Dr Ksenia Matlawska-Wasowska
GAMIDI, Maria Curie Experienced Researcher
Pathogenic Mechanisms Group
Department of Microbiology
National University of Ireland, Galway
EDUCATION
2002 M.Sc. (Hons) Genetics/Phisiology
University of Lodz, Poland
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2006 Ph.D. (Hons) Genetics
University of Lodz, Poland
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SKILLS
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human and animal cells culture in vitro (tumor and regular)
Drosophila melanogaster cultures
plant test systems in vivo
MTT assay
single cell gel electrophoresis “comet assay”
assay”
TUNEL assay
caspases
caspases assays
DNA degradation assay „DNA ladder”
ladder”
DNA isolation and purification
PCR, RT-PCR-techniques
fluorescence and light microscopy (fluorochromes
(fluorochromes:: orange acridine,
acridine,
ethidium bromide,
bromide, Hoechst 33258,
33258, propidium iodide, DAPI, etc.)
etc.)
cytogenetics tests: micronuclei test,
test, chromosomal aberration test,
test, sister
chromatid exchange, etc.
GAMIDI AIMS
1. Identify the pathogenic mechanism of Vibrio
parahaemoliticus, in particular the role of two Type
Three Secretion Systems
2. Look at the effects of V. parahaemolyticus on the human
epithelial intestinal cells resposnes, in particular focus
on the role of c-Jun N-terminal kinase (JNK) activation
in the modification of host cell behaviour by this
bacterium
Vibrio parahaemolyticus
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Gram negative,
rod shaped,
haliophilic
motile (polar flagellum)
non–sporulating
warm marine
environments
MECHANISM OF PATHOGENESIS OF
V. PARAHAEMOLYTICUS
-TDH (thermostable direct haemolysin)
-TRH (thermostable related haemolysin)
-Type III Secretion System:
TTSS1 and TTSS2
 syringe like protein structure
 insert effector proteins into host cells
 enable bacteria to persist within the host
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RESULTS OBTAINED SO FAR...
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Invasion of V. parahaemolyticus into
eucariotic cells
Role of TTSS of V. parahaemolyticus on
induction of mammalian cell death
Effect of TTSS of V. parahaemolyticus on
IL-8 chemokine production by human cells
MATERIALS
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human epithelial colon Caco-2 cells
bacterial strains:
-Vibrio parahaemolyticus wild type (RIMD2210633)
-TTSS1 mutant
-TTSS2 mutant
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METHODS
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Gentamicin protection assay was performed to determine how
the two TTSS affect the invasion of Vibrio parahaemolyticus into
Caco-2 cells
Lactate Dehydrogenase (LDH) assay was performed to
determine the cell cytotoxicity level by means of cell lysis
measurement
Thiazolyl Blue Tetrazolium Bromide assay (MTT) was used to
determine the cell cytotoxicity level by means of cell viability
measurement
Enzyme Linked Immunosorbent Assay (ELISA) was carried
out to investigate the effect of TTSS on the production of the
chemokine IL-8, which is secreted by epithelial cells after
recognition of foreign antigens. Undifferentiated and differentiated
Caco-2 cells were used.
RESULTS
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IRISH SOCIETY FOR
IMMUNOLOGY,
DUBLIN, SEPTEMBER
2007
CONCLUSIONS
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V. parahaemolyticus invades the Caco-2 cells
TTSS has no effect on invasion by V. parahaemolyticus
V. parahaemolyticus is cytotoxic to Caco-2 cells in concentration
and time dependent manner
TTSS1 is essential for cytotoxicity
possibility of specific complex reaction between TTSS1 and TTSS2
V. parahaemolyticus induces the production of chemokine IL-8
induction of IL-8 secretion increased with differentiation to cryptlike cells and villus-like cells
TTSS1 appears to inhibit IL-8 secretion
TTSS2 appears to have no role in IL-8 secretion
EXPERIMENTS IN PROGRESS...
#1. Effect of TTSS of V. parahaemolyticus on
induction of mammalian cell death (continuation)
- apoptosis/necrosis induction (fluorochrome staining, DNA fragmentation
assay, caspase assay)
EXPERIMENTS IN PROGRESS...
#2. Induction of mammalian cell death by TTSS
effector proteins from Vibrio parahaemolyticus
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transfection (Lipofectamine 2000 Reagent)
cytotoxicity assessment (LDH, MTT)
protein expression detection (SDS/PAGE; WESTERN BLOTT)
human epithelial kidney HEK293 cells
human epithelial colon Caco-2 cells (?)
expression plasmids (pcDNA3.1/V5-His-TOPO) containing specific gene
sequences encoding proteins from V. parahaemolyticus
CONSTRUCTED EXPRESSION PLASMIDS CONTAINING
SPECIFIC SEQUENCES FROM
V. PARAHAEMOLYTICUS
PLASMIDS
GENES
Other information
PROTEIN
SIZE(Da)
pABGS16
vpa1321
Vop C [CNF]
45057
pABGS18
vp1680
Cytotoxic to
epithelial cells
57891
pABGS20
vp1686
Cytotoxic to
macrophages
46575
pABGS22
vpa1364
? unknown
32252
pABGS24
vpa1327
Vop T
30770
pABGS26
vpa1346
Vop P/Vop A
37399
pABGS28
lacZ
positive control
119379
EXPERIMENTS IN PROGRESS...
# 3. Role of JNK/MAPK activation in the
modification of host cell behaviour by V.
parahaemolyticus
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characterisation of JNK/MAPK activation pathway
requirement of JNK/MAPK activation/inhibition for cytotoxicity
ability of transfection plasmids to induce JNK/MAPK (?)
(SDS/PAGE and WESTERN BLOTT)
human epithelial colon Caco-2 cells
human epithelial kidney HEK293 cells (?)
V. parahaemolyticus wild type, TTSS1 mutant, TTSS2 mutant,
TTSS1/TTSS2 mutant
expression plasmids containing specific gene sequences encoding proteins
from V. parahaemolyticus (?)
Acknowledgements
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Dr Aoife Boyd
MSc Sinead D’Arcy
Joshua Bell
Nicky O’Boyle
Aaron Griffin
Adrian Glynn