Transcript Analyzing the Increased and Decreased Expression of Microarray

Analyzing the Increased and Decreased
Expression of Microarray Data for Vibrio
cholerae
Merrell DS, Butler SM, Qadri F, Dolganov NA, Alam A,
Cohen MB, Calderwood SB, Schoolnik GK, and Camilli A.
Host-induced epidemic spread of the cholera bacterium.
Nature 2002 Jun 6; 417(6889) 642-5.
J’aime Moehlman
Amanda Wavrin
April 27th, 2010
BIOL 398/S10: Bioinformatics Lab
Outline
• Vibrio cholerae is a waterborne disease that is infectious
to humans
• Microarray data is used to measure gene expression
• Microarray data from the
Merrell et al. (2002) study
• Increased processes and organization
result in a more virulent strain
• Decreased overall organization
results in increased pathogenicity
• Significantly changed in
comparision to Merrell
http://www.zdravstvena.info/vsznj/wpcontent/uploads/2009/01/kolera.jpg
Vibrio cholerae is a waterborne disease
that is infectious to humans
• Organisms in the genus Vibrio are one of the most
common surface water organisms in the world.
• They can be found in both fresh and marine waters and
live in association with other animals.
• V. cholerae is noninvasive and affects the small
intestine.
• It produces a cholera toxin, which is responsible for the
characteristic diarrhea.
• This leads to dehydration, anuria, acidosis, shock,
cardiac complications, and circulatory failure.
DNA Microarray is used in expression
profiling
• Each probe contains a known gene fragment.
• The microarray is washed leaving behind the strongly
paired hybridized strands.
• The intensity of the probe
depends on the amount of
dye able to bind at each point.
http://www.webpages.uidaho.edu/
Microarray Analysis in the Merrel et al.
study
• The study was conducted in Dhaka, Bangladesh
• Samples were collected from three patients.
• The collected strain, O1 Inaba El Tor, was combined
with an in vitro strain and used to inoculate mice.
• The output ratios were corrected to represent the
competitive indices (CI) of the V. cholerae.
• A CI above 1 indicates increased infectivity.
• A CI below 1 indicates decreased infectivity.
The human-shed V. cholerae had a CI
above 1 indicating an enhanced infectivity
• V. cholerae that was cultured and purified in vitro did
not show enhanced infectivity
• RNA from each sample was used for DNA synthesis
• This was labeled with Cy5 and hybridized to the
microarray with a Cy3- labeled common reference
strain (exponential growing).
• A two-fold difference indicated differentially
expressed genes
GenMAPP was used to download the
correct V. cholerae gene database
• There were 772 errors detected when converting the raw
data.
• We customized our data by selecting new color sets.
– Pink showed increased expression
– Blue showed decreased expression
• Criterion 0 represented increased.
• Criterion 1 represented decreased.
• Statistical Criteria:
–
–
–
–
Z Score > 2
P value < 0.05
Number Changed Ranged from <3-5
% Changed Ranged < 15-25
Increased Gene Expression for Infectivity
Increased Metabolic Processes and
Organization resulted in more virulent strain
• Metabolic Processes
– Amino acid
– Cellular
– Nitrogen
• Amino Acid Processes
Cellular Metabolic Process
GO Term
– Are relative to cell metabolism
• Movement via Flagellum
– Process carried out at cellular level which controls
assembly, arrangement, or disassembly of flagellum
Decreased Gene Expression for Infectivity
Decreased in Overall Organization
• Nucleotide binding
• Protein functions
– Folding
– Binding
• Molecular binding
– Zinc
– Cation
– Vitamin
Protein Folding GO Term
Increases in metabolic, amino acid
processes and flagellum organization show
overall virulence in V. cholerae
• Increased metabolic processes play a role in energy
distribution within the bacteria.
• Increase amino acid metabolic processes present
increase in storage molecules.
• Increased movement via flagellum plays a role in
increased overall virulence of the bacterial cells.
• Amino acid processes effect protein structure, which
has an overall effect on protein function.
• These increases focus on the pathogenicity of V.
cholerae and the structural component of the cells.
Decreased overall organization of the cell
results in increased pathogenicity
• Decreased protein binding will effect the protein
structures which has an overall effect on the protein
function.
• Utilizes the increased storage molecules to decrease
protein synthesis.
• Decreases molecular binding in order to focus on
increasing its’ virulence.
Significantly changed genes in comparison
to Merrell et al. (2002)
• According to the Merrell et al. study, these genes were
significantly changed
– VC0028, VC0941, VC0869, VC0051, VC0647, VC0468, VC2350,
VCA0583
• We found that only 2 of these 8 genes showed to be
significantly changed in our results.
• These genes were VC0647 and VCA0583
– VC0647: mRNA catabolic processes, RNA processing, cytoplasm,
RNA binding, 3'5'exoribonuclease activity, transferase activity,
nucleotidyltransferase polyribonucleotide
nucleotidyltransferase activity
– VCA0583: transport, cell envelope outermembrane bounded
periplasmic space, transport activity
References
• Merrell DS, Butler SM, Qadri F, Dolganov NA, Alam A,
Cohen MB, Calderwood SB, Schoolnik GK, and Camilli
A. Host-induced epidemic spread of the cholera
bacterium. Nature 2002 Jun 6; 417(6889) 642-5.
• Todar, Kenneth. Online Textbook of Bacteriology “Vibrio
cholerae” http://www.textbookof
bacteriology.net/cholera.html. 25 April 2010.