1. V. parahaemolyticus Sodium transport genes & Osmoregulatory pumps Andrea, Saikumar, Stacey, & Cesar Andrea, Saikumar, Stacey, & Cesar (Kozo, et. al, 2002)

2. Intro to V. parahaemolyticus Gram negative bacterium, curved rod shaped with single flagellum Gram negative bacterium, curved rod shaped with single flagellum Part of bacterial Vibrionaceae family Part of bacterial Vibrionaceae family Thrives in salt water (halophilic) obligate heterotrophs Thrives in salt water (halophilic) obligate heterotrophs Found predominately in marine and estuary communities Found predominately in marine and estuary communities (Research in Microbiology, 2004; http://en.wikipedia.org/wiki/Vibrio_parahemolyticus)

3. Pathogenesis of V. parahaemolyticus Mutualistic state with oysters/shellfish Mutualistic state with oysters/shellfish –Concentrated in gills of oysters due to filtering Pathogenic state with humans/mammals. Pathogenic state with humans/mammals. –Causes gastrointestinal problems –Major cause of food poisoning from consuming raw/undercooked seafood ( Research in Microbiology, 2004) ( Research in Microbiology, 2004)

4. Genome of V. Parahaemolyticus Genome similar to V. cholerae Genome similar to V. cholerae Two chromosomes (conserved vs. non- conserved genes) Two chromosomes (conserved vs. non- conserved genes) Where would we expect to find Na+/H+ genes and how do we determine the chromosomal location of theses genes? Where would we expect to find Na+/H+ genes and how do we determine the chromosomal location of theses genes? (FEMS Microbiology Review, 2001)

5. Osmoregulatory Pumps Specific genes that allows plasticity in marine hosts (non infectious) to human hosts (infectious) Specific genes that allows plasticity in marine hosts (non infectious) to human hosts (infectious) Location of these genes within the genome Location of these genes within the genome Antiporter regulation effects on virulence Antiporter regulation effects on virulence The problem of the chicken and the egg The problem of the chicken and the egg Evolutionary patterns Evolutionary patterns

6. Na+/H+ Antiporter Na+/H+ Antiporter is a transport protein used to maintain gradients across the cell membrane Na+/H+ Antiporter is a transport protein used to maintain gradients across the cell membrane http://upload.wikimedia.org/wikipedia/en/7/71/Antiporter.jpg

7. Location of the Genes of Interest  Na+/H+ are essential for survival  Expect to be found on conserved regions of the chromosome  FISH  fluorescent in situ hybridization (http://www.genome.gov/glossary.cfm?key=fluorescence%20in%20situ%20hybridization%20%28FISH%29)

8. Genome Map Genes encoding sodium pumps are highly conserved closely together mostly on chromosome one Genes encoding sodium pumps are highly conserved closely together mostly on chromosome one –Gene VP2449 –Gene VP1092 Kozo, et. al, 2002) (Kozo, et. al, 2002)

9. Genes, Con’t… If Na+/H+ antiporter genes were found on non conserved regions this would indicate that the genes were not necessary for survival If Na+/H+ antiporter genes were found on non conserved regions this would indicate that the genes were not necessary for survival Genes evolved with a specific purpose and can be easily manipulated without killing the bacteria Genes evolved with a specific purpose and can be easily manipulated without killing the bacteria

10. Na+/H+ Antiporter Expression in changing Environment Experiment Experiment –Growing the bacteria under: –Optimum conditions (pH, salinity, temperature, food) –Decreased temperature –Decreased food –Varying pH Cultures obtained and stain with the appropriate dye Cultures obtained and stain with the appropriate dye Antiporters will be visualized and counted Antiporters will be visualized and counted

11. Expected Results Under stressful conditions Under stressful conditions –pH extremes and varying salinity – up regulation or down regulation of antiporter proteins – up regulation or down regulation of antiporter proteins –Varying Temperature and Food supply –Expression of antiporters would not be affected but cell proliferation would be greatly affected

12. Rabbit Model for Pathogenicity V. parahaemolyticus collected from bivalves V. parahaemolyticus collected from bivalves Grown in cell broths Grown in cell broths Inoculate rabbits with a fixed dosage of broth Inoculate rabbits with a fixed dosage of broth Rabbits were sacrificed 24 hours post infection Rabbits were sacrificed 24 hours post infection Post mortem cell cultures Na+/H+ antiporter proteins stained Post mortem cell cultures Na+/H+ antiporter proteins stained Cells visualized and compared with cell cultures from bivalves. Cells visualized and compared with cell cultures from bivalves. (Lexomboon 2000)

13. Expected Results Original hypothesis: Due to a change in environmental conditions, there should be an up regulation of Na+/H+ antiporter proteins Original hypothesis: Due to a change in environmental conditions, there should be an up regulation of Na+/H+ antiporter proteins The number of Na+/H+ antiporter proteins will remain relatively constant. The number of Na+/H+ antiporter proteins will remain relatively constant. Side note: Side note: –This antiporter uses H+ concentrations to maintain Na+ gradients. –The digestive systems of animals have a high concentration of H+, thus enabling the antiporter to create a greater Na+ gradient causing osmotic diarrhea.

14. Evolutionary Patterns Environmental conditions – –Oxygen, temperature, and salinity have significant affects on virulence Higher salinity increases virulence towards shrimp Composition and metabolism of V. parahaemolyticus – –Altered for adaptation – –Results in increased pathogenicity

15. Evolutionary Patterns Con’t… Outer membrane proteins (OMP) – –Play key role in adaptation to changes in external environment – –Osmolarity location is outermost part of cell. Synthesis of OMPs – –Regulation when V. parahaemolyticus is transferred to different salinity environments

16. Did Pathogenesis Evolve from a Mutualist or Vice-Versa? Specific virulence factors exhibited in colonization by V. parahaemolyticus May be required for colonization Defense mechanisms of host must be conquered in either case

17. The problem of the Chicken and the egg One view: One view: –Pathogenicity evolve prior to mutualistic associations Common ancestral origin of many characteristics of host-tissue colonization? Most sensible for pathogen to lead to symbiont: allows host and attacker to survive. V. parahaemolyticus pathogenicity islands (PAI) on chromosomeII: 80kb of DNA. (http://jb.asm.org/cgi/reprint/190/5/1835.pdf)http://jb.asm.org/cgi/reprint/190/5/1835.pdf

18. A Different View Human host gives V. parahaemolyticus perfect environment – –Optimal temperature and nutrition allow for increase in proliferation and environment exploitation. Virulence results from a “perfect” host

19. References C. Xu, H. Ren, S. Wang, and X. Peng. “Proteomic analysis of salt-sensitive outer membrane proteins of Vibrio parahaemolyticus.” Research in Microbiology 155 (2004) 835-842. C. Xu, H. Ren, S. Wang, and X. Peng. “Proteomic analysis of salt-sensitive outer membrane proteins of Vibrio parahaemolyticus.” Research in Microbiology 155 (2004) 835-842. “Vibrio parahaemolyticus” Obtained from “Vibrio parahaemolyticus” Obtained from Kozo Makimo, et. al. “Genomic Map of V. parahaemolyticus.” “V. Parahaemolyticus Image” July 2004. Kozo Makimo, et. al. “Genomic Map of V. parahaemolyticus.” “V. Parahaemolyticus Image” July 2004. R. Sleator, and Colin Hill. “Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence.” FEMS Microbiology Reviews 26 (2001). 49-71. R. Sleator, and Colin Hill. “Bacterial osmoadaptation: the role of osmolytes in bacterial stress and virulence.” FEMS Microbiology Reviews 26 (2001). 49-71. Lexomboon, Udom. “The Infant Rabbit as a Model of Pathogenicity for Vibrio parahaemolyticus”, 2000, http://www.afrims.org/weblib/eapr/1971/APR71p178-181.pdf. Lexomboon, Udom. “The Infant Rabbit as a Model of Pathogenicity for Vibrio parahaemolyticus”, 2000, http://www.afrims.org/weblib/eapr/1971/APR71p178-181.pdf. http://www.afrims.org/weblib/eapr/1971/APR71p178-181.pdf T.Sugiyama, T.Iida, K.Izutsu, K.Park and T.Honda. “ Precise region and character of the pathogenecity island in clinical Vibrio parahaemolyticus strains.” Journal of Bacteriology 190(2007)1835-1837. T.Sugiyama, T.Iida, K.Izutsu, K.Park and T.Honda. “ Precise region and character of the pathogenecity island in clinical Vibrio parahaemolyticus strains.” Journal of Bacteriology 190(2007)1835-1837.