Document Detail


Screening Helicobacter pylori genes induced during infection of mouse stomachs.
MedLine Citation:
PMID:  22969195     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
AIM: 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.
CONCLUSION: 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:
Aparna Singh; Nathaniel Hodgson; Ming Yan; Jungsoo Joo; Lei Gu; Hong Sang; Emmalena Gregory-Bryson; William G Wood; Yisheng Ni; Kimberly Smith; Sharon H Jackson; William G Coleman
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Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, N.I.H., Extramural; Research Support, N.I.H., Intramural    
Journal Detail:
Title:  World journal of gastroenterology : WJG     Volume:  18     ISSN:  2219-2840     ISO Abbreviation:  World J. Gastroenterol.     Publication Date:  2012 Aug 
Date Detail:
Created Date:  2012-09-12     Completed Date:  2013-03-12     Revised Date:  2014-05-20    
Medline Journal Info:
Nlm Unique ID:  100883448     Medline TA:  World J Gastroenterol     Country:  China    
Other Details:
Languages:  eng     Pagination:  4323-34     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Line
DNA, Bacterial / genetics
Disease Models, Animal
Gene Expression Profiling / methods*
Gene Expression Regulation, Bacterial
Genes, Bacterial / genetics*
Helicobacter Infections / genetics*
Helicobacter pylori / genetics*,  isolation & purification
Macrophages / cytology,  microbiology
Male
Mice
Mice, Inbred C57BL
Stomach / microbiology
Stomach Diseases / genetics*
Grant Support
ID/Acronym/Agency:
Y3-DK-3521-07/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/DNA, Bacterial
Comments/Corrections

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