Transcript Document

28. 07. 2011
Blanka Florová
INTERACTION BETWEEN
PROKARYOTIC AND
EUKARYOTIC CELL
PROBIOTICS
• „Live
microorganisms
which
when
administered in adequate amounts confer a
health benefit on the host“ (FAO/WHO, 2001)
• Most of them are lactic acid bacteria, e.g.
genera Lactobacillus, Enterococcus,
Bifidobacterium
• They improve the colon microbiota after
antibiotic therapy, improve lactose
intolerance, stimulate immune system,
produce antimicrobial substances, etc.
AIMS OF THE PROJECT
• To evaluate and to quantify the rate of binding
between eukaryotic (Caco-2) and prokaryotic
(Lactobacillus mucosae D, Staphylococcus aureus)
cells
• To compare the degree of adhesivity between L.
mucosae D and S. aureus and their possible
interaction
• To determine the influence of trypsin, pronase E
and proteinase K on prokaryotic and eukaryotic cell
binding
• To image the prokaryotic (L. mucosae D,
Escherichia coli) and eukaryotic cell binding by
FESEM
ORIGIN OF CELL AND
BACTERIAL CULTURES
• Caco-2: human epitelial colon adenocarcinoma cells
(European Collection of Cell Cultures)
• Lactobacillus mucosae D was obtained from stomach
mucus of breast-fed lamb (Očová, Slovakia)
• Staphylococcus aureus CCM 4516 was purchased
from the Czech Collection of Microorganisms (Brno,
Czech Republic)
• Escherichia coli CNCTC 377/79 was purchased from
the Czech National Collection of Type Cultures (Prague,
Czech Republic)
MATERIALS AND CULTIVATION
CONDITIONS
• Caco-2 cells were cultivated in MEM medium
supplemented with foetal bovine serum, non-essential
aminoacids, antibiotics and antimycotics for two weeks
at 37ºC in 5% CO2 atmosphere
• L. mucosae D was cultivated in MRS medium for 24h
at 37ºC
• S. aureus and E. coli were cultivated in LB medium for
24h at 37°C
• Bacteria (1,5x108 CFU/ml) were co-cultivated (1, 8, 24h
or 2h) with Caco-2 cells in 5% CO2 atmosphere in 37°C
METHODS
• Optical microscopy –
cells were stained with
May-Grünwald and
Giemsa-Romanowski
dyes
• Field Emission
Scanning Electron
Microscopy (FESEM)
• Statistical evaluation
– evaluation of
difference of two
relative values
(Reisenauer, 1970)
EXCLUSION/DISPLACEMENT/
COMPETITION ASSAY
• EXCLUSION: Pre-incubation Caco-2 cells
with L. mucosae D for 2h, afterwards
added S. aureus
• DISPLACEMENT: Pre-incubation Caco-2
cells with S. aureus for 2h, afterwards
added L. mucosae D
• COMPETITION: L. mucosae D and S.
aureus were incubated simultaneously
with Caco-2 cells for 2h
RESULTS
% of Caco-2 cells adhered with at least one bacterial
cell
Exclusion, displacement and competition between
L. mucosae D and S. aureus on Caco-2 cells
80
70
60
50
competition
40
displacement
30
exclusion
20
10
0
L. mucosae D
S. aureus
RESULTS
Statistical evaluation
evaluation of difference of two relative values
D
D+Sa
SaD
DSa
D+Sa
***
***
SaD
***
Sa
DSa
D+Sa
SaD
DSa
D+Sa
***
***
SaD
***
DSa
FIELD EMISSION SCANNING
ELECTRON MICROSCOPY
Caco-2 cells
Caco-2 cells co-cultivated
with L. mucosae D
FIELD EMISSION SCANNING
ELECTRON MICROSCOPY
Caco-2 cells
Caco-2 cells co-cultivated
with E. coli
FIELD EMISSION SCANNING
ELECTRON MICROSCOPY
Caco-2 cells
co-cultivated
with L.
mucosae D
Play surface proteins key role in prokaryotic
and eukaryotic cell binding?
• Digestion (1h/37°C) of L. mucosae D by
three proteases: trypsin, proteinase K
and pronase E
• Following-up incubation with Caco-2 cells
for 1, 8 and 24h
RESULTS
Time - dependent ability of adherence of L. mucosae D
to Caco-2 cells
% of Caco-2 cells adhered with at least one
bacterial cell
70
60
50
D
40
DK
DE
30
DT
20
10
0
1
8
time (h)
24
RESULTS
Statistical evaluation
evaluation of difference of two relative values
1h D
D
 DE ***
 DK ***
- DT ns
8h D
D
 DE *
 DK ***
 DT **
24h



D
D
DE ***
DK ***
DT ***
CONCLUSIONS
• Overall, S. aureus adhered on surface of Caco-2
cells better than the L. mucosae D
• The greatest ability of L. mucosae D to rival
S. aureus was observed in the competition assay
• S. aureus has the highest ability to adhere on Caco2 cells in displacement (74,58%), the lowest in the
competition (19,73%)
• L. mucosae D has the highest ability to adhere on
Caco-2 cells in exclusion (34,13%), the lowest in
the competition (9,52%)
CONCLUSIONS
• L. mucosae D digested with trypsin had the overall best
ability to renew surface proteins
• Nondigested cells of L. mucosae D had the lowest ability
to bind Caco-2 cells.
• The highest ability to bind to surface of Caco-2 cells has L.
mucosae D digested with pronase E in 1sth
• The highest ability to bind to surface of Caco-2 cells has L.
mucosae D digested with proteinase K in 8thh
• The highest ability to bind to surface of Caco-2 cells L.
mucosae D digested with trypsin in 24thh
REFERENCES
•
Reisenauer, R. Metody matematické statistiky. Práce
SNTL, Praha, 1970.
• FAO/WHO. Health and nutritional properties of
probiotics in food including powder milk with live lactic
acid bacteria. 2001.
www.who.int/entity/foodsafety/publications/fs_managem
ent/probiotics2/en (20.7.2009)
• I would like to acknowledge to
my project leader Hanka Kiňová Sepová for
leading, precious advices and patience
Víťa Březina for his help
Monča Homolková, Pavlína Tláskalová and
Šárka Beranová for technical support
THANK YOU FOR YOUR ATTENTION