Isolation & Characterization of T gondii Virulence Genes

描述（由申请方提供）：球虫寄生虫弓形虫是全球范围内人类和牲畜的严重病原体。T.弓形虫是一种专性细胞内寄生虫，可导致胎儿神经发育异常和免疫功能低下患者的脑炎。现有的抗T.弓形虫是有毒的，必须混合服用，并且不能消除慢性感染。我们的目标是识别和功能表征T。弓形虫基因是建立小鼠慢性感染所必需的，但不是细胞培养生长所必需的。我们称这些毒力基因为。为了这个目标，我们已经调整了T。刚地。STM最初用于用独特的序列标签诱变沙门氏菌。通过比较接种物中代表的标签与从感染宿主中回收的细菌来分离毒力基因。一个4900 T.已经通过口服接种体外衍生的包囊筛选了弓形虫STM克隆在小鼠中建立慢性感染的能力。在这4900个突变体中，4500个突变来自化学诱变，400个突变来自插入诱变。我们发现了180种潜在突变体，它们在小鼠中引起慢性感染的能力存在缺陷。在180个STM菌株中，有7个是插入突变体。当在小鼠中重新测试时，四种插入突变体的毒性至少低100倍（通过LD 50测量）。这些突变体的插入位点侧翼的基因已经被分离出来，目前正在进行分析。针对预测蛋白质之一产生的抗血清仅在应激条件下识别120 KD蛋白质。另一个基因预测具有信号序列和几个跨膜结构域的蛋白质。然后，我们将使用分子遗传学，生物化学和细胞生物学方法的组合来表征这些基因的功能。大量的潜在突变体意味着许多基因参与毒力;这些基因将最容易通过扩大和筛选插入突变体库来分离。我们将产生并分析6000个新的插入突变体。这将是重要的调查，如果平行的毒力基因存在于其他致病性球虫，不容易在体外操作，但有基因组测序项目，如疟原虫，隐孢子虫。

DESCRIPTION (provided by the applicant): The coccidian parasite Toxoplasma gondii is a serious pathogen of humans and livestock worldwide. T. gondii is an obligate intracellular parasite that causes abnormal fetal neurological development and encephalitis in immunocompromised patients. The drugs available against T. gondii are toxic, must be taken in combinations, and are not capable of eliminating a chronic infection. Our goal is to identify and functionally characterize T. gondii genes that are essential for the establishment of a chronic infection in mice, but not necessary for cell culture growth. We are terming these virulence genes. For this goal, we have adapted signature-tagged mutagenesis (STM) for T. gondii. STM was originally used to mutagenize Salmonella with a unique sequence tag. Virulence genes were isolated by comparing the tags represented in the inoculum versus the bacteria recovered from an infected host. A library of 4900 T. gondii STM clones has been screened for their ability to establish a chronic infection in mice using oral inoculation of in vitro derived cysts. Of these 4900 mutants, 4500 have derived their mutation from chemical mutagenesis and 400 were generated by insertional mutagenesis. We have uncovered 180 potential mutants defective in their ability to cause a chronic infection in mice. Of the 180 STM strains, seven are insertional mutants. When retested in mice, four of the insertional mutants were at least 100-fold less virulent (as measured by LD50). The genes flanking the insertion site for these mutants have been isolated and are currently being analyzed. Antiserum generated against one of the predicted proteins recognizes a 120 KD protein only during stress conditions. Another gene predicts a protein with a signal sequence and several transmembrane domains. We will then use a combination of molecular genetics, biochemistry, and cell biological approaches to characterize the function(s) of these genes. The large numbers of potential mutants imply that many genes are involved in virulence; these will be most easily isolated by expanding and screening the library of insertional mutants. We will generate and analyze 6000 new insertional mutants. It will be important to investigate if parallel virulence genes exist in other pathogenic coccidians that are not as easy to manipulate in vitro, but that have genome sequencing projects such as Plasmodium, and Cryptosporidium.