Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
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