Symbiotic bacteria in animals

1. Symbiotic bacteria in animals • Oct 3 2006 • Nancy Moran • Professor, Ecology and Evolutionary Biology Reading: The gut flora as a forgotten organ by A. O’Hara and F Shanahan EMBO Reports. 2006

2. What is symbiosis? • Term typically used for a chronic association of members of more than one genetic lineage, without overt pathogenesis • Often for mutual benefit, which may be easy or difficult to observe • Exchange of nutrients or other metabolic products, protection, transport, structural integrity

3. Microbes in animal evolution • Bacteria present by 3.9 bya, Archaea and Eukaryota by >2 bya • The Earth is populated by ecologically diverse microbes • Animals appear about 1 bya • Animals evolved in microbial soup • “Innate” immune system probably universal among animal phyla: pathogenic infection was a constant selection pressure • But animals also evolved codependence on microbes, some of which are required for normal development and reproduction

4. evolutionary innovations through symbiosis: examples • Eukaryotic cell (mitochondria) • Photosynthesis in eukaryotes (plastids) • Colonization of land by plants (mycorrhizae) • Nitrogen fixation by plants (rhizobia) • Animal life at deep sea vents (chemoautotrophic life systems) • Use of many nutrient-limited niches by animal lineages

5. Why do hosts and symbionts cooperate so often? • Persistent association allows both to increase their persistence and replication. • Coinheritance • Long-term infection • Intimate metabolic exchange generating immediate beneficial feedback

6. Symbiosis- main variables • Route of infection (maternal, horizontal, mixture) • Mechanisms of benefiting or exploiting hosts • Location of symbionts in host body: • intracellular, between cells, in specialized organ or in other tissues, within gut lumen, etc. • Molecular mechanisms of invading host tissues or cells: similarities and differences between symbionts and pathogens

7. Termite with gut removed Diverse microbes in termite gut Routes of transmission • Vertical (parent to offspring) • Horizontal • May live in the environment (outside hosts), or not • Mixture of vertical and horizontal • Eg acquire from other individuals in the same family or colony (termites, humans… )

8. Vertical transmission (parent to offspring) • Infection of eggs, seeds, embryos, or babies • Usually maternal only • Has evolved in many invertebrate symbioses with bacteria, viruses and fungi • Can be transovariolar (within the mother’s body) or some other route (e.g. fecal-oral for gut inhabitants)

9. Ways that vertically transmitted microbes can increase in frequency • Increase host survival & reproduction (mutualism) • “Reproductive manipulation” • Turn presumptive male hosts into females • Cause all-female progeny so that all offspring are carriers (“son-killers”) • Cause hosts to be parthenogenetic (all female) • Cytoplasmic incompatibility: infected males sterilize uninfected females • All of these are known to occur--caused by bacterial symbionts in insects: “Wolbachia” and spiroplasmas

10. Ways that vertically transmitted microbes can increase in frequency • Increase host survival & reproduction (mutualism) • Very common Why might vertical transmission be associated with mutualistic effects on hosts? • Most famous cases are the lineages leading to organelles • Mitochondria evolved from the alpha-Proteobacteria about 2 billion years ago • Chloroplasts evolved from cyanobacteria about 1 billion years ago

11. Vertically transmitted symbiont can ultimately fuse with the host to form a “super-organism” --mutually obligate relationship --very unlike pathogens Eukaryotic genomes are littered with hundreds of genes from mitochondria and plastids--now apparent from plant and animal genome sequences.

12. (Phylogenetic evidence for gene transfer from organelles) Cyanobacteria Cyanobacteria Eukaryote- Plant Cyanobacteria Bacteria Bacteria Bacteria Bacteria Eukaryote-protozoan Eukaryote-protozoan Eukaryote-animal Eukaryote-fungal e.g. Arabidopsis genome has >1000 genes from cyanobacteria

13. Vertically transmitted bacteria in animal hosts--2 examples • Insect-nutritional mutualists (aphids and Buchnera) • Symbionts providing defense against natural enemies of hosts

14. Beneficial microbes in animal hosts--examples • Insect-nutritional mutualists (aphids & Buchnera) Many invertebrates have specialized intracellular associations with bacteria that make nutrients Examples: marine bivalves, leeches, many insects

15. Metazoa: ancestral loss of many genes underlying biosynthesis of compounds essential for metabolism, including many amino acids and many cofactors. -->dietary requirements. Little or no gene uptake Tree of Life, N. Pace

16. Aphids-Buchnera • Intracellular bacteria in specialized host cells • Vertically transmitted-mother to offspring • Infection dates to >100 million years • Rather closely related to E. coli, but genome much reduced (only 600 of ~4000 ancestral genes retained) • Provides nutrients to host, allowing use of a diet that otherwise would be inadequate.

17. maternal bacteriocytes containing symbionts late embryos early embryos with symbionts visible 1 mm J. Sandström

18. Buchnera aphidicola within pea aphid bacteriocyte 1mm J. White

19. Aphid eggs containing Buchnera from mother 0.5 mm A. Mira

20. ->Strict vertical transmission since ancient infection of ancestral host

21. % of total amino acids in phloem sap of 6 angiosperms ALA ASN ASP GLN GLU GLY PRO SER ARG HIS ILE LEU LYS MET CYS PHE TYR THR TRP VAL Essential nutrients for animals Aphid stylet sheaths in phloem sieve tubes Schizaphis graminum on barley

22. trpE trpG trpG trpE trpE trpG trpG trpE trp plasmid in Buchnera (Schizaphis graminum) = genomic adaptation to make more nutrients for hosts ori chorismate trpEG plasmid 14.3 kb ori anthranilate synthase ori ori anthranilate tryptophan chromosome trpD trpA trpC(F) trpB Lai, Baumann & Baumann PNAS 1994

23. The Buchnera gene set (570 genes) is a subset of that of E. coli (~4500 genes) Shigenobu et al 2000 Nature

24. Nonessential amino acid biosynthetic pathways tyrA tyrA hisC Chorisimate ---> ---> ---> TYR proB proA proC Glutamate ---> ---> ---> PRO serA serC serB 3-Phosphoglycerate --->---> ---> SER glyA Serine---> GLY cysE cysK Serine---> ---> CYS gtBD/gdhA 2-oxoglutarate ---> GLU glnA Glutamate ---> GLN aspC+tyrB Oxaloacetate ---> ASP asnB/asnA Aspartate ---> ASN alaB/avtA Pyruvate ---> ALA Essential amino acid biosynthetic pathways argA argB argC argD argE carAB argF argG argH Glutamate---> ---> ---> ---> ---> Ornithine ---> ---> ---> ---> ARG ilvHI ilvC ilvD ilvE Pyruvate ---> ---> ---> ---> VAL ilvA ilvHI ilvC ilvD ilvE Threonine ---> a-Ketobutyrate ---> ---> ---> --->ILE + Pyruvate ilvHI ilvC ilvD leuA leuCD leuB ilvE Pyruvate ---> ---> ---> ---> ---> ---> --->LEU aroH aroB aroD aroE aroK aroA aroC PEP+Erythrose ---> ---> ---> ---> ---> ---> ---> Chorismate 4-Phosphate pheA pheA hisC Chorismate ---> ---> ---> PHE trpEG trpD trpC trpC trpAB Chorismate ---> ---> ---> ---> ---> TRP thrA asd thrA thrB thrC Aspartate ---> ---> ---> Homoserine ---> ---> THR metB metC metE Homoserine ---> ---> ---> MET thrA asd dapA dapB dapD dapC dapE dapF lysA Aspartate ---> ---> ---> ---> ---> ---> ---> ---> ---> LYS hisG hisI hisA hisHF hisB hisC hisB hisD PRPP + ATP ---> ---> ---> ---> ---> ---> ---> ---> HIS GENE / product present in Buchnera GENE / product absent in Buchnera (based on Shigenobu et al 2000)

25. But other symbionts appear not to have not left a legacy of many genes transferred to host genomes, at least not in animals so far sequenced (e.g., Drosophila) Eukaryotic genomes contain many genes from organelles, apparent from eukaryotic genome sequences. Why this difference?

26. Heritable mutualistic bacteria (maternal transmission) • Mitochondria • Chloroplasts • Obligate “nutritional” symbionts (e.g. Buchnera in aphids) • Facultative maternally transmitted symbionts Not much like pathogens-host has taken over mechanisms of invading host cells and has coevolved to maintain the association Much more like pathogens--have to invade naïve hosts, overcome immune responses, but typically benefit hosts

27. Similarities between facultative symbionts and pathogens at the molecular level • Use of toxins that target eukaryotic cells and manipulate the cell cycle • Use of secretion systems that deliver effector molecules to the host cytoplasm, sometimes enable host cell invasion • Eg Type III Secretion Systems used by Salmonella and Yersinia pestis (mammalian pathogens) and by mutualistic symbionts of animals and plants • Similar trends in genome evolution: proliferation of insertion sequences (transposable elements) and inactivation of many ancestral genes

28. Experiments comparing pea aphids with the same genotype but differing in presence of secondary symbionts: lines established by microinjection and inherited in all descendants • Heat tolerance (Chen & Purcell 1997, Montllor et al. 2002, J. Russell & N. Moran 2006) • Defense against wasp parasitoids (K. Oliver et al. 2003) • Mutualistic effects of facultative symbionts on aphids

29. Hamiltonella defensa confers protection against parasitoid wasps Kill developing parasite larva within aphid body Increases aphid survival & reproduction Oliver, et al. PNAS 2003 & 2005

30. Other cases of vertically transmitted symbionts providing defense: Polyketides produced by symbionts of beetles • Many drug candidates from marine and terrestrial invertebrates are suspected metabolites of uncultured bacterial symbionts. • Polyketides used as anti-tumor drugs

31. Symbionts providing defense: Polyketides produced by symbionts of beetles and sponges Biosynthesis is encoded in a 75kb acquired chromosome fragment Used as anti-tumor drugs J Piel 2002 PNAS 99: 14002

32. Why are vertically transmitted symbionts rare in vertebrates? • Other animal phyla studied have maternally transmitted symbionts, often originating hundreds of times (eg arthropods, molluscs) • Acquired immunity system prohibits this type of symbiosis? • Vertebrates typically have very large numbers of bacterial taxa associated with surfaces and gut

33. Horizontally transmitted or “environmentally acquired” symbionts • Common and often clearly mutualistic • Examples: • squid and Vibrio fischeri: symbionts reacquired every day from seawater, special signalling system for recognizing the right bacteria • Termite gut microbes • Mammalian gut microbes • Mouth-in habiting bacteria

34. Commensal bacteria in mammalian guts- Case of humans In a person, bacterial cells outnumber somatic and germ cells by >10 fold Human intestinal microbiota: 500-1,000 different species, aggregate biomass of ~ 1.5 kg per person Number of genes in the human ‘microbiome’ may exceed number of human genes by 100-fold Xu & Gordon, PNAS, 2003

35. Recent research on the human gut microbiota Summarized in A. O’Hara and F. Shanahan, “The gut flora as a forgotten organ”

36. Bacteria in mammalian gut • Infected during birth • Big change in community at weaning, from mostly aerobes to mostly anaerobes • Differences between individuals that reinstate themselves following antibiotic treatment • Some common bacterial types across individuals • Some species with specialized communities

37. Digestive tract of a cow

38. Symbiotic bacteria in mammalian guts- Bacteroides thetaiotaomicron in Mouse JI Gordon lab (Washington University) Normally infection of the gut occurs at birth Gnotobiotic = germ-free from birth Infection of gnotobiotic mice with single strain of B. thetaiotaomicron(LV Hooper et al 2001 Science) Infection had major effects on expression of >100 mouse genes including genes modulating fundamental intestinal functions, some of these are affected similarly in zebra fish Major effects on development of intestine, vascularization

39. Commensal bacteria in mammalian guts- Bacteroides thetaiotaomicron DEVELOPMENT induction of capillary networks in intestine, etc. NUTRITION Absorption and processing of carbohydrates & lipids: germ-free mice require ~30% more calories IMMUNITY AND DEFENSE Neutralization of dietary toxins Mucosal barrier protects against infectious microbes Bacterial surface molecules affect immune system functioning and development

40. Intestinal vascularization of gut is dependent on presence of bacteria Germ-free conventional B. thetaiotamicron only

41. Commensal bacteria in mammalian guts- Bacteroides thetaiotaomicron genome Gene content of the bacterium reflects its nutritional role esp in carbohydrate metabolism 172 glycosylhydrolases for breaking down carbohydratess into easily absorbed sugars, many of these are secreted from bacterial cells) Clear capacity for continued gene turnover and acquisition of new DNA and genes (phage, etc. ). Symbionts, particularly consortia of commensal bacteria, can be a means of acquiring novel metabolic functions in eukaryotes

42. Undigested carbohydrate polymers bind to surface of Bt Much of Bt genome is devoted to making binding proteins plus surface-localized glycohydrolases that liberate simple sugars from the carbohydrates. Sugars available to be used by: host, Bt, other bacteria

43. B. thetaiotamicron upregulates a large set of its genes upon colonization of the mouse intestine 64 enzymes for digesting polysaccharides in dietary fiber Xylan, pectin, arabinose degrading enzymes. Many of these are secreted by the bacteria. Expression (transcription) is affected by mouse diet. Shows adaptation to the gut-bound lifestyle. Host mucous provides an endogenous source of glycans used by Bt when dietary supply is low. Bt embed in the mucosal layer (next slide)

44. Scanning electron microscope images showing distribution of B. thetaiotaomicron within its intestinal habitat. (A) Low-power view of the distal small intestine of B. thetaiotaomicron– monoassociated gnotobiotic mice, showing a villus (arrow) viewed from above. (B to D) Progressively higher power views showing B. thetaiotaomicron associated with luminal contents (food particles, shed mucus) [arrows in (B) and (C)] and embedded in the mucus layer overlying the epithelium [boxed region in (C), larger image in (D)]. Scale bars, 50 µm (A), 5 µm [(B) and (C)], 0.5 µm (D). Sonnenberg et al 2005 Science 307:1955

45. B. thetaiotamicron in mammalian guts • Represents an extended phenotype--uses genes for host benefit and regulates them adaptively in response to host environment (diet) • Retains capacity to acquire new genes, based on presence of integrases, phage; different strains differ in gene content.

46. Methanogens (Archaea) use hydrogen gas (generated by carb digestion) to make methane, thereby increasing efficiency of energy conversion Manipulation of microbial gut community could lower propensity for obesity?

47. Consequences of interfering with gut community? • Antibiotics-eradicate most bacteria in gut, followed by unusual progression back to original state • Gut bacteria are environmentally acquired--Overly hygienic conditions-may not develop full diversity of gut community • Association with Irritable Bowel Syndrome, Crohn’s disease • May affect development of immune system • Consequences for digestive efficiency, metabolism, tendency to fat deposition, obesity

48. Methanobrevibacter smithii (Archaea) Methanogen Determines efficiency of caloric uptake "Changes in microbial ecology prompted by Western diets, and/or differences in microbial ecology between individuals living in these societies, may function as an 'environmental' factor that affects predisposition toward energy storage and obesity.” Backhad et al. Proc Natl Acad Sci USA 2004; 101: 15718-15723