A model of host-microbial interactions in an open mammalian ecosystem

Science

Volumn 273, Issue 5280, 1996, Pages 1380-1383

  Bry, Lynn ^a   Falk, Per G ^a   Midtvedt, Tore ^a   Gordon, Jeffrey I ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; BACTERIAL COLONIZATION; BACTEROIDES; ECOSYSTEM; GERMFREE ANIMAL; HOST; INTESTINE FLORA; MAMMAL; NONHUMAN; PRIORITY JOURNAL; RNA ANALYSIS;

EID: 0029845965     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5280.1380     Document Type: Article

References (35)

1. D.C. Savage, in The Regulatory and Protective Role of the Normal Microflora, R. Grubb, T. Midtvedt, K. E. Norin, Eds. (Macmillan, London, 1989), pp. 3-18.
2. P. L. Stark, and A. Lee, J. Med. Microbiol. 15, 189 (1982); K. E. Norin et al., Acta Ped. Scand. 74, 207 (1985).
3. P. L. Stark, and A. Lee, J. Med. Microbiol. 15, 189 (1982); K. E. Norin et al., Acta Ped. Scand. 74, 207 (1985).
4. D. C. Savage, Annu. Rev. Microbiol. 31, 107 (1977).
5. D. van der Waaij, ibid. 43, 69 (1989).
6. J. I. Gordon and M. L. Hermiston, Curr. Opin. Cell Biol. 6, 795 (1994).
7. T. Midtvedt, Microecol. Ther. 16, 121 (1986).
8. P. Falk, K. A. Roth, J. I. Gordon, Am. J. Physiol. 266, G987 (1994); L. Bry, P. G. Falk, J. I. Gordon, Proc. Natl. Acad. Sci. U.S.A. 93, 1161 (1996) . These references describe the lectins and protocols used for immunohistochemical surveys of the intestine.
9. P. Falk, K. A. Roth, J. I. Gordon, Am. J. Physiol. 266, G987 (1994); L. Bry, P. G. Falk, J. I. Gordon, Proc. Natl. Acad. Sci. U.S.A. 93, 1161 (1996) . These references describe the lectins and protocols used for immunohistochemical surveys of the intestine.
10. Germ-free NMRI/KI mice (9) were housed in gnotobiotic isolators (10) under a strict 12-hour light cycle and fed an autoclaved chow diet (Lactamin, Vadstena, Sweden). Weekly fecal samples were checked for the presence of organisms by Gram stain and by incubation in thioglycollate medium under aerobic and anaerobic conditions. CONV mice were housed under the same conditions in a specified pathogen-free environment. GF animals were removed from the isolators and crossed to CONV mice every second generation.
11. T Granholm et al., Cytokine 4, 545 (1992).
12. B.E. Gustafsson, Ann. N. Y. Acad. Sci. 78, 17 (1959).
13. A. Varki, Glycobiology 3, 97 (1993).
14. D. Lenoir et al., Biochim. Biophys. Acta 1234, 29 (1995); Y. Umesaki et al., Microbiol. Immunol. 39, 555 (1995); A. Pusztai et al., Glyconj. J. 12, 22 (1995); M. C. Biol, A. Martin, P. Louisot, Biochemie 14, 13 (1994).
15. D. Lenoir et al., Biochim. Biophys. Acta 1234, 29 (1995); Y. Umesaki et al., Microbiol. Immunol. 39, 555 (1995); A. Pusztai et al., Glyconj. J. 12, 22 (1995); M. C. Biol, A. Martin, P. Louisot, Biochemie 14, 13 (1994).
16. D. Lenoir et al., Biochim. Biophys. Acta 1234, 29 (1995); Y. Umesaki et al., Microbiol. Immunol. 39, 555 (1995); A. Pusztai et al., Glyconj. J. 12, 22 (1995); M. C. Biol, A. Martin, P. Louisot, Biochemie 14, 13 (1994).
17. D. Lenoir et al., Biochim. Biophys. Acta 1234, 29 (1995); Y. Umesaki et al., Microbiol. Immunol. 39, 555 (1995); A. Pusztai et al., Glyconj. J. 12, 22 (1995); M. C. Biol, A. Martin, P. Louisot, Biochemie 14, 13 (1994).
18. A. A. Salyers and M. Pajeau, Appl. Environ. Microbiol. 55, 2572 (1989).
19. S. J. Hultgren et al., Cell 73, 887 (1993).
20. GF and CONV mice were killed between 1600 and 1800 hours at P1, 5, 7, 10, 14, 17, 21, 23, 25, 28, 42, and 90 (n = 4 to 10 mice per time point per experiment; n = 3 independent experiments).
21. H. Cheng, Am. J. Anat. 141, 521 (1974); M. E. Selsted et al., J. Cell Biol. 118, 929 (1992); L. Bry et al., Proc. Natl. Acad. Sci. U.S.A. 91, 10335 (1994).
22. H. Cheng, Am. J. Anat. 141, 521 (1974); M. E. Selsted et al., J. Cell Biol. 118, 929 (1992); L. Bry et al., Proc. Natl. Acad. Sci. U.S.A. 91, 10335 (1994).
23. H. Cheng, Am. J. Anat. 141, 521 (1974); M. E. Selsted et al., J. Cell Biol. 118, 929 (1992); L. Bry et al., Proc. Natl. Acad. Sci. U.S.A. 91, 10335 (1994).
24. Surveys of wholemounts [M. L. Hermiston and J. I. Gordon, J. Cell Biol. 129, 489 (1995)] and sections indicated that from P1 to P17, the lineage-specific, crypt-villus. and duodenal-ileal patterns of Ulex europaeus type 1 agglutinin (UEA1), Anguilla anguilla agglutinin (AAA), Aleuria aurantia (orange peel) agglutinin (OPA), and H type 2 monoclonal antibody (mAb 92FRA2, DAKO) binding were indistinguishable in GF and CONV small intestine.
25. Sections prepared along the duodenal-colonic axis of P28 to P90 CONV and GF NMRI/KI gut were stained with UEA1 (specificity: Fucα1,2Galβ). OPA [Fucα1,6/3N-acetylglucosamine (GlcNAc)], AAA (α-L-fucose), the H2 mAb (Fucα1,2Galβ1,4GlcNAc), Arachis hypogae agglutinin (Galβ1,3GalNAc), Dolichos biflorus agglutinin (GalNAcα1,3Gal), Maakia amurensis agglutinin (NeuAcα2.3Gal), and Griffonia simplicifolia type II agglutinin (GlcNAcα1,3Gal/Glc). Only the fucose-specific markers showed differences in binding. These differences were limited to the small intestinal epithelium.
26. The intact cecum and colon from CONV P42 mice were homogenized in 10 ml of sterile 0.9% NaCl. Samples (0.5 ml) were spread on the fur and inoculated into the mouth and rectum of P28 or P70 GF animals (n = 5 mice per time point per experiment). XGF mice were housed with CONV animals.
27. Three samples of luminal contents, recovered from duodenum, jejunum, ileum, and cecum of each animal, were inoculated individually into thioglycollate medium. Serial dilutions were made from this inoculum, samples plated onto blood agar, and CFU counted after a 3-day incubation at 37°C under anaerobic conditions.
28. Total cellular RNA was extracted with RNAzol B (Tel-Test, Houston, TX). RT-PCR reactions were carried out with 250 ng of RNA, and primers designed from a mouse α1,2-FT cDNA [P50 (5′-GCGAATATGCCACGCTGTTTGC-3′ and 5′-GCACGGGTATCCTGTGAAGCGC-3′)], under conditions suggested by the manufacturer of the GeneAmp Thermostable rTth kit (Perkin-Elmer). Actin primers were used as internal controls to detect actin mRNA and to exclude any genomic DNA contamination [5′-TGGAATCCTGTGGCATCCATGAAAC-3′ and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′; actin mRNA, 320-base pair (bp) product; actin gene. 470-bp product].
29. K. Shindo and K. Fukushima, Gastroenterol. Jap. 11, 167 (1976); T. Ushijima et al., Microbiol. lmmunol. 27, 985 (1983).
30. K. Shindo and K. Fukushima, Gastroenterol. Jap. 11, 167 (1976); T. Ushijima et al., Microbiol. lmmunol. 27, 985 (1983).
31. R) (13).
32. 2 = 10) - reject hypothesis (ii).
33. Bacteria were labeled with FITC as described (26) with the following modification: cecal contents were washed five times in 0.2 M sodium carbonate buffer (pH 9) and allowed to settle for 5 min between washes. The supernatant was taken for labeling. Methods for incubating Bouin's- or formalin-fixed or frozen sections of intestinal Swiss rolls (17) with labeled bacteria for in situ binding assays are described in (26).
34. P. Falk, T. Borén, D. Haslam, M. Caparon, Methods Cell Biol. 45, 165 (1994).
35. We thank J. Lowe (University of Michigan) for a mouse α1,2-FT cDNA (P50), A. Salyers (University of Illinois, Urbana) for B. thetaiotaomicron strains, and J. Bergstedt and A.-K. Persson for technical assistance. Supported by grants from the National Institutes of Health (DK30292 and 37960), Swedish Can cer Society (3523-B95-02XBB), and Medical Research Council (B96-16X-11595-01).