1. Host-induced epidemic spread of the cholera bacterium Merrell DS, Butler SM, Qadri F, Dolganov NA, Alam A, Cohen MB, Calderwood SB, Schoolnik GK, and Camilli A. Nature 2002 Jun 6; 417(6889) 642-5. doi:10.1038/nature00778 Journal Club Presentation BIOL398/S10: Bioinformatics Lab J’aime Moehlman & Amanda Wavrin April 13 th, 2010

2. Outline Vibrio cholerae is a highly infectious waterborne disease. The human-shed form of V. cholerae proves to be more pathogenic than a strain grown in vitro. Strains, sample collection and Microarray analysis. Further research can be done on the proteome.

3. Vibrio cholerae is a waterborne disease that is infectious to humans It produces a cholera toxin that acts on the mucosal epithelium and is responsible for diarrhea. Cholera is one of the most rapidly fatal illnesses. The disease can progress to shock in as little as 4-12 hours. Death can follow 18 hours to several days after the onset of symptoms. A healthy person who is infected may die within 2-3 hours if no treatment is provided.

4. Samples of the O1 Inaba El Tor strain were collected and another strain was grown in vitro The study took place in Dhaka, Bangladesh due to the commonality of outbreaks in a natural setting. The O1 Inaba El Tor strain is distinguished by its deletion of the lacZ gene. The two strains were combined and used to inoculate infant mice. The ratio of the mixed assays was 1:10, it was corrected to a 1:1 output ratio. Bacteria was recovered from the small intestine and was then plated on a medium.

5. Passage through the human GI tract increases the infectivity of cholera 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.

6. Passage of V. cholerae enhances infectivity in secondary hosts The samples were diluted in pond water that was free of V. cholerae. The pH of the two pond water samples used were 7- 7.5. They were then mixed with the in vitro grown competitor strain. When this mixture was infected into mice, the hyperinfectious state remained.

7. The competitive indices for human-shed V. cholerae shows an increase in infectivity

8. Microarray analysis ORFs were found and portions were amplified by polymerase chain reaction and spotted onto slides. V. cholerae RNA was collected from stool samples and DSM-V999 strain was grown overnight in vitro. DNAse treatment to remove DNA contamination was carried out. Equal concentrations of each test RNA and common reference RNA were used for reverse transcription reactions. Control arrays were also hybridized to identify potential affects of freezing the stools.

9. Transcriptional profiling using DNA microarray A spotted DNA microarray containing about 87% of the identified ORFs of the El Tor strain was used. Positive samples were attained from 3 patients. The samples were collected in beakers, filtered through cheese cloth, and frozen at 80° (C) Protocols were reviewed and approved by three different review committees.

10. The stool RNA was analyzed by agarose gel electrophoresis to ensure its integrity RNA from each sample was used for DNA synthesis The stool RNA was analyzed by agarose gel electrophoresis to ensure its integrity. This was labeled with Cy5 and hybridized to the microarray with a Cy3- labeled common reference strain (exponential growing). The samples were hybridized in quadruplicate and relative fluorescent intensities were determined. The data was quantified, normalized and corrected to yield intensity ratios.

11. SAM program was used to determine significant differences in the intensity ratios The in vitro strain was used as class I, and each individual sample as class II. They obtained these results: – 237 genes were differentially regulated, of these: – 44 were induced – 193 were repressed

12. Transcriptional profile of human-shed V. cholerae

13. Transcriptomes of the V. cholerae were similar to that of the cultured DSM-V99 strain It was consistent with bacterial growth conditions that were also found in rice-water stools. They proposed that V. cholerae moves from a nutrient rich environment in the small intestine to a nutrient poor lumenal fluid. This fluid is quickly removed.

14. Before being shed, V. cholerae turns off expression of specific genes This has the potential to be for dissemination to the environment or transmission to a new host. These genes are necessary for infection of humans and mice. These genes include those for the cholera toxin, and the Vibrio pathogenecity island. These results also suggest that increased expression of these genes is not necessary for the increased infectivity of cholera.

15. The role of chemotaxis during infectivity is unknown Some genes that are needed for chemotaxis are also required for expression of the cholera toxin. Within both cholera strains the genes required for chemotaxis were repressed while being shed. This suggests that the motile bacteria are non- chemotactic during dissemination, which could: – Increase the shedding from the GI tract – Increase infectivity

16. Opportunities for Further Research The next step would be making sense of the proteome of human-shed V. cholerae. Induction of the acid tolerance response (ATR) could be involved in the increased infectivity of human- shed V. cholerae. –If this is true, the mechanism of action is unknown. Discovering how the human host preps the bacteria for infection of additional humans can aid in the further study of human to human transmission of other microorganisms. The work done in this study could also aid in the development of a vaccine.

17. 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. 11 April 2010.http://www.textbookof bacteriology.net/cholera.html