A Global Quantitative Analysis of Operon/Gene Transcription in Bacillus Anthracis During Experimental Infection
Abstract
The objective is to perform the first comprehensive quantitative analysis of Bacillus anthracis operon/gene transcription during in viva experimental infection to enable the identification of additional virulence factors, potential vaccine candidates, and novel targets for therapeutic intervention. This study is planned to identity genes of interest by examining gene transcription in B. anthracis under several experimental conditions. This includes comparing in vitro vs. in viva grown organisms to identify those genes expressed exclusively in the host. Secondly, to identify those genes specifically expressed early after germination compared to those expressed during the terminal stages of infection. This will hopefully identify genes and targets that are specific to the earliest stage of the infectious process. The genes will be identified using quantitative RT-PCR by the Taqman assay. 1. Bioinformatic analysis of the B. anthracis genome has been completed and approximately 1230 ORFs have been selected for study and primer/probe sets have been obtained. 2. Analysis of gene expression during early stage B. anthracis infection requires delivery of 10(sub 8) spores intradermally followed by a 2 hour incubation during which significant spores germinate but outgrowth of the bacilli is minimized. 3. Empirical optimization of tissue handling, RNA extraction, eDNA synthesis, and Taqman assay conditions has significantly enhanced our ability to measure B. anthracis gene expression within host tissues. 4. Pre-amplification of cDNA derived from early-stage infected tissues will permit analysis of B. anthracis gene expression at this critical phase of infection. 5. eDNA from late stage infection in the guinea pig has been prepared. 6. eDNA from early stage infection is being isolated. 7. Upon receipt of nested primers, the comparison of gene expression during in vitro cultivation vs. early and late stage infection will begin.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jul 01, 2003
- Accession Number
- ADA417048
Entities
People
- Damien Mcdevitt
- Mick Gwynn