CELLULAR INVASION BY GROUP B STREPTOCOCCUS

B族链球菌（GBS）是细菌性脓毒症的最常见原因 和脑膜炎。虽然大量的文献 存在描述GBS感染的免疫学和流行病学， 新生儿，很少有信息存在的具体机制， 微生物用于诱发疾病。我们之前在一个 在亚人灵长类动物中由GBS诱导的新生儿败血症的子宫模型， 这种微生物能够侵入肺泡呼吸上皮细胞 细胞基于这些数据，我们开发了体外试验，用于研究 GBS引起的上皮细胞侵袭。在获得资助的头两年， 建议，我们证明了GBS能够侵入呼吸道， 上皮细胞，并确定了一些重要的细菌和 入侵所必需的细胞过程。今年，我们 研究了GBS穿越完整极地的能力， 上皮细胞单层。此外，我们已经开始产生 通过转座子诱变的同基因入侵突变体，以确定 该生物体细胞入侵的分子发病机制。 为了扩展我们以前的观察，我们建议继续使用 转座子诱变以获得GBS的同基因突变株， 改变了体外侵入上皮细胞的能力。的突变体 相对于亲本菌株，侵入较差，保持与 上皮细胞，并且在其表型方面没有改变，将被 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B中鉴定。枯草杆菌， 将通过核苷酸测序进行分析， 使用标准化的基因表达测定鉴定。的能力 将通过以下方法确定补充这些突变体的野生型侵入基因： 使用克隆载体和转化程序，我们最近 适应GBS。充分表征的入侵突变体的毒力将 测试它们诱导新生大鼠GBS感染的能力 感染模型与亲本菌株相比，证实了相关性 我们在试管中的发现突变对GBS表面的影响 组成或分泌的胞外产物，表面的能力， GBS的提取物，以增加入侵，并诱导蛋白质从 将在试点中研究细胞内状态下的GBS 为今后的研究做实验。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征， 新生儿B族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.