PHASE VARIATION IN THE PATHOGENESIS OF GBS INFECTION

GBS 感染发病机制的阶段变化

• 批准号: 3145709
• 负责人: DAVID G PRITCHARD
• 金额: $ 16.88万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3145709
• 关键词: Streptococcus agalactiae; Streptococcus infection; antibacterial antibody; bacterial capsules; density gradient ultracentrifugation; electron microscopy; electroporation; exo alpha sialidase; gene mutation; genetic manipulation; human tissue; laboratory mouse; microorganism culture; molecular cloning; mucins; plasmids; sialate; transposon /insertion element; virulence

Group B streptococci (GBS) are presently the most frequent cause of serious bacterial infections of neonates in the United States. The long-term goal of our studies is to find out why GBS, a commensal organism in about a third of the normal population of pregnant women, should cause serious, often fatal, infection in about 4 of every 1000 live-born infants. Our hypothesis is that GBS are not normally pathogenic but have pathogenic potential when as yet unknown environmental factors trigger a major phenotypic change that includes the production of increased amounts of capsular polysaccharide. The biochemical basis for capsule size variation in GBS will be studied. This will include determining if the type-specific polysaccharide chains are longer in large capsule strains, if there are more chains, or if there are other structural changes. Possible coordinate regulation of capsule size variation will be studied, looking specifically for concurrent changes in teichoic acid, neuraminidase, and other bacterial components. The molecular genetic basis of capsule size variation will be studied using transposon insertional mutagenesis to produce mutations in genes that affect capsule size. A new electroporation technique, along with a recently constructed plasmid suitable for use with tetracycline-resistant GBS strains, will be employed to yield mutants containing single transposon inserts. The DNA flanking the transposon inserts in mutants of interest will be cloned and the gene products involved in capsule size phase variation will be identified. Defined mutations will be made in some of the cloned genes which will then be used for the allelic replacement of the corresponding chromosomal genes by homologous recombination. The role of capsular size variation in GBS infection and in asymptomatic colonization will be studied. The hypothesis that disease causing strains possess large capsules that revert to small capsule forms upon culture will be tested by assessing capsule size by buoyant density centrifugation and electron microscopy Of organisms prior to culture on artificial media. Capsule size in asymptomatic vaginal colonization will be studied using two new complementary procedures. The first involves dissociation of GBS bound to vaginal epithelial cells using Triton X-100 prior to density gradient centrifugation. The second method involves electron microscopy of GBS in vaginal secretions selectively enriched for GBS on nickel grids coated with specific antibody. Capsule size will be correlated with levels of type-specific antibody in the vaginal secretions to determine if antibody plays a role in suppressing large-capsule forms of GBS. The effect of capsule size as a virulence factor will be studied in mouse protection tests, and the amount of antibody required for opsonization of large and small capsule strains will be studied in opsonophagocidal assays. Mucin enhances the virulence of certain GBS strains and appears to induce a phase shift. We plan to determine if the presence of sialic acid in mucin is essential for its ability to enhance virulence. The role of GBS neuraminidase in this process will be assessed using neuraminidase-negative mutants.

B群链球菌(GBS)是目前引起严重肺炎的最常见原因 美国新生儿的细菌感染。长期目标 我们的研究之一是找出为什么GBS，一种大约 第三，对正常人群的孕妇，应造成严重， 通常是致命的，每1000名活产婴儿中约有4人感染。我们的 假设GBS通常不是致病的，但有致病作用 潜在的，当未知的环境因素引发重大 表型变化，包括产生数量增加的 壳多糖。 将研究GBS中种皮大小变异的生化基础。 这将包括确定特定类型的多糖链是否 如果有更多的链，或者如果有 还有其他的结构性变化。胶囊可能的协调调节 将研究大小变化，专门寻找同时发生的变化 在磷壁酸、神经氨酸酶和其他细菌成分中。 胶囊大小变异的分子遗传学基础将用 转座子插入诱变以产生基因突变 影响胶囊大小。一种新的电穿孔技术，以及一种 新近构建的适用于四环素耐药的质粒 GBS菌株，将用于产生含有单一转座子的突变体 镶件。感兴趣突变体中转座子插入两侧的DNA 将被克隆的基因产物与包膜大小阶段有关 变异将被识别出来。定义的突变将在一些 克隆的基因随后将用于等位基因的替换 通过同源重组获得相应的染色体基因。 包膜大小变化在GBS感染和无症状中的作用 将对殖民进行研究。关于致病菌株的假设 拥有大胶囊，在培养过程中会还原为小胶囊形式 通过浮力密度离心法评估胶囊大小进行测试 人工培养前的有机体的电子显微镜。 无症状阴道定植的包膜大小将使用两种 新的补充程序。第一个涉及GBS束缚的解离 Triton X-100在密度梯度前对阴道上皮细胞的作用 离心法。第二种方法涉及GBS的电子显微镜检查。 在镀有GBS的镍网上选择性富含GBS的阴道分泌物 特异性抗体。胶囊大小将与血药浓度相关 阴道分泌物中的类型特异性抗体确定抗体 在抑制大胶囊形式的GBS方面发挥作用。的影响 胶囊大小作为毒力因素将在小鼠保护中进行研究 试验，以及大的和大的和调理所需的抗体量 小胶囊菌株将在吞噬视光试验中进行研究。粘蛋白 增强某些GBS菌株的毒力，并似乎诱导了一个阶段 换档。我们计划确定粘蛋白中唾液酸的存在是否 对其增强毒力的能力至关重要。GBS的角色 这一过程中的神经氨酸酶将使用神经氨酸酶阴性进行评估。 变种人。