Human intestinal microbiology and probiotics
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1 Human intestinal microbiology and probiotics Michiel Kleerebezem Wageningen Centre for Food Sciences Wageningen University NIZO food research P.O. Box BA Ede The Netherlands phone: +31-(0) fax: +31-(0)
2 Probiotics Live microorganisms administered in adequate amounts which confer a beneficial health effect on the host mainly Lactobacillus & Bifidobacterium food-grade bacteria transient; commercially attractive
3 Probiotics Live microorganisms administered in adequate amounts which confer a beneficial health effect on the host mainly Lactobacillus & Bifidobacterium food-grade bacteria transient; commercially attractive myriad of health effects claimed reduced infection risk anti-cancer immunomodulation detoxification constipation etc. etc. few DB-PC clinical trials show (positive) effect descriptive few molecular mechanisms proposed (proven)
4 Probiotics act in a complex niche diversity safety Probiotics health claims mechanism composition activity Microbiota disease health
5 How to explore molecular biology underlying probiotic in situ activity?
6 Probiotics, a genomics based approach Model microbe: Lactobacillus plantarum WCFS1 Human isolate (NCIMB 8826) Versatile and flexible LAB Survival in the human GI tract* Genome sequence available, 3.3 Mb ** Bioinformatics model support; advanced annotation, predictive gene-regulatory networks and global stochiometry-based and dedicated kinetic metabolic models*** *Vesa et al., APT, 2000 **Kleerebezem et al., PNAS, 2003 *** Teusink et al., COB, in press
7 Probiotics, a genomics based approach Model systems of mice and men Monitoring gene activity on both sides Molecular host microbe interaction models
8 GI-tract induced gene expression in L. plantarum WCFS1 R-IVET 72 ivi genes identified 54 functionally annotated
9 GI-tract induced gene expression in L. plantarum WCFS1 R-IVET 72 ivi genes identified 54 functionally annotated ~ 30 of these functions were previously identified in pathogen IVET screens Function in persistence / survival rather than virulence
10 GI-tract induced gene expression in L. plantarum WCFS1 72 ivi genes identified 54 functionally annotated 100 Validation qrt-pcr duodenum jejunum ileum colon Competitive persistence analysis ivi mutants vs wild-type C argg 1 pts14c 2 copa 3 lp_ lp_ Some L. plantarum ivi genes represent critical functions for persistence / survival in the GI-tract days
11 of Mice and Men probing L. plantarum gene activity in the human gut Consumption L. plantarum 299V Colon Surgery RNA isolation from mucosal scrapings Colon cancer patients cells of L. plantarum 299v per day for one week Study patient recovery after surgery L. plantarum among dominant adhered microbial population Collaboration: Lund University / Malmo Hospital, Sweden
12 of Mice and Men probing L. plantarum gene activity in the human gut Detection of highly expressed L. plantarum genes by DNA microarray analysis Tip of the iceberg ~ % signal detection, qrt-pcr validated ln (cy5/cy3) of 299v and spiked Reproducibility is good within patient Limited consistency between patients v Metabolic activity: sugar degradation pathways, amino acid / nucleotide biosynthesis, extracellular proteins spiked > 60 % of mouse ivi genes detected in human intestine! Parallel between mice and men
13 In situ genomics in the gastrointestinal tract Model systems of mice and men Monitoring gene activity on both sides Molecular host microbe interaction models
14 Probing host responses towards L. plantarum encounter Human in vivo perfusion + ileostomy models Germ free and conventional mouse models Human in vitro cell-line models catheter injection (10 ml/min) fluid sampling Collaboration with Jeff Gordon Gene chip Transcript profiling Affymetrix and Oligo-arrays
15 Human in vivo response towards L. plantarum encounter perfusion model, proximal SI healthy volunteers (n=8) placebo controlled 1.5 & 6 h perfusions (10 8 ml -1 ) catheter injection (10 ml/min) Biopsy taken before and after fluid sampling
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