STUDIES OF THE STRUCTURE AND FUNCTION OF PERTUSSIS TOXIN

对百日咳毒素（PT）的生物合成及其结构和作用机制进行了研究。我们已经启动了一项研究，以解决疫苗接种是否可以为PT的抗原变异提供选择性压力，如果是这样，抗原漂移是否可能导致无细胞百日咳疫苗的效力下降。我们将比较广泛使用无细胞百日咳疫苗前后获得的日本百日咳博德泰拉分离株的PT （ptx）基因序列。此外，我们将研究PT抗原变异是否会影响疫苗对百日咳的保护能力。为了做到这一点，我们将克隆来自B. bronchiseptica的PT基因，并将这些基因与百日咳中发现的基因交换。然后，我们将检查小鼠血清免疫百日咳原PT的能力，以中和支气管菌毒素。到目前为止，我们已经获得了1975年至今获得的临床分离株。我们已经开始从20世纪70年代获得的几个分离株和最近的几个分离株中克隆基因，以便我们可以获得ptx基因的序列信息。此外，我们已经克隆了结核分枝杆菌的ptx基因，并正在对结核分枝杆菌的ptx基因进行替换。我们正在继续对百日咳白咳的PT分泌进行分析。在此之前，我们确定了9个对毒素分泌至关重要的ptl基因。为了进一步了解分泌机制，我们研究了PtlC，其中包含一个假定的核苷酸结合区。我们发现PtlC对分泌至关重要。在该蛋白的假定核苷酸结合区域的突变表现出显性的负表型，这表明PtlC可能是一种提供能量的atp酶或一种信号通路打开的激酶。此外，显性负表型表明PtlC与自身或运输装置的其他关键组分相互作用。我们进一步研究了Ptl传输装置的结构。之前，我们发现PtlF与Pt1I相互作用。通过确定PtlF能够与另一个PtlF分子相互作用，我们扩展了我们对转运结构形态的认识。这项工作增加了我们对百日咳毒素分泌的认识，并可能有助于构建有效产生和分泌PT的百日咳B.菌株，这些菌株将对疫苗生产有用。此外，了解这种毒素的分泌机制可能有助于研制出分泌灭活百日咳毒素的减毒活疫苗，从而诱导产生有效的免疫反应。

Research was conducted to investigate the biosynthesis of pertussis toxin (PT) as well as the structure and mechanism of action of the toxin. We have initiated a study to address the question of whether vaccination can provide selective pressure for antigenic variation of PT and, if so, whether antigenic drift might result in decreased efficacy of acellular pertussis vaccine. We will compare the sequence of the PT (ptx) genes of Japanese isolates of Bordetella pertussis obtained both before and after wide-spread use of acellular pertussis vaccines. In addition, we will examine whether antigenic variation of PT can affect the ability of vaccine to protect against B. pertussis. In order to do this, we will clone the PT genes from B. bronchiseptica, and exchange these genes for those found in B. pertussis. We will then examine the ability of sera from mice immunized with PT originating from B. pertussis to neutralize the bronchiseptica toxin. To date, we have obtained clinical isolates that were obtained between 1975 and the present. We have begun to clone the genes from several of the isolates obtained in the 1970s and several of the recent isolates so that we can then obtain sequence information on the ptx genes. In addition, we have cloned the ptx genes from B. bronchiseptica and are in the process of replacing the ptx genes of the bronchiseptica genes. We are continuing our work on the analysis of the secretion of PT from B. pertussis. Previously, we identified nine ptl genes that are essential for the secretaion of the toxin. In order to gain further insight into the mechanisms of secretaion, we studied PtlC which contains a putative nucleotide-binding region. We found that PtlC is cricial for secretion. Mutations in the putative nucleotide-binding region of this protein exhibit a dominant negative phenotype suggesting that PtlC may be an ATPase that provides the energy or a kinase that signals the opening of a gate or channel. Furthermore, the dominant negative phenotype suggests that PtlC interacts either with itself or other critical components of the transport apparatus. We have further investigated the architecture of the Ptl transport apparatus. Previously, we showed that PtlF interacts with Pt1I. We have extended our knowledge of the morphology of the transport structure by determining that PtlF is capable of interacting with another PtlF molecule. This work increases our knowledge of the secretion of pertussis toxin and may aid in the construction of strains of B. pertussis which efficiently produce and secrete PT. Such strains would be useful for vaccine production. In addition, knowledge of the mechanism of secretion of this toxin might aid in the development of live-attenuated vaccines that secrete inactivated forms of pertussis toxin, thus inducing a productive immune response.