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Blanka Florová

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)

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Blanka Florová

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  1. 28. 07. 2011 Blanka Florová 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 4516was 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% 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

  6. METHODS • Opticalmicroscopy – cellswerestainedwithMay-Grünwald and Giemsa-Romanowskidyes • FieldEmissionScanningElectronMicroscopy (FESEM) • Statisticalevaluation – evaluationofdifferenceoftworelativevalues(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. aureuswere 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 Statisticalevaluation evaluationofdifferenceoftworelativevalues

  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 co-cultivated with E. coli Caco-2 cells

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

  13. Play surfaceproteinskey role in prokaryoticandeukaryotic cell binding? • Digestion (1h/37°C) of L. mucosae D bythree 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 Statisticalevaluation evaluationofdifferenceoftworelativevalues

  16. CONCLUSIONS • Overall, S. aureusadhered on surfaceof Caco-2 cellsbetterthantheL. mucosaeD • ThegreatestabilityofL. mucosaeD to rival S. aureuswasobserved in thecompetitionassay • S. aureushas thehighestability to adhere on Caco-2 cells in displacement(74,58%), thelowest in thecompetition(19,73%) • L. mucosaeD has thehighestability to adhere on Caco-2 cells in exclusion(34,13%), thelowest in thecompetition(9,52%)

  17. CONCLUSIONS • L. mucosaeD digestedwithtrypsin had theoverallbestability to renewsurfaceproteins • NondigestedcellsofL. mucosaeD had thelowestability to bind Caco-2 cells. • Thehighestability to bindtosurfaceof Caco-2 cells has L. mucosaeD digestedwithpronase E in 1sth • Thehighestability to bindtosurfaceof Caco-2 cells has L. mucosaeD digestedwithproteinase K in 8thh • Thehighestability to bindtosurfaceof Caco-2 cellsL. mucosaeD digestedwithtrypsin in 24thh

  18. 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_management/probiotics2/en (20.7.2009)

  19. I wouldlike to acknowledge to • my project leader Hanka KiňováSepováforleading, preciousadvicesandpatience • Víťa Březina for his help • Monča Homolková, Pavlína Tláskalová and Šárka Beranová fortechnical support

  20. THANK YOU FOR YOUR ATTENTION

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