1. 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 27 th, 2010 BIOL 398/S10: Bioinformatics Lab

2. 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 genes in comparison to Merrell http://www.zdravstvena.info/vsznj/wp- content/uploads/2009/01/kolera.jpg

3. 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.

4. DNA Microarray is used in expression profiling http://www.webpages.uidaho.edu/ 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.

5. 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.

6. 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.

7. 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

8. Increased Gene Expression for Infectivity

9. 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

10. Decreased Gene Expression for Infectivity

11. Decreased in Overall Organization Nucleotide binding Protein functions – Folding – Binding Molecular binding – Zinc – Cation – Vitamin Protein Folding GO Term

12. 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.

13. 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.

14. 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

15. 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.textbookof bacteriology.net/cholera.html