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 ShanahanEMBO Reports. 2006

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

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

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

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

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

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… )

Termite with gut removed

Diverse microbes in termite gut

•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)

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

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

Vertically transmitted symbiont can ultimately fusewith 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.

(Phylogenetic evidence for gene transfer from organelles)

CyanobacteriaCyanobacteriaEukaryote- PlantCyanobacteria BacteriaBacteriaBacteriaBacteriaEukaryote-protozoanEukaryote-protozoanEukaryote-animalEukaryote-fungal

e.g. Arabidopsis genome has >1000 genes from cyanobacteria

Vertically transmitted bacteria in animal hosts--2 examples

1 Insect-nutritional mutualists (aphids and Buchnera)

2 Symbionts providing defense against natural enemies of hosts

Beneficial microbes in animal hosts--examples

1 Insect-nutritional mutualists (aphids & Buchnera)Many invertebrates have specialized intracellular associations with bacteria that make nutrients

Examples: marine bivalves, leeches, many insects

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

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.

maternal bacteriocytes containing symbionts

early embryos with symbionts visible

late embryos

J. Sandström

1 mm

Buchnera aphidicola within pea aphid bacteriocyte

11µµmmJ. WhiteJ. White

Aphid eggs containing Aphid eggs containing BuchneraBuchnera from motherfrom mother

A. MiraA. Mira0.5 mm0.5 mm

colonizationof Asteraceae<20 Mya

ancestor ofextant aphids 100-200 Mya

Uroleucon & relatives

origin ofsymbiosis

host aphid gene phylogeny Buchnera gene phylogeny

AphididaePemphigus betae

Schlectendalia chinensis

Melaphis rhois

Chaitophorus viminalis

Mindarus kinseyi

Uroleucon sonchi

Acyrthosiphon pisum

Macrosiphum rosae

Myzus persicae

Rhopalosiphum padi

Schizaphis graminum

Rhopalosiphum maidis

Acyrthosiphon pisum

Macrosiphum rosae

Uroleucon erigeronense

Uroleucon caligatum

Uroleucon rurale

Uroleucon helianthicola

Uroleucon jaceicola

Uroleucon obscurum

Uroleucon rapunculoides

Uroleucon sonchi

Uroleucon solidaginis

Uroleucon jaceae

Uroleucon aeneum

Uroleucon rudbeckiae

Uroleucon astronomus

Uroleucon ambrosiae

-->Strict vertical transmission since ancient infection of ancestr>Strict vertical transmission since ancient infection of ancestral hostal host

Aphid stylet sheaths in phloem sieve tubesSchizaphis graminum on barley

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

broad beansbird cherrysonchusalfalfabarleybarley2wheat

ALA

ASN

ASP

GLN

GLU

GLY

PRO

SER

ARG

HIS ILE

LEU

LYS

ME

TC

YSPH

ETY

RTH

RTR

PVA

L

% of total amino acids in phloem sap of 6 angiosperms

Essential nutrients for animals

tryptophananthranilate

chorismate

trpD trpAtrpBtrpC(F)

trpEtrpG

trpE

trpG

trpE

trpG

trpE

trpG

trpEGplasmid14.3 kb

ori

anthranilatesynthase

ori

ori

ori

chromosome

trp plasmid in Buchnera (Schizaphis graminum)= genomic adaptation to make more nutrients for hosts

Lai, Baumann & Baumann PNAS 1994

The Buchnera gene set (570 genes) is a subset of that of E. coli (~4500 genes)

Shigenobu et al 2000 Nature

Nonessential amino acid biosynthetic pathways

tyrA tyrA hisCChorisimate ---> ---> ---> TYR

proB proA proCGlutamate ---> ---> ---> PRO

serA serC serB3-Phosphoglycerate ---> ---> ---> SER

glyASerine ---> GLY

cysE cysKSerine ---> ---> CYS

gtBD/gdhA2-oxoglutarate ---> GLU

glnAGlutamate ---> GLN

aspC+tyrBOxaloacetate ---> ASP

asnB/asnAAspartate ---> ASN

alaB/avtAPyruvate ---> ALA

Essential amino acid biosynthetic pathways

argA argB argC argD argE carAB argF argG argHGlutamate---> ---> ---> ---> ---> Ornithine ---> ---> ---> ---> ARG

ilvHI ilvC ilvD ilvEPyruvate ---> ---> ---> ---> VAL

ilvA ilvHI ilvC ilvD ilvEThreonine ---> a-Ketobutyrate ---> ---> ---> ---> ILE

+ Pyruvate

ilvHI ilvC ilvD leuA leuCD leuB ilvEPyruvate ---> ---> ---> ---> ---> ---> ---> LEU

aroH aroB aroD aroE aroK aroA aroCPEP+Erythrose ---> ---> ---> ---> ---> ---> ---> Chorismate4-Phosphate

pheA pheA hisCChorismate ---> ---> ---> PHE

trpEG trpD trpC trpC trpABChorismate ---> ---> ---> ---> ---> TRP

thrA asd thrA thrB thrCAspartate ---> ---> ---> Homoserine ---> ---> THR

metB metC metEHomoserine ---> ---> ---> MET

thrA asd dapA dapB dapD dapC dapE dapF lysAAspartate ---> ---> ---> ---> ---> ---> ---> ---> ---> LYS

hisG hisI hisA hisHF hisB hisC hisB hisDPRPP + ATP ---> ---> ---> ---> ---> ---> ---> ---> HIS GENE / product present in Buchnera

GENE / product absent in Buchnera(based on Shigenobu et al 2000)

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 containmany genes from organelles, apparent from eukaryotic genome sequences.

Why this difference?

Heritable mutualistic bacteria (maternal transmission)

Not much like pathogens-host has taken over mechanisms of invading host cells and has coevolved to maintain the association

• Mitochondria• Chloroplasts• Obligate “nutritional” symbionts (e.g.

Buchnera in aphids)

• Facultative maternally transmitted symbionts

Much more like pathogens--have to invade naïve hosts, overcome immune responses, but typically benefit hosts

Similarities between facultative symbiontsand pathogens at the molecular level

• Use of toxins that target eukaryotic cells and manipulate the cell cycle

• Use of secretion systems that deliver effectormolecules 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

Mutualistic effects of facultative symbionts on aphids 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 Heat tolerance (Chen & Purcell 1997, (Chen & Purcell 1997, MontllorMontllor et al. 2002, J. et al. 2002, J. Russell & N. Moran 2006)Russell & N. Moran 2006)

Defense against wasp parasitoids Defense against wasp parasitoids (K. Oliver et al. (K. Oliver et al. 2003)2003)

Hamiltonella defensa

confers protection against parasitoid wasps

Kill developing parasite larva within aphid body

Increases aphid survival & reproduction

Oliver, et al. PNAS 2003 & 2005

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

Symbionts providing defense: Polyketides produced by symbionts of beetles and sponges

Biosynthesis is encoded in a 75kbacquired chromosome fragment

Used as anti-tumor drugs

J Piel 2002 PNAS 99: 14002

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

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

CommensalCommensal bacteria in mammalian gutsbacteria in mammalian guts--Case of Case of humanshumans

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

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

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

Digestive tract of a cow

Symbiotic bacteria in mammalian guts-Bacteroides thetaiotaomicron in MouseJI Gordon lab (Washington University)

Normally infection of the gut occurs at birthGnotobiotic = 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

Commensal bacteria in mammalian guts-Bacteroides thetaiotaomicron

DEVELOPMENTinduction of capillary networks in intestine, etc.

NUTRITIONAbsorption and processing of carbohydrates & lipids: germ-free mice require ~30% more calories

IMMUNITY AND DEFENSENeutralization of dietary toxinsMucosal barrier protects against infectious microbesBacterial surface molecules affect immune system functioning

and development

Intestinal vascularization of gut is dependent on presence of bacteria

Germ-free conventional B. thetaiotamicron only

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. ).

SymbiontsSymbionts, particularly consortia of , particularly consortia of commensalcommensal bacteria, can be a bacteria, can be a means of acquiring novel metabolic functions in eukaryotesmeans of acquiring novel metabolic functions in eukaryotes

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

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)

Sonnenberg et al 2005 Science 307:1955

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).

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.

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?

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

Methanobrevibacter smithii(Archaea)

MethanogenDetermines 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