regulation of gene expression in Mannheimia haemolytica A1.

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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.