In the past several years, a significant need has arisen to analyze bacterial gene expression following interactions with host cells in vivo. In vivo whole genome expression analyses are seen as crucial for identifying novel bacterial targets for therapeutic intervention and for a comprehensive understanding of bacterial pathogenesis. This work will develop, improve, optimize, and validate methods for (1) the purification of bacterial mRNAs from complex mixtures of host cell and bacterial RNA and (2) the global amplification of bacterial mRNAs following their purification from complex RNA mixtures. The goals of this study are to maximize recovery and amplification of bacterial mRNAs while maintaining accurate gene representation in the final sample. Methods and procedures will be validated by microarray expression analysis studies. Optimized procedures will be incorporated into first- and second-generation kits that will comprise complete systems for the purification and amplification of bacterial mRNAs from in vitro and in vivo samples containing excessive amounts of eukaryotic host cell RNA. Results of the work performed here will enable scientists to capitalize on NIH investments in bacterial genome sequencing - by performing extensive analyses of bacterial gene expression during the host phase of the bacterial life cycle. These newly enable studies will assist in identifying genes and gene products important for initiating bacterial infections, for enabling bacterial replication within the host, and for evasion of host immune responses. Gene expression analyses with non-cultivable pathogens, insect-borne pathogens, and commensal organisms will also be enabled. With the technology developed here, new basic research will be focused on identifying novel vaccine candidates and expanding the number of potential targets for therapeutic intervention. ? ?
