1. Blanka Florová 28. 07. 2011 INTERACTION BETWEEN PROKARYOTIC AND EUKARYOTIC CELL

2. 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.

3. 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

4. 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)

5. 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% CO 2 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 1,5x10 8 CFU/ml)Bacteria (1,5x10 8 CFU/ml) were co-cultivated (1, 8, 24h or 2h) with Caco-2 cells in 5% CO 2 atmosphere in 37°C

6. METHODS Optical microscopy – cells were stained with May-Grünwald and Giemsa-Romanowski dyesOptical microscopy – cells were stained with May-Grünwald and Giemsa-Romanowski dyes Field Emission Scanning Electron Microscopy (FESEM)Field Emission Scanning Electron Microscopy (FESEM) Statistical evaluation – evaluation of difference of two relative values (Reisenauer, 1970)Statistical evaluation – evaluation of difference of two relative values (Reisenauer, 1970)

7. 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

8. RESULTS Exclusion, displacement and competition between L. mucosae D and S. aureus on Caco-2 cells

9. RESULTS Statistical evaluation evaluation of difference of two relative values DD+Sa SaD***SaD DSa*** DSa SaD+Sa SaD***SaD DSa*** DSa

10. FIELD EMISSION SCANNING ELECTRON MICROSCOPY Caco-2 cells co-cultivated with L. mucosae D Caco-2 cells

11. FIELD EMISSION SCANNING ELECTRON MICROSCOPY Caco-2 cells Caco-2 cells co-cultivated with E. coli

12. FIELD EMISSION SCANNING ELECTRON MICROSCOPY Caco-2 cells co-cultivated with L. mucosae D

13. 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

14. RESULTS Time - dependent ability of adherence of L. mucosae D to Caco-2 cells

15. RESULTS Statistical evaluation evaluation of difference of two relative values 1hDD  DE***  DK*** - DTns 8hDD  DE*  DK***  DT** 24hDD  DE***  DK***  DT***

16. CONCLUSIONS Overall, S. aureus adhered on surface of Caco-2 cells better than the L. mucosae DOverall, S. aureus adhered on surface of Caco-2 cells better than the L. mucosae D The greatest ability of L. mucosae D to rivalThe 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 Caco- 2 cells in displacement (74,58%), the lowest in the competition (19,73%)S. aureus has the highest ability to adhere on Caco- 2 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%)L. mucosae D has the highest ability to adhere on Caco-2 cells in exclusion (34,13%), the lowest in the competition (9,52%)

17. 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 1 st h The highest ability to bind to surface of Caco-2 cells has L. mucosae D digested with proteinase K in 8 th h The highest ability to bind to surface of Caco-2 cells L. mucosae D digested with trypsin in 24 th h

18. REFERENCES Reisenauer, R. Metody matematické statistiky. Práce SNTL, Praha, 1970. 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) 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)

19. I would like to acknowledge toI would like to acknowledge to my project leader Hanka Kiňová Sepová for leading, precious advices and patience my project leader Hanka Kiňová Sepová for leading, precious advices and patience Víťa Březina for his help Víťa Březina for his help Monča Homolková, Pavlína Tláskalová and Šárka Beranová for technical support Monča Homolková, Pavlína Tláskalová and Šárka Beranová for technical support

20. THANK YOU FOR YOUR ATTENTION