PHASE VARIATION IN THE PATHOGENESIS OF GBS INFECTION

GBS 感染发病机制的阶段变化

• 批准号: 3145708
• 负责人: DAVID G PRITCHARD
• 金额: $ 17.28万
• 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/3145708
• 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族链球菌（GBS）是目前最常见的严重 美国新生儿的细菌感染。 远景目标 我们的研究之一是找出为什么GBS，一种大约在 三是正常人群中的孕妇，应引起严重， 通常是致命的，每1000个活产婴儿中约有4个感染。 我们 假设GBS通常不是致病性，但具有致病性 当未知的环境因素引发重大的 表型变化，包括产生增加量的 荚膜多糖 将研究GBS中胶囊大小变化的生化基础。 这将包括确定类型特异性多糖链是否 在大胶囊菌株中，如果有更多的链， 还有其他的结构性变化。 胶囊的可能协调调节 将研究规模变化，特别是寻找并发的变化 磷壁酸、神经氨酸酶和其他细菌成分。 将使用以下方法研究荚膜大小变异的分子遗传基础： 转座子插入诱变以在基因中产生突变， 影响胶囊大小。 一种新的电穿孔技术，沿着 最近构建的适合用于四环素抗性的质粒 GBS菌株，将用于产生含有单个转座子的突变体 插入物。 转座子侧翼的DNA插入感兴趣的突变体中 将被克隆的基因产物与荚膜大小相关联 变化将被识别。 定义的突变将在一些 克隆的基因，然后将用于等位基因替换的 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GBS患者中的作用 殖民地将被研究。 致病菌株 具有大胶囊，在培养时恢复为小胶囊形式， 通过浮力密度离心法评估胶囊大小进行测试， 电子显微镜在人工培养基上培养之前的有机体。 无症状阴道定植中的包膜大小将使用两种 新的补充程序。 第一个涉及GBS结合的解离 在密度梯度之前使用Triton X-100对阴道上皮细胞 离心法 第二种方法涉及GBS的电子显微镜， 阴道分泌物在涂有GBS的镍网格上选择性富集 特异性抗体。 胶囊大小将与 型特异性抗体在阴道分泌物，以确定是否抗体 在抑制大胶囊形式的GBS中起作用。 的影响 将在小鼠保护中研究作为毒力因子的荚膜大小 测试，以及调理大的和 将在调理噬菌体测定中研究小荚膜菌株。 粘蛋白 增强了某些GBS菌株的毒力，并似乎诱导了一个阶段， 班 我们计划确定粘蛋白中唾液酸的存在是否是 这是其增强毒力的能力所必需的。 GBS的作用 神经氨酸酶在这一过程中将评估使用神经氨酸酶阴性 变种人