Transcript Effect of Flik mutation on the transcriptional activity

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/microarraysfa
b/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
• Divergent genes in CCUG
•In CGH Standard deviation of
may be present but their
hybridization values for this gene
sequences may be poorly
was very high  not reliable
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 RpoNdependent 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 be
turned off)
• It is hypothesized that
HP0957 acts as a posttranscriptional 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_hybridizat
ion. Nov 10, 2011.
• http://www.digizyme.com/portfolio/microarraysfab/robotic.ht
ml. Nov 10, 2011
• http://www.childrensmercy.org/stats/model/arrayNormalizatio
n.htm. Nov 10, 2011