PNEUMOCOCCAL TRANSFORMATION AND VIRULENCE

肺炎球菌转化和毒力

• 批准号: 2072738
• 负责人: H ROBERT MASURE
• 金额: $ 16.14万
• 依托单位: ROCKEFELLER UNIVERSITY
• 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/2072738
• 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.

肺炎链球菌是一种分布广泛的病原体，也是引起肺炎的主要原因。 肺炎、中耳炎、菌血症和脑膜炎。自当前以来 多价多糖疫苗未能保护最易受感染的人 对感染(2岁以下儿童和老年人)、身份识别 对肺炎球菌毒力重要的蛋白质的比例目前很高 新疫苗倡议的优先事项。的出现和在全球的传播 青霉素耐药肺炎球菌也对水平方向产生了兴趣。 通过自然转化进行的基因转移。我们最近报道了 首次使用随机翻译基因融合(PhoA突变)来 鉴定和改变革兰氏阳性生物体中的出口蛋白质。我们有 还开发了随机转录融合(LacZ突变)来评估 肺炎球菌的基因调控。使用这项新技术，我们拥有 描述了几个与已知序列相似的遗传基因座 出口蛋白质家族。同系物被发现依赖于蛋白质 多肽透皮，青霉素结合蛋白，CLP蛋白酶，两种- 组分传感器调节器和ABC转运体 负责RTX类细菌毒素的出口。这 技术首次使绘制表面蛋白质图谱成为可能 以系统和完整的方式对肺炎球菌进行研究。 鉴于我们的新基因融合发现了丰富的新蛋白质 技术，我们选择将这个项目的重点放在识别和 这些表面蛋白的特性是：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转化过程中的参与者。去做 因此，我们将评估我们的突变体库在这些步骤中的功能损失 肺炎球菌感染，特别是对上皮细胞和 内皮细胞，并在转化过程中。根据前期工作， 5个基因座的突变使肺炎球菌对II型的粘附性降低约60% 肺细胞或内皮细胞。我们将通过基因和分子决定 分析这些基因座是否直接编码粘连或它们是否中介 表达作为监管级联的一部分。这些基因座对家系的影响 毒力将在定植和感染的动物模型中进行评估。 参与黏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 确定新的蛋白质疫苗候选者的目标。在进一步的 初步工作，两种截然不同的突变会降低天然基因的效率 转变了大约90%。这些基因座是编码多肽的PLPA 渗透酶和REC，这是一个操纵子，编码输出的蛋白质和一个 RecA同源基因。我们将检验PLPA是一个调节点的假设 通过转运小分子寡肽来调节转化 细胞内的信使。REC操纵子，在 转化，是第一个被克隆的转化调控基因。 我们将确定顺式和反式作用元件，以调节 表达这个轨迹，以便开始组装一个模型 控制转换过程的信号级联。