验表明 OE 菌株是与 WT 相比，Sak_1753 能够显着更多地结合上皮细胞，并且 Sak_1753 的结合也显着更少。此外，sak_1753 KO 菌株表现出对胶原蛋白 1 和纤维蛋白原的显着降低。小鼠模型中的定植表明 Sak_1753 存在于细胞表面，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 的作用。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。进一步确定 Sak_1753 蛋白质印迹技术和免疫荧光的细胞定位。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。