Genomic Comparison of A. actinomycetemcomitans Strains

描述（由申请人提供）：假设细菌病原体的基因组由核心基因库和柔性基因库组成。稳定的核心基因库是克隆遗传的，几乎存在于一个物种的每一个品系中。相比之下，柔性库包含通过水平基因转移（HGT）获得的遗传元件。不同的细菌菌株具有不同的灵活基因群，这些基因群是根据它们特定的生活方式而定制的。基因组岛（Genomicislands，GEI）是细菌通过HGT获得的10- 100 kbp的DNA大片段，是细菌基因组柔性基因库的重要组成部分。病原菌和非病原菌在某些物种中的区别可以通过病原菌株获得GEI来解释。细菌也可以通过基因组缺失来增加其毒力。在某些情况下，基因组缺失消除了毒力因子的抑制剂，并允许致病性的完全表达。伴放线杆菌（Actinobacillusactinomycetemcomitans，Aa）是引起牙周炎的主要致病菌。Aa的单个克隆似乎表现出不同程度的致病性，甚至可能采用不同的毒力机制。我们的实验室已经获得了初步的证据，GEI和较小的基因组胰岛（未知功能）在不同的Aa菌株的存在。GEI和SLET的插入导致一些Aa菌株中基因的截短和/或失活。在这里，我们测试的假设，这两个GEI/胰岛和基因组缺失是常见的Aa菌株。此外，基因的获得和丢失可能会改变Aa的表型和毒力。确定了4个特定目的：I）基于测序的Aa菌株HK 1651，通过DNA微阵列进行Aa分离物的基因组比较，II）通过PCR基因组定位，然后克隆和测序，鉴定Aa中的非HK 1651 GEI/胰岛，III）检查Aa GEI/胰岛的表达及其对Aa基因表达谱的影响，IV）通过阵列评估Aa基因组随时间的稳定性。该研究计划预计将（i）生成不同Aa菌株基因组中GEI/胰岛和大DNA缺失的完整目录，（ii）获得表达的证据，（iii）在存在或不存在GEI/胰岛的情况下检查Aa的基因表达谱，（iv）获得Aa在短时间（数年）内基因组不稳定性的证据，（v）鉴定Aa的新的候选毒力因子和（vi）阐明Aa的表型和毒力变异的遗传基础。本研究结果有助于牙周病发病机制的研究。

DESCRIPTION (provided by applicant): It is hypothesized that the genomes of bacterial pathogens are comprised of a core gene pool and a flexible gene pool. The stable core gene pool is inherited clonally and is found in virtually every strain of a species. In contrast, the flexible pool contains genetic elements that are acquired by horizontal gene transfer (HGT). Individual bacterial strains have different constellations of flexible genes that are tailored to their specific life style. Genomic islands (GEIs) are large DNA blocks (10-100kbp) acquired by bacteria via HGT en bloc and constitute important components of the flexible gene pool of bacterial genome. The distinctions between pathogenic and non-pathogenic bacteria in some species have been explained by the acquisition of GEIs by pathogenic strains. Bacteria may also increase their virulence by genomic deletions. In some instances, genomic deletions eliminate inhibitors of virulence factors and allow a full expression of pathogenicity. The Gram-negative facultative bacillus Actinobacillus actinomycetemcomitans (Aa) is a major pathogen in human periodontitis. Individual clones of Aa appear to exhibit differing degrees of pathogenicity and might even employ different virulence mechanisms. Our laboratory has obtained preliminary evidence for the presence of GEIs and smaller genomic islets (of unknown functions) in diverse Aa strains. The insertions of GEIs and slets have lead to truncation and/or inactivation of genes in some Aa strains. Here we test the hypothesis that both GEIs/islets and genomic deletions are common in Aa strains. Furthermore, the gain and loss of genes may change the phenotypes and virulence of Aa. 4 Specific Aims are identified: I) Perform genomic comparison of Aa isolates by DNA microarrays based on the sequenced Aa strain HK1651, II) identify non-HK1651 GEIs/islets in Aa by PCR genomic mapping followed by cloning and sequencing, III) Examine the expression of Aa GEIs/islets and their impact to the gene expression profiles of Aa, IV) Assess the stability of Aa genome over time by arrays. The research plan is expected to (i) generate a thorough catalogue of GEIs/islets and large DNA deletions in the genomes of diverse Aa strains, (ii) obtain evidence for the expression (and possibly the functions) of GEIs/islets, (iii) examine the gene expression profiles of Aa with or without the presence of GEIs/islets, (iv) obtain evidence for the genomic instability of Aa over short periods of time (years), (v) identify new candidate virulence factors of Aa and (vi) elucidate the genetic basis of the phenotype and virulence variations of Aa. The results are likely to further the progress in the field of periodontal disease pathogenesis.