title: Screening Helicobacter pylori genes induced during infection of mouse stomachs.
abstract: OBJECTIVE
  To investigate the effect of in vivo environment on gene expression in Helicobacter pylori (H. pylori) as it relates to its survival in the host.
  
  
  METHODS
  In vivo expression technology (IVET) systems are used to identify microbial virulence genes. We modified the IVET-transcriptional fusion vector, pIVET8, which uses antibiotic resistance as the basis for selection of candidate genes in host tissues to develop two unique IVET-promoter-screening vectors, pIVET11 and pIVET12. Our novel IVET systems were developed by the fusion of random Sau3A DNA fragments of H. pylori and a tandem-reporter system of chloramphenicol acetyltransferase and beta-galactosidase. Additionally, each vector contains a kanamycin resistance gene. We used a mouse macrophage cell line, RAW 264.7 and mice, as selective media to identify specific genes that H. pylori expresses in vivo. Gene expression studies were conducted by infecting RAW 264.7 cells with H. pylori. This was followed by real time polymerase chain reaction (PCR) analysis to determine the relative expression levels of in vivo induced genes.
  
  
  RESULTS
  In this study, we have identified 31 in vivo induced (ivi) genes in the initial screens. These 31 genes belong to several functional gene families, including several well-known virulence factors that are expressed by the bacterium in infected mouse stomachs. Virulence factors, vacA and cagA, were found in this screen and are known to play important roles in H. pylori infection, colonization and pathogenesis. Their detection validates the efficacy of these screening systems. Some of the identified ivi genes have already been implicated to play an important role in the pathogenesis of H. pylori and other bacterial pathogens such as Escherichia coli and Vibrio cholerae. Transcription profiles of all ivi genes were confirmed by real time PCR analysis of H. pylori RNA isolated from H. pylori infected RAW 264.7 macrophages. We compared the expression profile of H. pylori and RAW 264.7 coculture with that of H. pylori only. Some genes such as cagA, vacA, lpxC, murI, tlpC, trxB, sodB, tnpB, pgi, rbfA and infB showed a 2-20 fold upregulation. Statistically significant upregulation was obtained for all the above mentioned genes (P < 0.05). tlpC, cagA, vacA, sodB, rbfA, infB, tnpB, lpxC and murI were also significantly upregulated (P < 0.01). These data suggest a strong correlation between results obtained in vitro in the macrophage cell line and in the intact animal.
  
  
  CONCLUSIONS
  The positive identification of these genes demonstrates that our IVET systems are powerful tools for studying H. pylori gene expression in the host environment.
authors.affiliation: Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Aparna
authors.initials: A
authors.lastname: Singh
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Hodgson
authors.forename: Ming
authors.initials: M
authors.lastname: Yan
authors.forename: Jungsoo
authors.initials: J
authors.lastname: Joo
authors.forename: Lei
authors.initials: L
authors.lastname: Gu
authors.forename: Hong
authors.initials: H
authors.lastname: Sang
authors.forename: Emmalena
authors.initials: E
authors.lastname: Gregory-Bryson
authors.forename: William G
authors.initials: WG
authors.lastname: Wood
authors.forename: Yisheng
authors.initials: Y
authors.lastname: Ni
authors.forename: Kimberly
authors.initials: K
authors.lastname: Smith
authors.forename: Sharon H
authors.initials: SH
authors.lastname: Jackson
authors.forename: William G
authors.initials: WG
authors.lastname: Coleman
mesh_terms: D000818:Animals; D002460:Cell Line; D004269:DNA, Bacterial / Q000235:genetics; D004195:Disease Models, Animal; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D016481:Helicobacter Infections / Q000235:genetics*; D016480:Helicobacter pylori / Q000235:genetics* / Q000302:isolation & purification; D008264:Macrophages / Q000166:cytology / Q000382:microbiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D013270:Stomach / Q000382:microbiology; D013272:Stomach Diseases / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial