Samantha Hurndon Isaiah Castañeda

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Effect of Flik mutation on the transcriptional activity of the 54 sigma factor RpoN in Helicobacter pylori Douillard, Francois, Ryan Kieran, Jason Hinds, and Paul O'Toole. Samantha Hurndon Isaiah Castaeda. Outline. - PowerPoint PPT Presentation

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<ul><li><p>Effect of Flik mutation on the transcriptional activity of the 54 sigma factor RpoN in Helicobacter pylori </p><p> Douillard, Francois, Ryan Kieran, Jason Hinds, and Paul O'Toole</p><p>Samantha Hurndon Isaiah Castaeda</p></li><li><p>Outline Helicobacter pylori is a bacterium that may cause a variety of gastrointestinal disordersThe FliK protein plays an important role in H. pylori motility Microarrays constructed and confirmed with Comparative genomic hybridizationResults indicate that mutations in FliK affect transcriptionThe knowledge of FliK mechanisms have expanded, but more experiments are underway for a more complete understanding</p></li><li><p>Helicobacter pylori is a motile Gram-negative bacterium that persists in the human gastric mucosaH. Pylori causes an infection responsible for gastrointestinal disorders</p><p>Peptic and duodenal ulcers</p><p>Predisposing factor for gastric adenocarcinoma and B cell MALT lymphoma</p><p>Developing countries are the most severely infected</p></li><li><p>Motility is a key feature of H. pylori and is required for colonization and persistenceMotile bacteria flagella contribute to:Motility adhesion inflammatory response by host cells 80, 54, 28 control the transcription of flagellar genesThere are three classes (stages) of regulation Class I: 80 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 (28 ) and an anti sigma factor (FlgM)</p></li><li><p>Outline Helicobacter pylori is a bacterium that may cause a variety of gastrointestinal disordersThe FliK protein plays an important role in H. pylori motility Microarrays constructed and confirmed with Comparative genomic hybridizationResults indicate that mutations in FliK affect transcriptionThe knowledge of FliK mechanisms have expanded, but more experiments are underway for a more complete understanding</p></li><li><p>FliK is the gene that controls hook length during flagellar assembly Microarry analysis was done on fliK-null mutant Under the control of the RpoN there is an increase in transcription of genes </p><p>Suggested that the FliK protein is required to turn off the RpoN regulation during flagellar assembly</p></li><li><p>Outline Helicobacter pylori is a bacterium that may cause a variety of gastrointestinal disordersThe FliK protein plays an important role in H. pylori motility Microarrays constructed and confirmed with Comparative genomic hybridizationResults indicate that mutations in FliK affect transcriptionThe knowledge of FliK mechanisms have expanded, but more experiments are underway for a more complete understanding</p></li><li><p>NCTC26695 and J99 was used to analyze the genetic conservation between CCUG178774 CGH was performed to improve the subsequent type II array experiment analyses Genes were classified into 3 groups: Absent (highly divergent)Uncertain Absolutely present 7 Genes from these groups were analyzed by PCR using genomic DNA from the test strain (CCUG) and the reference strain (NCTC) </p></li><li><p>H. Pylori strain 17874 was used from the University of Gothenburgs Culture Collection</p><p>Mutants were cultured on Columbia agar base plates with an antibiotic</p><p>After 2 days, genomic DNA was isolated from the cultures using a DNeasy tissue kit from Qiagen</p></li><li><p>H. Pylori cells were then harvested &amp; centrifuged for 15 seconds at 10,000 g</p><p>The resulting cell pellets were suspended in 750 L of Bacteria RNA Protect reagent from Qiagen</p><p>Qiagens RNeasy Mini kit was used to isolate RNA from the H. pylori</p></li><li><p>H. Pylori microarray design &amp; construction was performed at the Bacterial Microarray Group facility at St. Georges University in LondonProducts of PCR represent all ORFs</p><p>These products were robotically spotted in duplicate using an automated microarrayer from BioRoboticshttp://www.digizyme.com/portfolio/microarraysfab/robotic.html</p></li><li><p>Comparative genomic hybridization was used to study the macrodiversity of the H. pylori strainThe wild type CCUG17874 DNA was labeled with dCTP Cy3Cy3-dCTP is a fluorescent cyan dyeWild type NCTC26695 DNA was labeled with dCTP Cy5Cy5-dCTP is a fluorescent red dyeThe mixture was incubated at 37C for 90 mins in the dark</p></li><li><p>The array hybridizations were performed in duplicate </p><p>Quantile normalization was usedQuantile normalization improves consistency &amp; reduces bias </p><p>Three lists were produced:Divergent genesUncertain genesPresent genes</p></li><li><p>The diagram below gives an overview of CGH</p></li><li><p>Type II microarray analysis was performed to compare wild type strains to mutant strainsCy5-labeled cDNA was co-hybridized to a reference Cy3-labeled cDNA</p><p>This was done by mixing the labeled nucleic acids &amp; purifying them using Qiagens MinElute PCR Purification kitAfter being mixed in a hybridization solution, they were washed in sodium dodecyl sulfate &amp; saline sodium citrateThey were then dried with a centrifuge and scanned by a dual-laser scanner</p></li><li><p>Array hybridizations were performed in triplicateThe data was then exported to Microsoft Excel</p><p>Genes listed as missing or uncertain were removedTriplicates were averagedThe data consists of ratios comparing mutant and wild type DNAOne-way ANOVA was used to calculate statistical significance</p></li><li><p>Real-time PCR was used to confirm presence of genes</p><p>Primers were designed using a software called Primer3</p><p>Reactions were carried out in technical triplicateAt least 2 independent RNA preparations were used</p></li><li><p>Outline Helicobacter pylori is a bacterium that may cause a variety of gastrointestinal disordersThe FliK protein plays an important role in H. pylori motility Microarrays constructed and confirmed with Comparative genomic hybridizationResults indicate that mutations in FliK affect transcriptionThe knowledge of FliK mechanisms have expanded, but more experiments are underway for a more complete understanding</p></li><li><p>PCR confirmed the CGH results with one exception</p><p>flgI was shown to be absent in CGH however was shown to be present in PCR </p><p>flgI is an essential structural component of the flagellar superstructure</p><p>In CGH Standard deviation of hybridization values for this gene was very high not reliable</p><p> Divergent genes in CCUG may be present but their sequences may be poorly conserved in comparison to NCTC </p></li><li><p>14.25% of NCTC Genes were absent in CCUG genome</p></li><li><p>Genes Identified as missing revealed 5 large regions have been disrupted in the CCUG genome Outer rings show strand location of genes with missing genes in red </p></li><li><p>Gene expression has been significantly changed after mutation on fliK </p></li><li><p>Flik mutation affected the transcription of almost all class II genes in flagellar regulon Shown above is the differential expression ratios of all known Flagellar genes in the fliK mutant relative to the wild-type </p></li><li><p>Results suggest that HP0114 and flaB are not co-transcribed Identified a potential terminator located downstream of flaB followed immediately by a U-rich region These two regions are not completely co-transcribed RT-PCR analysis of the flaB operon structure. The fold changes were in agreement with the microarray data</p></li><li><p>The fold-changes obtained were in good agreement with the microarray data Four genes were analysed by qRT-PCR </p><p>RpoN control transcription of flaB and flaE </p><p> Mutation of flgE caused a significant increase in rpoN and flaB transcription </p></li><li><p>Outline Helicobacter pylori is a bacterium that may cause a variety of gastrointestinal disordersThe FliK protein plays an important role in H. pylori motility Microarrays constructed and confirmed with Comparative genomic hybridizationResults indicate that mutations in FliK affect transcriptionThe knowledge of FliK mechanisms have expanded, but more experiments are underway for a more complete understanding</p></li><li><p>Previous studies on flagellum regulatory mechanisms looked at strain-specific effectsIn this study, the gene of interest itself was identifiedInactivating fliK led to an increase in RpoN-dependent genes (Class II)HP0870 (flagellar hook)HP0115 (minor flagellin)Similar to previous studies, HP0114 does not appear to be in class II</p></li><li><p>Upregulation of RpoN-dependent gene in fliK activates transcriptionAfter the hook is completed, transcription is FliA-dependent- By an unknown mechanism (FliK-dependent)RpoN regulon would beturned off)</p><p>It is hypothesized that HP0957 acts as a post-transcriptional regulator</p></li><li><p>ReferencesDouillard, 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. 21.4 (2009): 1901-1911. </p><p>http://en.wikipedia.org/wiki/Comparative_genomic_hybridization. Nov 10, 2011.</p><p>http://www.digizyme.com/portfolio/microarraysfab/robotic.html. Nov 10, 2011</p><p>http://www.childrensmercy.org/stats/model/arrayNormalization.htm. Nov 10, 2011</p></li></ul>