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Analyzing the Increased and Decreased Expression of Microarray Data for Vibrio cholerae. J’aime Moehlman Amanda Wavrin April 27 th , 2010 BIOL 398/S10: Bioinformatics Lab. Outline. Vibrio cholerae is a waterborne disease that is infectious to humans
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Analyzing the Increased and Decreased Expression of Microarray Data for Vibrio cholerae 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 • Statistical analysis of the microarray data • Microarray data from the Merrell et al. (2002) study • Increased expression in microarray • Decreased expression in microarray
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.
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 Metabolic Processes and Organization resulted in more virulent strain • Metabolic Processes • Amino acid • Cellular • Nitrogen • Amino Acid Processes • Are relative to cell metabolism • Movement via Flagellum • Process carried out at cellular level which controls assembly, arrangement, or disassembly of flagellum Cellular Metabolic Process GO Term
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. choleraeand 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. • http://www.zdravstvena.info/vsznj/wp-content/uploads/2009/01/kolera.jpg