Transcript Presentation- Group C

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
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

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 similar to V. cholerae
Two chromosomes (conserved vs. nonconserved 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





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 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
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

Genes encoding
sodium pumps are
highly conserved
closely together mostly
on chromosome one
– Gene VP2449
– Gene VP1092
(Kozo, et. al, 2002)
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

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

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






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 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

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…

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?



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




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

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

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=htt
p://genome.naist.jp/bacteria/vpara/index.html&h=540&w=600&sz=51&hl=en&start=1&um=1&usg=__EzFOOX4e
2KQnHXnW7NqCC8ifylM=&tbnid=B1q8ILaobUJhkM:&tbnh=122&tbnw=135&prev=/images%3Fq%3Dv.%2Bparaha
emolyticus%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.