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V. parahaemolyticus. Sodium transport genes & Osmoregulatory pumps Andrea, Saikumar, Stacey, & Cesar. (Kozo, et. al, 2002). Intro to V . parahaemolyticus. Gram negative bacterium, curved rod shaped with single flagellum Part of bacterial Vibrionaceae family

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v parahaemolyticus

V. parahaemolyticus

Sodium transport genes & Osmoregulatory pumps

Andrea, Saikumar, Stacey, & Cesar

(Kozo, et. al, 2002)

intro to v parahaemolyticus
Intro to V. parahaemolyticus
  • Gram negative bacterium, curved rod shaped with single flagellum
  • Part of bacterial Vibrionaceae family
  • Thrives in salt water (halophilic) obligate heterotrophs
  • Found predominately in marine and estuary communities

(Research in Microbiology, 2004; http://en.wikipedia.org/wiki/Vibrio_parahemolyticus)

pathogenesis of v parahaemolyticus
Pathogenesis of V. parahaemolyticus
  • Mutualistic state with oysters/shellfish
    • Concentrated in gills of oysters due to filtering
  • Pathogenic state with humans/mammals.
    • Causes gastrointestinal problems
    • Major cause of food poisoning from consuming raw/undercooked seafood

(Research in Microbiology, 2004)

genome of v parahaemolyticus
Genome of V. Parahaemolyticus
  • Genome similar to V. cholerae
  • 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?
  • (FEMS Microbiology Review, 2001)
osmoregulatory pumps
Osmoregulatory Pumps
  • Specific genes that allows plasticity in marine hosts (non infectious) to human hosts (infectious)
  • Location of these genes within the genome
  • Antiporter regulation effects on virulence
  • The problem of the chicken and the egg
  • Evolutionary patterns
na h antiporter
Na+/H+ Antiporter
  • 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

location of the genes of interest
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)

genome map
Genome Map
  • Genes encoding sodium pumps are highly conserved closely together mostly on chromosome one
    • Gene VP2449
    • Gene VP1092

(Kozo, et. al, 2002)

genes con t
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

Genes evolved with a specific purpose and can be easily manipulated without killing the bacteria

na h antiporter expression in changing environment
Na+/H+ Antiporter Expression in changing Environment
  • 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
  • Antiporters will be visualized and counted
expected results
Expected Results
  • Under stressful conditions
    • pH extremes and varying salinity

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

(Lexomboon 2000)

expected results1
Expected Results
  • 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.
  • 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.
evolutionary patterns
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
evolutionary patterns con t
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
did pathogenesis evolve from a mutualist or vice versa
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
the problem of the chicken and the egg
The problem of the Chicken and the egg
  • 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)

a different view
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
references
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.
  • “Vibrio parahaemolyticus” Obtained from <http://en.wikipedia.org/wiki/Vibrio_parahemolyticus.>
  • Kozo Makimo, et. al. “Genomic Map of V. parahaemolyticus.” “V. Parahaemolyticus Image” July 2004.

<http://images.google.com/imgres?imgurl=http://genome.naist.jp/bacteria/vpara/images/vpem.jpg&imgrefurl=http://genome.naist.jp/bacteria/vpara/index.html&h=540&w=600&sz=51&hl=en&start=1&um=1&usg=__EzFOOX4e2KQnHXnW7NqCC8ifylM=&tbnid=B1q8ILaobUJhkM:&tbnh=122&tbnw=135&prev=/images%3Fq%3Dv.%2Bparahaemolyticus%26um%3D1%26hl%3Den%26sa%3DN.>

  • 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.
  • 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.