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
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 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.
(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
Vertically transmitted bacteria
in animal hosts--2 examples
1
2
Insect-nutritional mutualists (aphids and
Buchnera)
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.
late embryos
maternal bacteriocytes
containing symbionts
early embryos with
symbionts visible
1 mm
J. Sandström
Buchnera aphidicola within pea aphid bacteriocyte
1mm
J. White
Aphid eggs containing
Buchnera from mother
0.5 mm
A. Mira
host aphid gene phylogeny
Buchnera gene phylogeny
Aphididae
Uroleucon & relatives
Pemphigus betae
Ac yrthosi phon pi sum
origin of
symbiosis
Sc hl ec tendalia c hinensi s
Mac ros iphum ros ae
Uroleuc on eri geronense
Mel aphis rhois
Uroleuc on caligatum
Chaitophorus viminalis
Uroleuc on rurale
Uroleuc on helianthic ol a
Mindarus ki ns eyi
Uroleuc on jaceicola
Uroleuc on sonc hi
Uroleuc on ob sc urum
Uroleuc on rapunculoides
Ac yrthosi phon pi sum
Uroleuc on sonc hi
Mac ros iphum ros ae
Myzus pers ic ae
colonization
of Asteraceae
<20 Mya
Uroleuc on solidaginis
Uroleuc on jaceae
Uroleuc on aeneum
Rhopalosi phum padi
ancestor of
extant aphids
100-200 Mya
Uroleuc on rudbeck iae
Sc hi zaphis grami num
Rhopalosi phum mai dis
Uroleuc on as tronomus
Uroleuc on ambrosi ae
->Strict vertical transmission since ancient infection of ancestral host
Aphid stylet sheaths
in phloem sieve tubes
Schizaphis graminum on barley
70.0%
60.0%
% of total amino acids in phloem
sap of 6 angiosperms
50.0%
broad beans
40.0%
bird cherry
sonchus
alfalfa
barley
30.0%
barley2
Essential nutrients for animals
20.0%
10.0%
VAL
TRP
THR
TYR
PHE
CYS
MET
LYS
LEU
ILE
HIS
ARG
SER
PRO
GLY
GLU
GLN
ASP
ASN
ALA
0.0%
wheat
trp plasmid in Buchnera (Schizaphis graminum)
= genomic adaptation to make more nutrients for hosts
trpG
chorismate
ori
trpE
trpG
anthranilate
synthase
trpEG
plasmid
14.3 kb
trpE
ori
trpE
trpG
trpE
ori
anthranilate
chromosome
trpD
ori
trpC(F)
trpG
tryptophan
trpB
trpA
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
Essential amino acid biosynthetic pathways
argA argB argC argD argE
carAB argF argG argH
Glutamate---> ---> ---> ---> ---> Ornithine ---> ---> ---> ---> ARG
ilvHI ilvC ilvD ilvE
tyrA tyrA hisC
Chorisimate ---> ---> ---> TYR
proB proA proC
Pyruvate ---> ---> ---> ---> VAL
ilvA
Nonessential amino acid biosynthetic pathways
Glutamate ---> ---> ---> PRO
serA serC serB
ilvHI ilvC ilvD ilvE
3-Phosphoglycerate ---> ---> ---> SER
Threonine ---> a-Ketobutyrate ---> ---> ---> ---> ILE
+ Pyruvate
glyA
Serine ---> GLY
ilvHI ilvC ilvD leuA leuCD leuB ilvE
Pyruvate ---> ---> ---> ---> ---> ---> ---> LEU
cysE cysK
aroH aroB aroD aroE aroK aroA aroC
PEP+Erythrose ---> ---> ---> ---> ---> ---> ---> Chorismate
4-Phosphate
Serine ---> ---> CYS
gtBD/gdhA
2-oxoglutarate ---> GLU
pheA pheA hisC
Chorismate ---> ---> ---> PHE
glnA
Glutamate ---> GLN
trpEG trpD trpC trpC trpAB
Chorismate ---> ---> ---> ---> --->
aspC+tyrB
TRP
Oxaloacetate --->
thrA asd thrA
thrB thrC
asnB/asnA
Aspartate ---> ---> ---> Homoserine ---> ---> THR
Aspartate --->
metB metC metE
Homoserine ---> ---> --->
ASP
MET
thrA asd dapA dapB dapD dapC dapE dapF lysA
ASN
alaB/avtA
Pyruvate ---> ALA
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)
Eukaryotic genomes contain
many genes from organelles,
apparent from eukaryotic
genome sequences.
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)
Why this difference?
Heritable mutualistic bacteria
(maternal transmission)
•
•
•
Mitochondria
Chloroplasts
Obligate “nutritional” symbionts (e.g.
Buchnera in aphids)
Not much like pathogens-host has
taken over mechanisms of invading
host cells and has coevolved to
maintain the association
•
Facultative maternally transmitted
symbionts
Much more like pathogens--have
to invade naïve hosts, overcome
immune responses, but typically
benefit hosts
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
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 (Chen & Purcell 1997, Montllor et al. 2002, J.
Russell & N. Moran 2006)
Defense against wasp parasitoids (K. Oliver et al.
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 75kb
acquired 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
Commensal bacteria in mammalian gutsCase 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
Recent research on the human gut microbiota
Summarized 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 gutsBacteroides 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
Commensal bacteria in mammalian gutsBacteroides 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
Intestinal vascularization of gut
is dependent on presence of bacteria
Germ-free
conventional
B. thetaiotamicron only
Commensal bacteria in mammalian gutsBacteroides 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
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)
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
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)
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