Investigation of Microarray Data from Douillard’s Heliocobacter pylori Experiment Samantha Hurndon Isaiah Castañeda

Outline Francois Douillard and his colleagues published a paper in 2009 about the Flik gene of H. pylori Statistical data was gathered to determine which genes in this experiment underwent the greatest changes MAPPFinder was used to determine the most upregulated and downregulated genes Data indicated that many of the top 10 most significant genes dealt with transcription Data shows that RpoN dependent genes were not among the most significantly changed

Motility is a key feature of H. pylori and is required for colonization and persistence There are three classes (stages) of regulation –Class I: transcription of the early flagellar genes –Class II: RpoN regulation of the middle flagellar structural genes. (fliK encodes hook-length-control pro) –Class III: FliA and an anti sigma factor (FlgM) FliK is the gene that controls hook length during flagellar assembly

CCUG17874 & NCTC26695 Strains were used for the microarray dataset The wild type CCUG17874 DNA was labeled with dCTP Cy3 –Cy3-dCTP is a fluorescent cyan dye Wild type NCTC26695 DNA was labeled with dCTP Cy5 –Cy5-dCTP is a fluorescent red dye

Microarray chips were hybridized Cy5 hp0906KO (C1) with Cy3 CCUG17874 (A3) → Hyb:32525 Cy3 CCUG17874 (A3) with Cy5 CCUG17874 (C1) → Hyb:32519 Cy5 hp0906KO (B1) with Cy3 CCUG17874 (A2) → Hyb:32524 Cy3 CCUG17874 (A1) with Cy5 hp0906KO (A1) → Hyb:32523 Cy5 CCUG17874 (B1) with Cy3 NCTC26695 (B1) → Hyb:32528 Cy3 CCUG17874 (A5) with Cy5 hp0958 KO (B1) → Hyb:32521 Cy3 NCTC26695 (A1) with Cy5 CCUG17874 (A1) → Hyb:32526 Cy5 hp0958 KO (A1) with Cy3 CCUG17874 (A4) → Hyb:32520 Cy3 CCUG17874 (A2) with Cy5 CCUG17874 (B2) → Hyb:35218 Cy3 CCUG17874 (A1) with Cy5 CCUG17874 (A1) → Hyb:32517 Cy3 CCUG17874 (A6) with Cy5 hp0958 KO (C1) → Hyb:32522

FliK protein is required to turn off the RpoN regulation during flagellar assembly Under the control of the RpoN there is an increase in transcription of genes Gene expression has been significantly changed after mutation on fliK Flik mutation affected the transcription of almost all class II genes in flagellar regulon

Outline Francois Douillard and his colleagues published a paper in 2009 about the Flik gene of H. pylori Statistical data was gathered to determine which genes in this experiment underwent the greatest changes MAPPFinder was used to determine the most upregulated and downregulated genes Data indicated that many of the top 10 most significant genes dealt with transcription Data shows that RpoN dependent genes were not among the most significantly changed

Statistical analysis was performed on the microarray dataset Data was gathered from the European Bioinformatics Institute website There were 3 experiments –hp0958KO –hp0906KO –CCUG17874 Averages, Tsats, and P-values were calculated for every gene of each experiment

HP0906 KO had the highest amount significantly changed genes Gene P-Values HP0958 KOHP0906 KOCCUG17874 < < < <

Outline Francois Douillard and his colleagues published a paper in 2009 about the Flik gene of H. pylori Statistical data was gathered to determine which genes in this experiment underwent the greatest changes MAPPFinder was used to determine the most upregulated and downregulated genes Data indicated that many of the top 10 most significant genes dealt with transcription Data shows that RpoN dependent genes were not among the most significantly changed

GenMAPP was used to find significant increases in gene expression

Using GenMAPP, a list of the most significantly suppressed genes was compiled

Outline Francois Douillard and his colleagues published a paper in 2009 about the Flik gene of H. pylori Statistical data was gathered to determine which genes in this experiment underwent the greatest changes MAPPFinder was used to determine the most upregulated and downregulated genes Data indicated that many of the top 10 most significant genes dealt with transcription Data shows that RpoN dependent genes were not among the most significantly changed

Many of the most significant genes deal with transcription HP1555 – Elongation Factor Associates with the EF-Tu.GDP complex and induces the exchange of GDP to GTP. It remains bound to the aminoacyl-tRNA.EF-Tu.GTP complex up to the GTP hydrolysis stage on the ribosome HP Flagellar hook-basal body protein FliE HP0912 – Predicted to be an outer membrane protein HP Transcription antitermination protein HP0601- Flagellin A Part of the Flagellar assembly and affects motility HP1429 – Predicted to be a polysialic acid capsule expression protein HP Acyl carrier protein HP1427 – Predicted to be a histidine and glutamine-rich metal binding protein HP Serine acetyltransferase HP0264 – Predicted to be an ATP-dependent chaperone protein ClpB

Douillard found that Flik mutation affected the transcription of almost all class II genes in flagellar regulon Shown above are the differential expression ratios of all known Flagellar genes in the fliK mutant relative to the wild-type

Outline Francois Douillard and his colleagues published a paper in 2009 about the Flik gene of H. pylori Statistical data was gathered to determine which genes in this experiment underwent the greatest changes MAPPFinder was used to determine the most upregulated and downregulated genes Data indicated that many of the top 10 most significant genes dealt with transcription Data shows that RpoN dependent genes were not among the most significantly changed

None of the most significantly changed genes were RpoN-dependent genes Our Perhaps there was more significant transcriptional activity than Class II genes

References Douillard, Francois, Ryan Kieran, Jason Hinds, and Paul O'Toole. "Effect of FliK mutation on the transcriptional activity of the sigma factor RpoN in Heliobacter pylori." Microbiology (2009): http://