Molecular Characterization of Host-Specific Biofilm Formation in a Vertebrate Gut Symbiont

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Although vertebrates harbor bacterial communities in their gastrointestinal tract whose composition is host-specific, little is known about the mechanisms by which bacterial lineages become selected. The goal of this study was to characterize the ecological processes that mediate host-specificity of the vertebrate gut symbiont Lactobacillus reuteri, and to systematically identify the bacterial factors that are involved. Experiments with monoassociated mice revealed that the ability of L. reuteri to form epithelial biofilms in the mouse forestomach is strictly dependent on the strain's host origin. To unravel the molecular basis for this host-specific biofilm formation, we applied a combination of transcriptome analysis and comparative genomics and identified eleven genes of L. reuteri 100-23 that were predicted to play a role. We then determined expression and importance of these genes during in vivo biofilm formation in monoassociated mice. This analysis revealed that six of the genes were upregulated in vivo, and that genes encoding for proteins involved in epithelial adherence, specialized protein transport, cell aggregation, environmental sensing, and cell lysis contributed to biofilm formation. Inactivation of a serine-rich surface adhesin with a devoted transport system (the SecA2-SecY2 pathway) completely abrogated biofilm formation, indicating that initial adhesion represented the most significant step in biofilm formation, likely conferring host specificity. In summary, this study established that the epithelial selection of bacterial symbionts in the vertebrate gut can be both specific and highly efficient, resulting in biofilms that are exclusively formed by the coevolved strains, and it allowed insight into the bacterial effectors of this process.

Vyšlo v časopise: Molecular Characterization of Host-Specific Biofilm Formation in a Vertebrate Gut Symbiont. PLoS Genet 9(12): e32767. doi:10.1371/journal.pgen.1004057
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004057

Souhrn

Zdroje

1. MoranNA (2006) Symbiosis. Curr Biol 16: R866–871.

2. MandelMJ (2010) Models and approaches to dissect host-symbiont specificity. Trends Microbiol 18 : 504–511.

3. MoranNA (2007) Symbiosis as an adaptive process and source of phenotypic complexity. Proc Natl Acad Sci U S A 104 (Suppl 1) 8627–8633.

4. MoranNA, McCutcheonJP, NakabachiA (2008) Genomics and Evolution of Heritable Bacterial Symbionts. Annu Rev Genet 42 : 165–190.

5. NyholmSV, McFall-NgaiMJ (2004) The winnowing: establishing the squid-vibrio symbiosis. Nat Rev Microbiol 2 : 632–642.

6. McFall-NgaiM, Heath-HeckmanEA, GilletteAA, PeyerSM, HarvieEA (2012) The secret languages of coevolved symbioses: insights from the Euprymna scolopes-Vibrio fischeri symbiosis. Semin Immunol 24 : 3–8.

7. DaleC, MoranNA (2006) Molecular interactions between bacterial symbionts and their hosts. Cell 126 : 453–465.

8. HerreEA, KnowltonN, MuellerUG, RehnerSA (1999) The evolution of mutualisms: exploring the paths between conflict and cooperation. Trends Ecol Evol 14 : 49–53.

9. BackhedF, LeyRE, SonnenburgJL, PetersonDA, GordonJI (2005) Host-bacterial mutualism in the human intestine. Science 307 : 1915–1920.

10. WalterJ, BrittonRA, RoosS (2011) Host-microbial symbiosis in the vertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm. Proc Natl Acad Sci U S A 108 (Suppl 1) 4645–4652.

11. LeyRE, HamadyM, LozuponeC, TurnbaughPJ, RameyRR, et al. (2008) Evolution of mammals and their gut microbes. Science 320 : 1647–1651.

12. OchmanH, WorobeyM, KuoCH, NdjangoJB, PeetersM, et al. (2010) Evolutionary relationships of wild hominids recapitulated by gut microbial communities. PLoS Biol 8: e1000546.

13. MartinezI, MullerCE, WalterJ (2013) Long-term temporal analysis of the human fecal microbiota revealed a stable core of dominant bacterial species. PLoS One 8: e69621.

14. SchlossPD, SchubertAM, ZackularJP, IversonKD, YoungVB, et al. (2012) Stabilization of the murine gut microbiome following weaning. Gut Microbes 3 : 383–393.

15. OhPL, BensonAK, PetersonDA, PatilPB, MoriyamaEN, et al. (2010) Diversification of the gut symbiont Lactobacillus reuteri as a result of host-driven evolution. ISME J 4 : 377–387.

16. FreseSA, BensonAK, TannockGW, LoachDM, KimJ, et al. (2011) The Evolution of Host Specialization in the Vertebrate Gut Symbiont Lactobacillus reuteri. PLoS Genet 7: e1001314.

17. SavageDC, BlumershineRV (1974) Surface-surface associations in microbial communities populating epithelial habitats in the murine gastrointestinal ecosystem: scanning electron microscopy. Infect Immun 10 : 240–250.

18. YukiN, ShimazakiT, KushiroA, WatanabeK, UchidaK, et al. (2000) Colonization of the stratified squamous epithelium of the nonsecreting area of horse stomach by lactobacilli. Appl Environ Microbiol 66 : 5030–5034.

19. SuegaraN, MorotomiM, WatanabeT, KawalY, MutaiM (1975) Behavior of microflora in the rat stomach: adhesion of lactobacilli to the keratinized epithelial cells of the rat stomach in vitro. Infect Immun 12 : 173–179.

20. WalterJ (2008) Ecological role of lactobacilli in the gastrointestinal tract: implications for fundamental and biomedical research. Appl Environ Microbiol 74 : 4985–4996.

21. TannockGW, GhazallyS, WalterJ, LoachD, BrooksH, et al. (2005) Ecological behavior of Lactobacillus reuteri 100-23 is affected by mutation of the luxS gene. Appl Environ Microbiol 71 : 8419–8425.

22. WalterJ, LoachDM, AlqumberM, RockelC, HermannC, et al. (2007) D-alanyl ester depletion of teichoic acids in Lactobacillus reuteri 100-23 results in impaired colonization of the mouse gastrointestinal tract. Environ Microbiol 9 : 1750–1760.

23. FullerR, BarrowPA, BrookerBE (1978) Bacteria associated with the gastric epithelium of neonatal pigs. Appl Environ Microbiol 35 : 582–591.

24. SavageDC, DubosR, SchaedlerRW (1968) The gastrointestinal epithelium and its autochthonous bacterial flora. J Exp Med 127 : 67–76.

25. FullerR, BrookerBE (1974) Lactobacilli which attach to the crop epithelium of the fowl. Am J Clin Nutr 27 : 1305–1312.

26. WesneyE, TannockGW (1979) Association of rat, pig, and fowl biotypes of lactobacilli with the stomach of gnotobiotic mice . Microb Ecol 5 : 35–42.

27. BaylesKW (2007) The biological role of death and lysis in biofilm development. Nat Rev Microbiol 5 : 721–726.

28. FeltcherME, BraunsteinM (2012) Emerging themes in SecA2-mediated protein export. Nat Rev Microbiol 10 : 779–789.

29. ZhouM, WuH (2009) Glycosylation and biogenesis of a family of serine-rich bacterial adhesins. Microbiology 155 : 317–327.

30. WalterJ, ChagnaudP, TannockGW, LoachDM, Dal BelloF, et al. (2005) A high-molecular-mass surface protein (Lsp) and methionine sulfoxide reductase B (MsrB) contribute to the ecological performance of Lactobacillus reuteri in the murine gut. Appl Environ Microbiol 71 : 979–986.

31. Hall-StoodleyL, CostertonJW, StoodleyP (2004) Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2 : 95–108.

32. SonnenburgJL, AngenentLT, GordonJI (2004) Getting a grip on things: how do communities of bacterial symbionts become established in our intestine? Nat Immunol 5 : 569–573.

33. BollingerRR, BarbasAS, BushEL, LinSS, ParkerW (2007) Biofilms in the normal human large bowel: fact rather than fiction. Gut 56 : 1481–1482.

34. JohanssonME, PhillipsonM, PeterssonJ, VelcichA, HolmL, et al. (2008) The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Proc Natl Acad Sci U S A 105 : 15064–15069.

35. GohYJ, KlaenhammerTR (2010) Functional roles of aggregation-promoting-like factor in stress tolerance and adherence of Lactobacillus acidophilus NCFM. Appl Environ Microbiol 76 : 5005–5012.

36. TurnerMS, HafnerLM, WalshT, GiffardPM (2004) Identification and characterization of the novel LysM domain-containing surface protein Sep from Lactobacillus fermentum BR11 and its use as a peptide fusion partner in Lactobacillus and Lactococcus. Appl Environ Microbiol 70 : 3673–3680.

37. BuistG, SteenA, KokJ, KuipersOP (2008) LysM, a widely distributed protein motif for binding to (peptido)glycans. Mol Microbiol 68 : 838–847.

38. NovickRP, GeisingerE (2008) Quorum sensing in staphylococci. Annu Rev Genet 42 : 541–564.

39. Sharma-KuinkelBK, MannEE, AhnJS, KuechenmeisterLJ, DunmanPM, et al. (2009) The Staphylococcus aureus LytSR two-component regulatory system affects biofilm formation. J Bacteriol 191 : 4767–4775.

40. MacKenzieDA, JeffersF, ParkerML, Vibert-ValletA, BongaertsRJ, et al. (2010) Strain-specific diversity of mucus-binding proteins in the adhesion and aggregation properties of Lactobacillus reuteri. Microbiology 156 : 3368–3378.

41. SchluterJ, FosterKR (2012) The evolution of mutualism in gut microbiota via host epithelial selection. PLoS Biol 10: e1001424.

42. LeeSM, DonaldsonGP, MikulskiZ, BoyajianS, LeyK, et al. (2013) Bacterial colonization factors control specificity and stability of the gut microbiota. Nature 501 : 426–429.

43. FrankSA (1996) Host-symbiont conflict over the mixing of symbiotic lineages. Proc Biol Sci 263 : 339–344.

44. BerberovEM, ZhouY, FrancisDH, ScottMA, KachmanSD, et al. (2004) Relative importance of heat-labile enterotoxin in the causation of severe diarrheal disease in the gnotobiotic piglet model by a strain of enterotoxigenic Escherichia coli that produces multiple enterotoxins. Infect Immun 72 : 3914–3924.

45. AbramoffMD, MagalhaesPJ, RamSJ (2004) Image processing with ImageJ. Biophotonics international 11 : 36–42.

46. WalterJ, SchwabC, LoachDM, GanzleMG, TannockGW (2008) Glucosyltransferase A (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 contribute to cell aggregation, in vitro biofilm formation, and colonization of the mouse gastrointestinal tract. Microbiology 154 : 72–80.

47. GentlemanRC, CareyVJ, BatesDM, BolstadB, DettlingM, et al. (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5: R80.

48. LangmeadB, TrapnellC, PopM, SalzbergSL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10: R25.

49. FengJ, MeyerCA, WangQ, LiuJS, Shirley LiuX, et al. (2012) GFOLD: a generalized fold change for ranking differentially expressed genes from RNA-seq data. Bioinformatics 28 : 2782–2788.

50. RozenS, SkaletskyH (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132 : 365–386.

51. PfafflMW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45.

52. PerkinsDN, PappinDJ, CreasyDM, CottrellJS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20 : 3551–3567.

53. McConnellM, MercerA, TannockGW (1991) Transfer of plasmid pAMBl between members of the normal microflora inhabiting the murine digestive tract and modification of the plasmid in a Lactobacillus reuteri host. Microbial Ecol Health Dis 4 : 343–355.

54. HeavensD, TailfordLE, CrossmanL, JeffersF, MacKenzieDA, et al. (2011) Genome sequence of the vertebrate gut symbiont Lactobacillus reuteri ATCC 53608. J Bacteriol 193 : 4015–4016.

55. HammonsS, OhPL, MartinezI, ClarkK, SchlegelVL, et al. (2010) A small variation in diet influences the Lactobacillus strain composition in the crop of broiler chickens. Syst Appl Microbiol 33 : 275–281.