Isolation & Characterization of T gondii Virulence Genes

简介（由申请人提供）：弓形虫球虫寄生虫是世界范围内人类和牲畜的严重病原体。弓形虫是一种专性细胞内寄生虫，可导致免疫功能低下患者的胎儿神经发育异常和脑炎。现有的抗弓形虫药物是有毒的，必须联合服用，并且不能消除慢性感染。我们的目标是鉴定和功能表征弓形虫基因，这些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。我们将这些毒力基因命名为。为了实现这一目标，我们对弓形虫进行了特征标记诱变（STM）。STM最初用于用一个独特的序列标签诱变沙门氏菌。通过比较接种物中所代表的标签与从感染宿主中恢复的细菌，分离出毒力基因。对4900个弓形虫STM克隆库进行了筛选，以确定它们能够通过口服接种体外来源的囊肿在小鼠中建立慢性感染。在这4900个突变体中，4500个突变体来自化学诱变，400个突变体来自插入诱变。我们已经发现了180种潜在的突变体，它们在引起小鼠慢性感染的能力上存在缺陷。在180株STM菌株中，有7株是插入突变体。当在小鼠中重新测试时，其中四个插入突变体的毒性至少降低了100倍（根据LD50测量）。这些突变体插入位点两侧的基因已经被分离出来，目前正在进行分析。针对其中一种预测蛋白产生的抗血清仅在应激条件下识别120 KD蛋白。另一个基因预测蛋白质的信号序列和几个跨膜结构域。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。大量的潜在突变意味着许多基因参与了毒力；通过扩展和筛选插入突变体库，这些突变体将最容易分离。我们将生成并分析6000个新的插入突变体。研究其他致病球虫（如疟原虫和隐孢子虫）是否存在类似的毒力基因将是重要的，这些球虫在体外不容易操作，但有基因组测序项目。

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.