Blanka Florová

28. 07. 2011

INTERACTION BETWEEN INTERACTION BETWEEN PROKARYOTIC AND PROKARYOTIC AND EUKARYOTIC CELLEUKARYOTIC CELL

PROBIOTICSPROBIOTICS

• „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 PROJECTAIMS 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 ORIGIN OF CELL AND BACTERIAL CULTURESBACTERIAL 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 MATERIALS AND CULTIVATION CONDITIONSCONDITIONS

• 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 (1,5x101,5x1088 CFU/ml) CFU/ml) were co-cultivated (1, 8, 24h

or 2h) with Caco-2 cells in 5% CO2 atmosphere in 37°C

METHODSMETHODS• Optical microscopyOptical microscopy – –

cells were stained with cells were stained with May-GrMay-Grüünwald and nwald and Giemsa-Romanowski Giemsa-Romanowski dyesdyes

• Field Emission Field Emission Scanning Electron Scanning Electron MicroscopyMicroscopy (FESEM) (FESEM)

• Statistical evaluationStatistical evaluation – evaluation of – evaluation of difference of two difference of two relative values relative values (Reisenauer, 1970)(Reisenauer, 1970)

EXCLUSION/EXCLUSION/DISPLACEMENTDISPLACEMENT//

COMPETITION ASSAYCOMPETITION 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

RESULTSRESULTSExclusion, displacement and competition between Exclusion, displacement and competition between

L. mucosaeL. mucosae D and D and S. aureusS. aureus on Caco-2 cells on Caco-2 cells

RESULTSRESULTSStatistical evaluationStatistical evaluation

evaluation of difference of two relative valuesevaluation of difference of two relative values

D D+Sa D+Sa      SaD *** SaD    DSa *** *** DSa

Sa D+Sa D+Sa      SaD *** SaD    DSa *** *** DSa

FIELD EMISSION SCANNING FIELD EMISSION SCANNING ELECTRON MICROSCOPYELECTRON MICROSCOPY

Caco-2 cells co-cultivated Caco-2 cells co-cultivated with with L. mucosae L. mucosae DD

Caco-2 cells Caco-2 cells

FIELD EMISSION SCANNING FIELD EMISSION SCANNING ELECTRON MICROSCOPYELECTRON MICROSCOPY

Caco-2 cells Caco-2 cells Caco-2 cells co-cultivated Caco-2 cells co-cultivated

with with E. coliE. coli

FIELD EMISSION SCANNING FIELD EMISSION SCANNING ELECTRON MICROSCOPYELECTRON MICROSCOPY

Caco-2 cells Caco-2 cells co-cultivated co-cultivated

with with L. L. mucosae mucosae DD

Play surface proteins key role in prokaryotic Play surface proteins key role in prokaryotic and eukaryotic cell binding?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

RESULTSRESULTSTime - dependent ability of adherence of Time - dependent ability of adherence of L. mucosaeL. mucosae D D

to Caco-2 cellsto Caco-2 cells

RESULTSRESULTSStatistical evaluationStatistical evaluation

evaluation of difference of two relative valuesevaluation 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 ***

CONCLUSIONSCONCLUSIONS

• Overall, Overall, S. aureus S. aureus adhered on surface of Caco-2 adhered on surface of Caco-2 cells better than the cells better than the L. mucosae L. mucosae DD

• The greatest ability of The greatest ability of L. mucosae L. mucosae D D to rival to rival S. aureusS. aureus was observed in the was observed in the competitioncompetition assay assay

• S. aureus S. aureus has the highest ability to adhere on Caco-has the highest ability to adhere on Caco-2 cells in 2 cells in displacement displacement (74,58%), the lowest in the (74,58%), the lowest in the competition competition (19,73%)(19,73%)

• L. mucosae L. mucosae D D has the highest ability to adhere on has the highest ability to adhere on Caco-2 cells in Caco-2 cells in exclusion exclusion (34,13%), the lowest in (34,13%), the lowest in the the competition competition (9,52%)(9,52%)

CONCLUSIONSCONCLUSIONS

• 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

REFERENCESREFERENCES• Reisenauer, R.Reisenauer, R. Metody matematické statistikyMetody matematické statistiky. Práce . Práce

SNTL, Praha, 1970. SNTL, Praha, 1970.

• FAO/WHO.FAO/WHO. Health and nutritional properties of Health and nutritional properties of probiotics in food including powder milk with live lactic probiotics in food including powder milk with live lactic acid bacteriaacid bacteria. 2001. . 2001. www.who.int/entity/foodsafety/publications/fs_managemwww.who.int/entity/foodsafety/publications/fs_management/probiotics2/en (ent/probiotics2/en (2020..77.200.20099) )

• I would like to acknowledge to I would like to acknowledge to

my project leader my project leader Hanka Kiňová Sepová Hanka Kiňová Sepová for for leading, precious advices and patienceleading, precious advices and patience

Víťa Březina for his helpVíťa Březina for his helpMonča Homolková, Pavlína Tláskalová and Monča Homolková, Pavlína Tláskalová and

Šárka Beranová for technical supportŠárka Beranová for technical support

THANK YOU FOR YOUR ATTENTIONTHANK YOU FOR YOUR ATTENTION