1. Host-induced epidemic spread of the cholera bacterium Theresa Graebener, Salomon Garcia, Claudia Campos Journal Club Presentation Biological Databases 367-01 October 19, 2010

2. Outline Background on Vibrio cholerae Look at the experimental analysis – Strains responsible for cholera Results and Conclusions – In depth look at figures and DNA microarrays References

3. Background on Vibrio cholerae It’s a gram negative bacterium Has polar flagellum Comma shaped Main cause for cholera V. cholerae has 2 circular chromosomes of unequal size that encode for an approximate of 3885 genes.

4. The experiment itself… For this study human subjects were gathered from the International Centre for Diarrhoeal Disease Research (ICDDR) Subjects were from Dhaka, Bangladesh mainly due to bacterium being in it’s natural habitat Serological analysis was done in order to identify if stool samples that were positive for V.cholerae 01 Inaba El Tor

5. Bacterial Strain responsible for the spread of V.cholerae Strain (DSM-V984) was strain that had been previously isolated and is marked by the deletion of the lacZ gene. It allows for the enumeration of in vitro and the stool samples Strain (DSM-V984) was mixed with V.cholerae in stool Mixture was injected to 3-5 day old Swiss Webster mice. Mice were euthanized after 20-24hrs and bacteria was recovered

6. Testing of the human shed V.cholerae occurred in order to test if the hyperinfectious phenotype was maintatined. V.cholerae samples that were freshly shed were diluted in pond water that was free from contaminants of V.cholerae Incubation at room temperature for 5 hrs, then diluted samples were mixed with in vitro grown competitor strains This mixture was injected to mice Hyperinfectious state remained the same the only difference being human passage of the bacteria enhances water bourne spread which lowers the dose to secondary individuals

7. Transcriptional profiling of human shed V.cholerae Spotted DNA microarray with 87% of known open reading frames Samples of the 3 patients were taken then later frozen in order to prepare for total RNA. RNA analyzed using agarose gel electrophoresis Strain DSM-V999 from one of the 3 samples, RNA was isolated after in vitro growth

8. Statistical Analysis reveals significant genes are responsible for gene expressions 237 genes were differentially regulated 44 genes were induced 193 genes were repressed in human stool samples Transcriptomes were similar to strain DSM- V999, 3120 out of 3357 open reading frames that were examined were expressed

9. On A samples mixed directly with in-vitro grown DSM- V984 On B samples incubated in local pond water for 5 hours Competition assays take advantage of concentration differences

10. The Data points represent the output ratio of stool- derived V. cholerae to in-vitro grown competitor strain after correction for deviations in input rations. Each data point represents output from a single animal

11. The geometric mean is indicated by horizontal bar (n) is the total number of animals per experiment

12. The transcription profiles were used to compare genetic similarities and differences The cluster diagram shows the whole- genome expression profile of V. cholerae recovered from the stools of three ICDDR patients

13. Patients labeled A B or C (in blue) Replicate arrays are numbered 1-4 (in orange)

14. Right panel is cluster diagram showing consistent differential regulation from patients A B and C Fold changed relative to V. cholerae grown in vitro calculated using the avg values from the quadruplicate arrays on left

15. Red indicated a minimum twofold increase in expression Green represents a minimum twofold reduction in expression Representative genes listed and their relative location indicated by arrow

16. Differences found between human shed V.cholerae and strain DSM-V999 Increased expression of genes required for biosynthesis of amino acids, iron uptake systems, ribosomal proteins, and formation of periplasmic nitrate reductase complex V. cholerae moves from rich nutrient environment to poor environment which is purged.

17. Several genes were found to be differentially expressed in stool samples These genes were found to be factors for infection of mice and humans VC0468 strain was found to play a significant role in the ability of V.cholerae to tolerate acid

18. Cholera epidemics may be propagated by human hosts Perhaps other epidemic pathogens are spread in a similar manner. Agent is more infectious after leaving its primary host Provides new targets for antimicrobial therapy

19. References Merrell D. Scott, Butler Susan M., QadriFirdausi, Dolganov Nadia A., Alam Ahsfaqui, Cohen Mitchell B., Calderwood Stephen B., Schoonik Gary K., & Camilli Andrew. Host-induced epidemic spread of the cholera bacterium. Nature. 2002. Vol, 417,