Journal Club Presentation BIOL398/S10: Bioinformatics Lab J’aime Moehlman & Amanda Wavrin

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 13th, 2010

2. Outline • Introduction to Vibrio cholerae • Results • Discussion • Methods • Microarray analysis • Further research • References

3. Vibriocholeraeis a waterborne disease, that is infectious humans. • It produces a cholera toxin that acts on the mucosal epithelium and is responsible for the characteristic diarrhea. • Cholera is one of the most rapidly fatal illnesses known. • A healthy person who is infected may die within 2-3 hours if no treatment is provided. • Usually, the disease progresses to shock in 4-12 hours, with death following in 18 hours to several days after the onset of symptoms.

4. Is stomach acid a factor that contributes to the epidemic spread of cholera? • The study took place in Dhaka, Bangladesh because of the common outbreaks of cholera in its natural setting. • They collected stool samples at the ICDDR and identified those positive with the O1 Inaba El Tor strain (marked by the deletion of the lacZ gene). • This strain was mixed with a strain grown in vitro; and then was used to inoculate infant mice. • Bacteria was recovered from the small intestine and was then plated on a medium.

5. 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. choleraehad a CI above 1 indicating an enhanced infectivity. • V. choleraethat was cultured and purified in vitro did not show enhanced infectivity. • These results suggest that passage through the human GI tract will increase the infectivity of the cholera.

6. Testing the hyperinfectious phenotype after incubation in pond water. • The samples were diluted in pond water that was free of V. cholerae. • They were then mixed with the in vitro grown competitor strain. • When this mixture was infected into mice, the hyperinfectious state remained. • From this they proposed that passage of V. choleraeenhances infectivity by lowering the infectious dose in secondary hosts.

7. Representation of the competitive indices for human-shed V. cholerae.

8. 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 and were then filtered and frozen. • The stool RNA was analyzed by agarose gel electrophoresis to ensure its integrity.

9. 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). • The samples were hybridized in quadruplicate and relative fluorescent intensities were determined. • The data was quantified, normalized and corrected to yield intensity ratios.

10. The Statisical Analysis for Microarrays 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

11. Transcriptional profile of human-shed V. cholerae.

12. The transcriptomes of the V. cholerae were similar to that of the cultured DSM-V99 strain. • The transcriptome is consistent with bacterial growth conditions that was also found in rice-water stools. • With these observations they propsed that V. cholerae moves from a nutrient rich environment in the small intestine to a nutrient poor lumenal fluid that is quickly removed.

13. Before being shed, V. choleraeturns 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 Vibriopathogenecity island. • These results also suggest that increased expression of these genes is not necessary for the increased infectivity of cholera.

14. The role of chemotaxis in infectivity is unknown. • There are some genes that are needed for chemotaxis that are also known to be required for expression of the cholera toxin. • Many of the genes required for chemotaxis were repressed when being shed. • This suggests that the motile bacteria are non-chemotactic during dissemination into the environment, which could: • Increase the shedding from the GI tract. • Increase infectivity.

15. Strains, sample collection and competetion assays • Fresh stool samples were collected in beakers, filtered through cheese cloth, and frozen at 80 degrees Celsius • Protocols were reviewed and approved by the Research Reviem Committee, the Ethical Review Committee, and the Institutional Review Board • Competition assays were done by mixing DSM-V984 grown overnight with stool bacteria in a ratio of 1:10 • It was given to 3-5 day old mice by gavage • The mice were then euthanized and the small intestines removed • Output ratios were corrected • The PH of the two pond water samples used were 7-7.5

16. Microarray analysis • ORFs were found using and ORF-finding program and portions of the ORFs 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. • Labelling reactions were done on two separate days which resulted in quadruplicate arrays for each strain. • Control arrays were also hybridized to identify potential affects of freezing the stools.

17. Opportunities for Further Research • Induction of the acid tolerance response (ATR) could potentially be involved in the increased infectivity of human-shed V. cholerae. • If this is true, the mechanism of action is unknown. • Transcriptional profiling only provides a small look at the gene expression right at the time of dissemination, the next step would be making sense of the proteome of human-shed V. cholerae. • By discovering how the human host preps the bacteria for infection of additional humans, it can aid it 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.

18. 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.