regulation of gene expression in Mannheimia haemolytica A1.

溶血性曼海姆病A1 （Mh）是引起牛肺炎巴氏杆菌病的重要细菌病原体。这种呼吸道疾病每年给饲养场养牛业造成数百万美元的经济损失。因此，人们对了解这种细菌的发病机制以及制定有效的措施来保护动物免受感染一直很感兴趣。20多年来，我的实验室已经成功地对细菌的许多毒力因子的编码基因进行了表征。其中一些毒力因子已作为重组抗原作为疫苗成分或在口服给药系统的开发中从转基因植物中表达。另一方面，调控这些毒力基因表达的机制尚不清楚。对于这些毒力基因在感染过程的不同阶段何时表达，我们所知甚少。此外，对调节这些毒力基因表达的环境信号一无所知。本提案的目的是：i)研究这些基因在动物体内感染期间的表达谱；ii)研究调控毒力基因表达的机制。将进行时间过程感染实验，其中几只动物将感染Mh并监测其肺炎体征。每隔一段时间，要么收集鼻腔样本，要么牺牲一头小牛。收集肺洗液和肺组织样本提取总RNA。该RNA将用于实时RT-PCR，使用Mh特异性引物来检测特定基因的表达。根据其编码产物在发病过程中的活性，预计一些基因在感染早期表达，而另一些基因在感染后期表达。第二个目标是检查在毒力基因的调控双组分调控系统的贡献。我们已经从Mh基因组序列中确定了几对系统。突变体将在两个同源蛋白中构建，以检查对蛋白表达的影响以及响应双组分系统的调控途径。

Mannheimia haemolytica A1 (Mh) is an important bacterial pathogen that causes bovine pneumonic pasteurellosis. This respitatory disease results in millions of dollars in economic loss annually to the feedlot cattle industry. Hence, there are continuing interest in understanding the pathogenesis of the bacterium as well as to develop effecive measures to protect animals from the infection. My laboratory has been successful on the characterization of genes that code for many of the virulence factors of the bacterium for more than 20 years. Some of these virulence factors have been produced as recombinant antigens as vaccine components or expressed from transgenic plants in the development of an oral delivery system. On the other hand, the mechanisms that regulate expressin of these virulence genes are not understood. Very little is known about when these virulence genes are expressed during different stages of the infection process. In addition, nothing is known about the enviromental signals that regulate expression of these virulence genes. The objective of this proposal are: i) investigate expression profiles of these genes during an infection in vivo in the animals; and ii) to investigate the mechanisms that regulate expression of the virulence genes. Time course infection experiments will be carried out in which several animals will be infected with Mh and monitored for pneumonic signs. At daily intervals, either nasal samples will be collected or a calf will be sacrificed. Lung washings and lung tissue samples will be collected for extraction of total RNA. The RNA will be used in real-time RT-PCR using Mh specific primers to examine expression of specific genes. It is anticipated that some genes will be expressed early during the infection and some later, depending on the activity of their encoded products in pathogenesis. A second objective is examine the contribution of two-component regulatory systems in regulation of the virulence genes. We have identified several pairs of there systems from the Mh genome sequence. Mutants will be constructed in each of the two cognate proteins to examine the effects on protein expression and the regulatory pathway in response to the two-component system.