Effect of Flik mutation on the transcriptional activity of the σ54 sigma

factor RpoN in Helicobacter pylori

Douillard, Francois, Ryan Kieran, Jason Hinds, and Paul O'Toole

Samantha Hurndon Isaiah Castañeda

Outline

• Helicobacter pylori is a bacterium that may cause a variety of gastrointestinal disorders

• The FliK protein plays an important role in H. pylori motility

• Microarrays constructed and confirmed with Comparative genomic hybridization

• Results indicate that mutations in FliK affect transcription

• The knowledge of FliK mechanisms have expanded, but more experiments are underway for a more complete understanding

Helicobacter pylori is a motile Gram-negative bacterium that persists in the human gastric

mucosa• H. Pylori causes an

infection responsible for gastrointestinal disorders

• Peptic and duodenal ulcers

• Predisposing factor for gastric adenocarcinoma and B cell MALT lymphoma

• Developing countries are the most severely infected

Motility is a key feature of H. pylori and is required for colonization and persistence

• Motile bacteria flagella contribute to:– Motility– adhesion – inflammatory response by host cells

• σ80, σ54, σ28 control the transcription of flagellar genes• There are three classes (stages) of regulation – Class I: σ80 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 (σ28 ) and an anti sigma factor (FlgM)

Outline

• Helicobacter pylori is a bacterium that may cause a variety of gastrointestinal disorders

• The FliK protein plays an important role in H. pylori motility

• Microarrays constructed and confirmed with Comparative genomic hybridization

• Results indicate that mutations in FliK affect transcription

• The knowledge of FliK mechanisms have expanded, but more experiments are underway for a more complete understanding

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

• Suggested that the FliK protein is required to turn off the RpoN regulation during flagellar assembly

Outline

• Helicobacter pylori is a bacterium that may cause a variety of gastrointestinal disorders

• The FliK protein plays an important role in H. pylori motility

• Microarrays constructed and confirmed with Comparative genomic hybridization

• Results indicate that mutations in FliK affect transcription

• The knowledge of FliK mechanisms have expanded, but more experiments are underway for a more complete understanding

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)

H. Pylori strain 17874 was used from the University of Gothenburg’s Culture

Collection

• Mutants were cultured on Columbia agar base plates with an antibiotic

• After 2 days, genomic DNA was isolated from the cultures using a DNeasy tissue kit from Qiagen

H. Pylori cells were then harvested & centrifuged for 15 seconds at 10,000 g

• The resulting cell pellets were suspended in 750 µL of Bacteria RNA Protect reagent from Qiagen

• Qiagen’s RNeasy Mini kit was used to isolate RNA from the H. pylori

H. Pylori microarray design & construction was performed at the

Bacterial Microarray Group facility at St. George’s University in London

• Products of PCR represent all ORFs

• These products were robotically spotted in duplicate using an automated microarrayer from BioRobotics– http://www.digizyme.com/portfolio/microarraysf

ab/robotic.html

Comparative genomic hybridization was used to study the macrodiversity

of the H. pylori strain

• 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

• The mixture was incubated at 37°C for 90 mins in the dark

The array hybridizations were performed in duplicate

• Quantile normalization was used– Quantile normalization improves consistency &

reduces bias

• Three lists were produced:– Divergent genes– Uncertain genes– Present genes

The diagram below gives an overview of CGH

Type II microarray analysis was performed to compare wild type

strains to mutant strains• Cy5-labeled cDNA was co-hybridized to a

reference Cy3-labeled cDNA

– This was done by mixing the labeled nucleic acids & purifying them using Qiagen’s MinElute PCR Purification kit

– After being mixed in a hybridization solution, they were washed in sodium dodecyl sulfate & saline sodium citrate

– They were then dried with a centrifuge and scanned by a dual-laser scanner

Array hybridizations were performed in triplicate

• The data was then exported to Microsoft Excel

– Genes listed as missing or uncertain were removed

– Triplicates were averaged– The data consists of ratios comparing mutant and

wild type DNA– One-way ANOVA was used to calculate statistical

significance

Real-time PCR was used to confirm presence of genes

• Primers were designed using a software called Primer3

• Reactions were carried out in technical triplicate– At least 2 independent RNA preparations were

used

Outline

• Helicobacter pylori is a bacterium that may cause a variety of gastrointestinal disorders

• The FliK protein plays an important role in H. pylori motility

• Microarrays constructed and confirmed with Comparative genomic hybridization

• Results indicate that mutations in FliK affect transcription

• The knowledge of FliK mechanisms have expanded, but more experiments are underway for a more complete understanding

PCR confirmed the CGH results with one exception

•flgI was shown to be absent in CGH however was shown to be present in PCR

•flgI is an essential structural component of the flagellar superstructure

•In CGH Standard deviation of hybridization values for this gene was very high not reliable

• Divergent genes in CCUG may be present but their sequences may be poorly conserved in comparison to NCTC

14.25% of NCTC Genes were absent in CCUG genome

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

Gene expression has been significantly changed after mutation on fliK

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

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

The fold-changes obtained were in good agreement with the microarray data

• Four genes were analysed by qRT-PCR

•RpoN control transcription of flaB and flaE

• Mutation of flgE caused a significant increase in rpoN and flaB transcription

Outline

• Helicobacter pylori is a bacterium that may cause a variety of gastrointestinal disorders

• The FliK protein plays an important role in H. pylori motility

• Microarrays constructed and confirmed with Comparative genomic hybridization

• Results indicate that mutations in FliK affect transcription

• The knowledge of FliK mechanisms have expanded, but more experiments are underway for a more complete understanding

Previous studies on flagellum regulatory mechanisms looked at

strain-specific effects• In this study, the gene of interest itself was

identified• Inactivating 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

Upregulation of RpoN-dependent gene in fliK activates transcription

• After the hook is completed, transcription is FliA-dependent

- By an unknown mechanism (FliK-dependent)RpoN regulon would beturned off)

• It is hypothesized that HP0957 acts as a post-transcriptional regulator

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. 21.4 (2009): 1901-1911.

• http://en.wikipedia.org/wiki/Comparative_genomic_hybridization. Nov 10, 2011.

• http://www.digizyme.com/portfolio/microarraysfab/robotic.html. Nov 10, 2011

• http://www.childrensmercy.org/stats/model/arrayNormalization.htm. Nov 10, 2011