PNEUMOCOCCAL TRANSFORMATION AND VIRULENCE

肺炎链球菌是一种广泛分布的病原体，也是导致肺炎的主要原因。 肺炎、中耳炎、菌血症和脑膜炎。由于目前 多价多糖疫苗不能保护那些最易感的人 感染（2岁以下儿童和老年人），识别 对肺炎球菌毒力重要的蛋白质目前很高， 优先考虑新的疫苗倡议。的出现和全球传播 青霉素耐药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）， 鉴定和改变革兰氏阳性生物体中输出蛋白质。我们有 我还开发了随机转录融合（LacZ诱变），以评估 肺炎球菌的基因调控利用这项新技术， 表征了几个与已知的序列相似的遗传位点， 输出蛋白质的家族。同源物被发现是蛋白依赖性的 肽渗透酶、青霉素结合蛋白、Clp蛋白酶、两种- 组件传感器调节器和ABC（ATP结合盒）转运蛋白 负责出口RTX类细菌毒素。这 这项技术首次使绘制表面蛋白成为可能， 对肺炎球菌进行系统而全面的研究 鉴于我们的新基因融合鉴定出的新蛋白质丰富 技术，我们选择把这个项目的重点放在识别和 这些表面蛋白的表征是：1]毒力 决定簇，因此可以作为缀合物疫苗的候选物， 2]参与外源DNA转化过程。做 所以，我们将评估我们的突变体库， 肺炎球菌感染，特别是粘附于上皮细胞， 内皮细胞和转化过程中。在前期工作的基础上， 5个基因座的突变使肺炎球菌粘附减少约60%至II型 肺细胞或内皮细胞。我们将通过基因和分子 分析这些基因座是否直接编码粘连，或者它们是否介导 表达作为调节级联的一部分。这些基因座对 将在动物模型中评估毒力的定殖和感染。 参与粘附或其他步骤的表面蛋白质 将评估发病机制的免疫原性和保护活性， 目的是鉴定新的蛋白质疫苗候选物。进一步 初步工作中，两种不同的突变降低了自然 约90的转化。这些基因座是编码肽的plpA， 通透酶和rec，rec是编码输出蛋白质的操纵子， RecA同源物。我们将检验plpA是一个调控位点的假设， 通过转运小寡肽调节转化， 细胞内信使rec操纵子在 这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， 为了开始组装一个模型， 控制转化过程的信号级联。

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.