CELLULAR INVASION BY GROUP B STREPTOCOCCUS

B组链球菌(GBS)是细菌性败血症最常见的原因 以及新生儿的脑膜炎。尽管这是一部内容广泛的文献 描述GBS感染的免疫学和流行病学的现有文献 新生儿，关于这一具体机制的信息很少 有机体用来引发疾病。我们之前已经演示过，在一个 亚人灵长类动物GBS致新生儿败血症子宫模型的建立 这种微生物能够侵袭肺泡呼吸道上皮 细胞。基于这些数据，我们开发了用于研究的体外检测方法。 GBS侵袭上皮细胞。在被资助的头两年 提案中，我们证明了GBS能够入侵呼吸道 上皮细胞，并鉴定了一些重要的细菌和 入侵所必需的细胞过程。于本年度内，我们 已经研究了GBS穿越完整极地的能力 体外培养上皮单分子层。此外，我们已经开始产生 转座子诱变等基因入侵突变体的研究 这种生物入侵细胞的分子发病机制。 为了扩大我们之前的观察，我们建议继续使用 转座子诱变获得GBS等基因突变株 改变了在体外侵袭上皮细胞的能力。突变体 与亲本菌株相比侵袭能力较差，仍然依附于 和其他表型不变的上皮细胞，将是 进一步分析。对入侵决定因素很重要的基因， 通过转座子诱变或基因组克隆到枯草杆菌中进行鉴定， 将通过核苷酸测序进行分析，基因产物 使用标准化的基因表达分析进行鉴定。美国人的能力 补充这些突变体的野生型入侵基因将由 使用我们最近获得的克隆载体和转化程序 改编为GBS。特性良好的入侵突变体的毒力将 检测其诱导新生大鼠GBS感染的能力 将感染模型与亲本菌株进行比较，确认了相关性 我们的体外研究结果。突变对GBS表面的影响 胞外产物的组成或分泌，表面的能力 GBS的提取物增强侵袭，以及对来自 GBS在细胞内状态下，将在试点中进行研究 为将来的研究做实验。我们预计这些研究应该开始 以确定在早期步骤中重要的细菌特性 新生儿B群链球菌感染的发病机制

Group B streptococci (GBS) are the most common cause of bacterial sepsis and meningitIs in the newborn infant. Although an extensive literature exists describing the immunology and epidemiology of GBS infections in neonates, very little information exists on the specific mechanisms this organism uses to induce disease. We had previously demonstrated, in an in utero model of neonatal sepsis induced by GBS in subhuman primates, that this organism is capable of invading alveolar respiratory epithelial cells. Based on this data, we developed in vitro assays for investigating epithelial cell invasion by GBS. During the first two years of the funded proposal, we demonstrated that GBS were capable of invading respiratory epithelial cells and identified some of the important bacterial and cellular processes necessary for invasion. During the current year, we have investigated the ability of GBS to traverse an intact polar epithelial monolayer in vitro. In addition, we have begun to generate isogenic invasion mutants by transposon mutagenesis in order to define the molecular pathogenesis of cellular invasion by this organism. To extend our previous observations, we propose to continue to use transposon mutagenesis to derive isogenic mutant strains of GBS with altered abilities to invade epithelial cells in vitro. Mutants which invade poorly relative to the parent strain, remain as adherent to epithelial cells and are otherwise unchanged in their phenotype, will be analyzed further. The genes important for invasion determinants, identified by transposon mutagenesis or genomic cloning into B. subtilis, will be analyzed by nucleotide sequencing, and the gene products identified using standardized gene expression assays. The ability of the wild type invasion genes to complement these mutants will be determined by using cloning vectors and transformation procedures we have recently adapted for GBS. The virulence of well characterized invasion mutants will be tested for their ability to induce GBS infections in neonatal rat GBS infection models compared to the parent strain, confirming the relevance of our in vitro findings. Effects of the mutations on GBS surface composition or secretion of extracellular products, the ability of surface extracts of GBS to augment invasion, and the induction of proteins from GBS during the intracellular state, will be investigated in pilot experiments for future studies. We anticipate these studies should begin to identify the bacterial traits important in the early steps in the pathogenesis of neonatal infections caused by group B streptococci.