H. pylori chronically infects gastric mucosa and is implicated in the pathogenesis of human gastritis, peptic ulcer disease, and gastric cancer. This proposal outlines a functional genomics-based approach designed to identify and characterize H. pylori virulence genes associated with human gastric disease. The proposed experiments will provide a functional map of the H. pylori genome which complements the complete sequences of two fully sequenced H. pylori genomes. A method termed """"""""kinetically monitored reverse transcriptase-initiated PCR""""""""; (kRT-PCR) forms the basis for the experimental plan. This method was developed in the laboratory of the principal investigator, and permits high throughput transcript quantitation. Genome-wide transcript profiling is proposed for H. pylori in certain benchmark states. The effects of growth phase, pH shift, iron, and human epithelial cells on H. pylori gene expression will be analyzed. These growth conditions, as well as knockout mutations within the cag pathogenicity island and within specific transcription factor genes, will be tested for their effects on the H. pylori transcriptome. Candidate virulence genes identified from the genome sequence include those involved in molecular mimicry, LPS biosynthesis, outer membrane proteins, candidate """"""""phase variation"""""""" genes, and several hundred H. pylori-specific genes identified by comparisons of H. pylori genome sequences with those of other pathogenic and non-pathogenic bacteria. The H. pylori transcriptome will be organized into operons. Operon structures and coordinate expression profiles should reveal potential functions for unknown ORFs. Gene expression profiles for H. pylori in vivo will be obtained using a non-human primate model. Transcript profiles obtained under a variety of in vitro conditions will be organized into relational data sets and analyzed to reveal transcriptional paths and networks operative in H. pylori.
| Graham, Kenneth J; Holland, Michael J (2005) PrimerSelect: a transcriptome-wide oligonucleotide primer pair design program for kinetic RT-PCR-based transcript profiling. Methods Enzymol 395:544-53 |
