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Category: Clinical Microbiology
Application of Microarray Analysis to the Investigation of Host-Pathogen Interactions, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555817848/9781555812614_Chap08-1.gif /docserver/preview/fulltext/10.1128/9781555817848/9781555812614_Chap08-2.gifAbstract:
Researchers have traditionally identified pathogenesis-related bacterial genes using molecular fingerprinting techniques such as restriction fragment length polymorphisms (RFLP) and random arbitrarily primed PCR (RAP PCR) to compare virulent and avirulent isolates. A more comprehensive approach to understanding the different pathogenic effects of diverse bacteria on their hosts requires that each gene within the bacterial genome be interrogated simultaneously. The availability of 95 complete genome sequences from 83 different microbial species and the development of microarrays containing representations of genes for many of these pathogens provide both of the prerequisites required for this approach. Helicobacter pylori induces gastric inflammation in all hosts, and such gastritis increases the risk for peptic ulceration, distal gastric adenocarcinoma, and gastric mucosal lymphoproliferative disease. H. pylori have now been completely sequenced, and genomic comparison has revealed that between 6 and 7% of the genes are strain-specific, reflecting a high level of genetic diversity. Contact between a host and a specific microbial pathogen not only alters bacterial gene expression but also results in dramatic changes in eukaryotic gene expression. Experiments involving exposure of bacteria to conditions that are similar to those encountered in vivo followed by assessment of the transcriptional responses using microarrays will undoubtedly become more common as investigators continue to elucidate bacterial signaling pathways that are involved in pathogenic host-microbial interactions.
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Identification of differences in bacterial genomic content (A) or gene expression following host-microbial interactions (B). (A) Genomic DNA isolated from a reference and a clinical strain is labeled by incorporating one of two fluorescent nucleotide analogs (Cy3 or Cy5) into the DNA. Differentially labeled DNA samples are then mixed and cohybridized to a whole-genome microbial microarray. Resulting signal intensities from each fluorophore are then compared for each ORF represented on the array, thus allowing one to identify differences in gene content between strains. (B) RNA is isolated from broth-exposed and cocultured bacteria and host cells, reverse transcribed into cDNA, and fluorescently labeled with Cy3 or Cy5. Differentially labeled cDNA samples are then mixed and cohybridized to either a microbial or eukaryotic microarray. Resulting signal intensities from each fluorophore are then compared to identify differences in pathogen and host gene expression that occur following contact.
Identification of differences in bacterial genomic content (A) or gene expression following host-microbial interactions (B). (A) Genomic DNA isolated from a reference and a clinical strain is labeled by incorporating one of two fluorescent nucleotide analogs (Cy3 or Cy5) into the DNA. Differentially labeled DNA samples are then mixed and cohybridized to a whole-genome microbial microarray. Resulting signal intensities from each fluorophore are then compared for each ORF represented on the array, thus allowing one to identify differences in gene content between strains. (B) RNA is isolated from broth-exposed and cocultured bacteria and host cells, reverse transcribed into cDNA, and fluorescently labeled with Cy3 or Cy5. Differentially labeled cDNA samples are then mixed and cohybridized to either a microbial or eukaryotic microarray. Resulting signal intensities from each fluorophore are then compared to identify differences in pathogen and host gene expression that occur following contact.
Relationship of H. pylori-induced gastric inflammation with variable disease outcomes. OR, odds ratios.
Relationship of H. pylori-induced gastric inflammation with variable disease outcomes. OR, odds ratios.
Hypothetical model by which H. pylori cag+ strains activate multiple pathways following contact with gastric epithelial cells. Following adherence, CagA is internalized and phosphorylated within the host cell by c-Src. Phospho-CagA then binds to and activates the host phosphatase SHP-2 and induces actin polymerization. A CagA-independent consequence of cag-mediated cellular interactions is translocation of an unknown factor (“?”), which then regulates pathways involved in the activation of transcription factors, such as AP-1 and NF-?B, which subsequently stimulate production of IL-8, a proinflammatory cytokine.
Hypothetical model by which H. pylori cag+ strains activate multiple pathways following contact with gastric epithelial cells. Following adherence, CagA is internalized and phosphorylated within the host cell by c-Src. Phospho-CagA then binds to and activates the host phosphatase SHP-2 and induces actin polymerization. A CagA-independent consequence of cag-mediated cellular interactions is translocation of an unknown factor (“?”), which then regulates pathways involved in the activation of transcription factors, such as AP-1 and NF-?B, which subsequently stimulate production of IL-8, a proinflammatory cytokine.
Absence (black) and presence (gray) of ORF in archival and recent H. pylori strain J99 isolates as determined by microarray analysis. DNA from 13 recent isolates was compared with that from archival strain ?99 by hybridization to a whole-genome microarray. Variably present ORFs are shown vertically for each of the isolates.
Absence (black) and presence (gray) of ORF in archival and recent H. pylori strain J99 isolates as determined by microarray analysis. DNA from 13 recent isolates was compared with that from archival strain ?99 by hybridization to a whole-genome microarray. Variably present ORFs are shown vertically for each of the isolates.
Summary of published studies using microarray analysis to investigate host-pathogen interactions
Summary of published studies using microarray analysis to investigate host-pathogen interactions