V. V. parahaemolyticusparahaemolyticus

Sodium transport genes & Sodium transport genes & Osmoregulatory pumpsOsmoregulatory pumps

Andrea, Saikumar, Stacey, & CesarAndrea, Saikumar, Stacey, & Cesar(Kozo, et. al, 2002)

Intro to Intro to VV. parahaemolyticus. parahaemolyticus

Gram negative bacterium, curved rod Gram negative bacterium, curved rod shaped with single flagellum shaped with single flagellum

Part of bacterial Vibrionaceae familyPart of bacterial Vibrionaceae family Thrives in salt water (halophilic) Thrives in salt water (halophilic)

obligate heterotrophsobligate heterotrophs Found predominately in marine and Found predominately in marine and

estuary communitiesestuary communities

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

Pathogenesis of Pathogenesis of V. parahaemolyticusV. parahaemolyticus

Mutualistic state with oysters/shellfishMutualistic state with oysters/shellfish– Concentrated in gills of oysters due to filteringConcentrated in gills of oysters due to filtering

Pathogenic state with humans/mammals.Pathogenic state with humans/mammals.

– Causes gastrointestinal problemsCauses gastrointestinal problems– Major cause of food poisoning from consuming Major cause of food poisoning from consuming

raw/undercooked seafoodraw/undercooked seafood

((Research in Microbiology, 2004)Research in Microbiology, 2004)

Genome of Genome of V. V.

ParahaemolyticusParahaemolyticus Genome similar to Genome similar to V. choleraeV. cholerae

Two chromosomes (conserved vs. Two chromosomes (conserved vs. non-conserved genes)non-conserved genes)

Where would we expect to find Where would we expect to find Na+/H+ genes and how do we Na+/H+ genes and how do we determine the chromosomal determine the chromosomal location of theses genes?location of theses genes?

(FEMS Microbiology Review, 2001)(FEMS Microbiology Review, 2001)

Osmoregulatory Osmoregulatory Pumps Pumps

Specific genes that allows plasticity in Specific genes that allows plasticity in marine hosts (non infectious) to human marine hosts (non infectious) to human hosts (infectious)hosts (infectious)

Location of these genes within the Location of these genes within the genomegenome

Antiporter regulation effects on Antiporter regulation effects on virulencevirulence

The problem of the chicken and the eggThe problem of the chicken and the egg Evolutionary patterns Evolutionary patterns

Na+/H+ AntiporterNa+/H+ Antiporter

Na+/H+ Antiporter is a transport Na+/H+ Antiporter is a transport protein used to maintain gradients protein used to maintain gradients across the cell membraneacross the cell membrane

http://upload.wikimedia.org/wikipedia/en/7/71/Antiporter.jpg

Location of the Genes of Location of the Genes of InterestInterest

Na+/H+ are Na+/H+ are essential for survival essential for survival

Expect to be Expect to be found on found on conserved regions conserved regions of the of the chromosomechromosome

FISH FISH fluorescent in situ fluorescent in situ hybridizationhybridization

(http://www.genome.gov/glossary.cfm?key=fluorescence%20in%20situ%20hybridization%20%28FISH%29)

Genome MapGenome Map

Genes encoding Genes encoding sodium pumps are sodium pumps are highly conserved highly conserved closely together closely together mostly on mostly on chromosome onechromosome one

– Gene VP2449Gene VP2449 – Gene VP1092Gene VP1092

(Kozo, et. al, 2002)Kozo, et. al, 2002)

Genes, Con’t…Genes, Con’t…

If Na+/H+ antiporter genes were found If Na+/H+ antiporter genes were found on non conserved regions this would on non conserved regions this would indicate that the genes were not indicate that the genes were not necessary for survivalnecessary for survival

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

Na+/H+ Antiporter Na+/H+ Antiporter Expression in changing Expression in changing

EnvironmentEnvironment ExperimentExperiment

– Growing the bacteria under:Growing the bacteria under:– Optimum conditions (pH, salinity, temperature, Optimum conditions (pH, salinity, temperature,

food)food)– Decreased temperatureDecreased temperature– Decreased foodDecreased food– Varying pHVarying pH

Cultures obtained and stain with the Cultures obtained and stain with the appropriate dyeappropriate dye

Antiporters will be visualized and countedAntiporters will be visualized and counted

Expected ResultsExpected Results

Under stressful conditionsUnder stressful conditions– pH extremes and varying salinitypH extremes and varying salinity

– – up regulation or down regulation of up regulation or down regulation of antiporter proteinsantiporter proteins

– Varying Temperature and Food Varying Temperature and Food supplysupply

– Expression of antiporters would not be Expression of antiporters would not be affected but cell proliferation would be affected but cell proliferation would be greatly affectedgreatly affected

Rabbit Model for Rabbit Model for Pathogenicity Pathogenicity

V. V. parahaemolyticus parahaemolyticus collected from bivalves collected from bivalves Grown in cell broths Grown in cell broths Inoculate rabbits with a fixed dosage of brothInoculate rabbits with a fixed dosage of broth Rabbits were sacrificed 24 hours post infectionRabbits were sacrificed 24 hours post infection Post mortem cell cultures Na+/H+ antiporter Post mortem cell cultures Na+/H+ antiporter

proteins stained proteins stained Cells visualized and compared with cell cultures Cells visualized and compared with cell cultures

from bivalves.from bivalves.

(Lexomboon 2000)

Expected ResultsExpected Results

Original hypothesis: Due to a change in Original hypothesis: Due to a change in environmental conditions, there should be an environmental conditions, there should be an up regulation of Na+/H+ antiporter proteinsup regulation of Na+/H+ antiporter proteins

The number of Na+/H+ antiporter proteins The number of Na+/H+ antiporter proteins will remain relatively constant.will remain relatively constant.

Side note: Side note: – This antiporter uses H+ concentrations to maintain This antiporter uses H+ concentrations to maintain

Na+ gradients. Na+ gradients. – The digestive systems of animals have a high The digestive systems of animals have a high

concentration of H+, thus enabling the antiporter to concentration of H+, thus enabling the antiporter to create a greater Na+ gradient causing osmotic create a greater Na+ gradient causing osmotic diarrhea.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 Evolutionary Patterns Con’t…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 Did Pathogenesis Evolve from a Mutualist or Vice-from a Mutualist or Vice-

Versa?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 The problem of the Chicken and the eggChicken and the egg

One view: One view: – Pathogenicity evolve prior to mutualistic Pathogenicity evolve prior to mutualistic

associationsassociations 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

ReferencesReferences C. Xu, H. Ren, S. Wang, and X. Peng. “C. Xu, H. Ren, S. Wang, and X. Peng. “Proteomic analysis of salt-sensitive outer membrane Proteomic analysis of salt-sensitive outer membrane

proteins of proteins of Vibrio parahaemolyticusVibrio parahaemolyticus.” Research in Microbiology 155 (2004) 835-842. .” Research in Microbiology 155 (2004) 835-842.

““Vibrio parahaemolyticus” Obtained from Vibrio parahaemolyticus” Obtained from <http://en.wikipedia.org/wiki/Vibrio_parahemolyticus.><http://en.wikipedia.org/wiki/Vibrio_parahemolyticus.>

Kozo Makimo, et. al. “Genomic Map of Kozo Makimo, et. al. “Genomic Map of V. parahaemolyticusV. parahaemolyticus.” “V. Parahaemolyticus Image” July .” “V. Parahaemolyticus Image” July 2004. 2004.

<http://images.google.com/imgres?imgurl=http://genome.naist.jp/bacteria/vpara/images/<http://images.google.com/imgres?imgurl=http://genome.naist.jp/bacteria/vpara/images/vpem.jpg&imgrefurl=http://genome.naist.jp/bacteria/vpara/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=&tindex.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%26umbnid=B1q8ILaobUJhkM:&tbnh=122&tbnw=135&prev=/images%3Fq%3Dv.%2Bparahaemolyticus%26um%3D1%26hl%3Den%26sa%3DN.>%3D1%26hl%3Den%26sa%3DN.>

R. Sleator, and Colin Hill. “R. Sleator, and Colin Hill. “Bacterial osmoadaptation: the role of osmolytes in bacterial stress Bacterial osmoadaptation: the role of osmolytes in bacterial stress and and virulencevirulence.” FEMS Microbiology Reviews 26 (2001). 49-71..” FEMS Microbiology Reviews 26 (2001). 49-71.

Lexomboon, Udom. “Lexomboon, Udom. “The Infant Rabbit as a Model of Pathogenicity for Vibrio The Infant Rabbit as a Model of Pathogenicity for Vibrio parahaemolyticusparahaemolyticus”, 2000, ”, 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 T.Sugiyama, T.Iida, K.Izutsu, K.Park and T.Honda. “ Precise region and character of the pathogenecity island in clinical pathogenecity island in clinical Vibrio parahaemolyticusVibrio parahaemolyticus strains.” Journal of Bacteriology strains.” Journal of Bacteriology 190(2007)1835-1837.190(2007)1835-1837.