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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 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. 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. 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. 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. 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 77.5. • They were then mixed with the in vitro grown competitor strain. • When this mixture was infected into mice, the hyperinfectious state remained. The competitive indices for human-shed V. cholerae shows an increase in infectivity 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. 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. 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. 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 Transcriptional profile of human-shed V. cholerae 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. 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. 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 nonchemotactic during dissemination, which could: – Increase the shedding from the GI tract – Increase infectivity 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 humanshed 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. 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.