PNEUMOCOCCAL TRANSFORMATION AND VIRULENCE

肺炎球菌转化和毒力

• 批准号: 2672372
• 负责人: Elaine I Tuomanen
• 金额: $ 16.25万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2672372
• 关键词: SDS polyacrylamide gel electrophoresis; Streptococcus pneumoniae; adhesin; biological signal transduction; cell adhesion; disease /disorder model; epithelium; fusion gene; gene expression; gene mutation; genetic library; genetic regulation; genetic transduction; laboratory rat; molecular cloning; nucleic acid sequence; permease; protein structure function; transcription factor; virulence

Streptococcus pneumoniae is a widespread pathogen and a leading cause of pneumonia, otitis media, bacteremia and meningitis. Since the current multivalent polysaccharide vaccines fail to protect those most susceptible to infection (children under the age of 2 and older adults), identification of proteins important to pneumococcal virulence is currently a high priority for new vaccine initiatives. The emergence and global spread of penicillin resistant pneumococci has also focused interest on horizontal gene transfer by natural transformation. We have recently reported the first use of random translational gene fusions (PhoA mutagenesis) to identify and alter exported proteins in a gram positive organism. We have also developed random transcriptional fusions (LacZ mutagenesis) to assess gene regulation in pneumococcus. Using this new technology, we have characterized several genetic loci with sequence similarity to known families of exported proteins. Homologs were found to protein-dependent peptide permeases, penicillin binding proteins, Clp proteases, two- component sensor regulators and ABC (ATP binding cassette) transporters responsible for the export of the RTX class of bacterial toxins. This technology makes it possible for the first time to map the surface proteins of the pneumococcus in a systematic and complete manner. Given the wealth of new proteins identified by our new gene fusion technology, we choose to focus this project on the identification and characterization of those surface proteins which are: 1] virulence determinants and thus could serve as candidates for a conjugate vaccine and 2] participants in the process of transformation by exogenous DNA. To do so, we will assess our banks of mutants for a loss of function in the steps of pneumococcal infection, particularly adherence to epithelia and endothelia and in the process of transformation. Based on preliminary work, mutations in 5 loci decrease pneumococcal adhesion by about 60% to Type II lung cells or endothelial cells. We will determine by genetic and molecular analysis if these loci directly encode adhesions or if they mediate expression as part of a regulatory cascade. The impact of these loci on virulence will be assessed in animal models for colonization and infection. Surface proteins that participate in adherence or other steps in pathogenesis will be assessed for immunogenic and protective activity with the aim of identifying new protein vaccine candidates. In further preliminary work, two distinct mutations decrease the efficiency of natural transformation by about 90. These loci are plpA which encodes a peptide permease and rec which is an operon that encodes an exported protein and a RecA homolog. We will test the hypothesis that plpA is a regulatory locus modulating transformation by transporting small oligopeptides that serve as intracellular messengers. The rec operon, is upregulated during transformation and is the first transformation-regulated gene to be cloned. We will identify both cis and trans acting elements that regulate expression of this locus in order to begin to assemble a model of the signaling cascade that controls the process of transformation.

肺炎链球菌是一种广泛传播的病原体，也是引起肺炎的主要原因 肺炎、中耳炎、菌血症和脑膜炎。自当前 多价多糖疫苗无法保护最易感人群 感染（2岁以下儿童和老年人）、识别 目前对肺炎球菌毒力重要的蛋白质的数量很高 新疫苗计划的优先事项。的出现和全球传播 青霉素耐药性肺炎球菌也将兴趣集中在水平 通过自然转化进行基因转移。我们最近报道了 首次使用随机翻译基因融合（PhoA 诱变） 识别并改变革兰氏阳性生物体中的输出蛋白质。我们有 还开发了随机转录融合（LacZ 诱变）来评估 肺炎球菌的基因调控。利用这项新技术，我们 表征了与已知序列相似的几个遗传位点 输出蛋白质家族。发现了蛋白质依赖性的同系物 肽通透酶、青霉素结合蛋白、Clp 蛋白酶、二元酶 成分传感器调节器和 ABC（ATP 结合盒）转运蛋白 负责RTX类细菌毒素的输出。这 技术首次使得绘制表面蛋白质图谱成为可能 系统、完整地了解肺炎球菌。 鉴于我们的新基因融合鉴定出大量新蛋白质 技术，我们选择将这个项目的重点放在识别和 这些表面蛋白的特征是： 1] 毒力 决定因素，因此可以作为结合疫苗的候选者 2]外源DNA转化过程的参与者。要做的事 因此，我们将评估突变体库的功能丧失情况 肺炎球菌感染，特别是与上皮细胞的粘附和 内皮细胞和转化过程中。在前期工作的基础上， 5 个位点的突变使 II 型肺炎球菌的粘附力降低约 60% 肺细胞或内皮细胞。我们将通过遗传和分子来确定 分析这些基因座是否直接编码粘连或它们是否介导 表达作为监管级联的一部分。这些位点的影响 将在动物模型中评估定植和感染的毒力。 参与粘附或其他步骤的表面蛋白 将评估发病机制的免疫原性和保护活性 目的是确定新的候选蛋白质疫苗。在进一步 初步工作中，两种不同的突变降低了自然的效率 约 90 的转化。这些位点是 plpA，编码肽 permease和rec是一个操纵子，编码输出的蛋白质和 RecA 同系物。我们将检验 plpA 是调控位点的假设 通过运输小寡肽来调节转化 细胞内信使。的rec操纵子，在期间被上调 转化，并且是第一个被克隆的转化调控基因。 我们将识别调节顺式和反式作用的元件 表达这个轨迹，以便开始组装一个模型 控制转化过程的信号级联。