the human microbiome talk by jonathan eisen @phylogenomics for scifoo 2007
DESCRIPTION
Presentation by Jonathan Eisen on the human microbiome for SciFoo 2007TRANSCRIPT
Humans are Microbial Carrying Vessels
Some Microbial Trivia
• Microbes rule the planet• There are 100x more microbial cells in the
human body than human cells• Microbes are really small• Human health may be determined more by
microbes than environment or genes• Most microbes in/on humans are beneficial
- known as commensals
Top Known Benefits of Human Commensals
• Preventing infection by pathogens
• Development of the immune system
• Digestion of food
• Vitamin production
• Toxin degradation
• Appearance and odor
Why Study Commensals?
• Diagnostic for health status• Disturbed by antibiotics• Colonization disrupted by C-sections• Imbalances may cause disease (autoimmune, IBS,
obesity)• Probiotics and prebiotics could improve health• Likely have many as of yet unknown functions
Microbes are Very Small
• Makes them hard to study• Even when examined in microscopes -
appearance is not a reliable indicator of microbial type or biology
• Culturing allows biology to be studied in detail in the lab
• Genome sequencing of cultured species very informative
Studying the microbiome - series of eras
Studying Microbiome Era IMicroscopy
Human Commensals
• Skin• Conjunctiva• Oral cavity• Intestinal tract• Upper respiratory
tract• Urogenital tract
Table 14.1c
Studying Microbiome Era II - Culturing
Guide to the Normal Bacterial Flora of Humans
Clostridia
• Members of the genus Clostridium
• Found in the the intestinal tract
Clostridium difficile. Clostridia are anaerobic endospore-forming bacteria, found mainly in the large intestine.
BACTERIA COMMONLY FOUND ON THE SURFACES OF THE HUMAN BODY
BACTERIUM Skin Conjunctiva Nos e Pharynx Mouth Lower Intestine
Anterior urethra
Vagina
Staphylococcus epidermidis (1)
++ + ++ ++ ++ + ++ ++
Staphylococcus aureus* (2)
+ +/- + + + ++ +/- +
Streptococcus miti s
+ ++ +/- + +
Streptococcus salivarius
++ ++
Streptococcus mutans* (3)
+ ++
Enterococcus faecalis * (4)
+/- + ++ + +
Streptococcus pneumoniae* (5)
+/- +/- + + +/-
Streptococcus pyogenes* (6)
+/- +/- + + +/- +/-
Neisseria sp. (7) + + ++ + + +
Neisseria meningitidis * (8)
+ ++ + +
Veillonella e sp. + +/-
Enterobacteriaceae* (Escherichia col i) (9)
+/- +/- +/- + ++ + +
Proteus sp. +/- + + + + + +
BACTERIA COMMONLY FOUND ON THE SURFACES OF THE HUMAN BODY
BACTERIUM Skin Conjunctiva Nos e Pharynx Mouth Lower Intestine
Anterior urethra
Vagina
Pseudomon as aeruginosa* (10)
+/- +/- + +/-
Haemophilus influenzae* (11)
+/- + + +
Bacteroides sp.*
++ + +/-
Bifidobacterium bifidum (12)
++
Lactobacillus sp. (13)
+ ++ ++ ++
Clostridium sp.* (14)
+/- ++
Clostridium tetani (15)
+/-
Corynebacteria (16)
++ + ++ + + + + +
Mycobacteria + +/- +/- + +
Actinomycetes + +
Spirochetes + ++ ++
Mycoplasmas + + + +/- +
Once Cultured ….
• Experimental biology
• Place on a tree of life
• Genome sequencing
• Genetic engineering
alanine/glycine
Na+
alanine/glycine
Na+
alanine/glycine
Na+
proline/betaine
H+
proline/betaine
H+WD0168
WD0414
WD1046
WD1047
WD0330
Na+
glutamate/aspartate
Na+
WD0211
WD0229
glutamate/aspartate
ornithine
putrescineWD0957
H+ Na+H+ Na+
WD0316 WD0407
H+
WD1107WD1299WD1300WD1391WD0816WD0765
Mg2+
WD0375
H+ Zn2+/Cd2+
WD1042
ATPADP
Zn2+
WD0362WD0938WD0937
ATPADP
Fe3+
WD1136WD0153WD0897
glycerol-3-phosphate/hexose-6-phosphate
phosphateWD0619
H+
drugs
H+
drugs
WD0056
WD0248
H+
drugs
H+
drugs
WD1320
WD0384
H+
?
H+
?
WD0621
WD0099
H+
metabolite?
WD0470
H+
metabolite?
WD1033
H+WD0249
metabolite?
ATPADP
heme
WD0411WD1093WD0340
K+
WD1249
Na+H+
drugsATP
ADP WD0400
phosphate
ATPADP
ORF00100ORF00714ORF00927ORF00940
(2?)
H+
F-type ATPase
ATP ADP
WD1233WD0203WD0204WD0427WD0428WD0429WD0655WD0656
phosphoenolpyruvate
1,3-bisphosphoglycerate
3-phosphoglycerate
2-phosphoglycerate
pyruvate
acetyl-CoA
citrate
isocitrate
oxaloacetate
suc-CoAsuccinate
fumarate
malate
oxaloacetate
TCA CYCLE
glyceraldehyde-3P
fructose-1,6-P2
dihydroxyacetone-P
WD1238
WD0091
WD0451
WD1167
WD0868
WD0494
WD0690
WD0105
WD0791
WD1309WD0544WD0751
WD1209WD1210
WD0437WD0727WD1221WD1222
WD0492
WD1121
mannose-1P mannose-6PWD0695
MALATE WD0488 WD1177WD0416WD0473WD0751WD0325
Non-oxidative Pentose Phosphate Pathway
xylulose-5P
glyceraldehyde-3P
sedoheptulose-7P
fructose-6P
ribose-5P
ribulose-5P
glyceraldehyde-3P
WD0551WD0387
WD0387
WD0712
erythrose-4P
WD1151
glycerol-3P
WD0731
Amino Acid catabolism
GLUTAMATE glutamineWD1322
GLUTAMINE glutamateWD0535
CYSTEINE alanineWD0997
THREONINE glycineWD0617,WD0617
PROLINE glutamateWD0103
SERINE glycineWD1035
Fatty Acid Biosynthesis WD0985, WD0650, WD1083, WD1170, WD0085
PRPP
WD0036
Thiamine metabolismWD1109,WD0763,WD0029,WD0913,WD1018,WD1024
AMP,ADP,dAMP, dADP,ATP,dATP,ITP,dITP,IMP,XMP,GMP,GDP,dGDP,dGTP,dGMP
WD1142WD1305WD1023WD0786WD0867WD0337WD0786
WD0661WD1183WD0197WD0089WD0195WD0439WD0197
adenylosuccinate WD0786
Purine Metabolism
UMPUDP
WD0684WD1295WD0895WD0230WD1239WD0228WD0461
aspartate semialdehydeaspartateWD1029 WD0960 WD0954
Wu et al., PLoS Biology 2004
Limitations of Culturing
• Can’t perfectly mimic niche
• Can’t mimic communities
• Great plate count anomaly
The Uncultured Majority
• Vast majority of microbes have never been cultured (total numbers and phylogenetic diversity)
• Particularly true for endosymbionts and extremophiles
• Main questions– Who is out there?– What are they doing?– Connect who and what.
Great Plate Count Anomaly
Culturing Microscope
CountCount
Great Plate Count Anomaly
Culturing Microscope
CountCount <<<<
Great Plate Count Anomaly Solution
Culturing Microscope
CountCount <<<<
DNA
PCR Saves the Day
rRNA Phylotyping
• Extract DNA
• Run rDNA PCR
• Sequence products
• Infer evolutionary tree
rRNA and Uncultured Microbes
Eisen et al. 1992
Majority of Microbes are “Uncultured”Numbers and Diversity
Phylotyping Can Be Used to Count
Phylotyping Diversity Indices
Bohannan and Hughes 2003
Hugenholtz 2002
Zoetendal, Erwin G., Vaughan, Elaine E. & de Vos, Willem M.A microbial world within us.Molecular Microbiology ハ 59 ハ (6), ハ 1639-1650.doi: 10.1111/j.1365-2958.2006.05056.x
Rawls et al. 2006
Palmer et al 2007
Palmer et al. 2007
Mouth Diversity
GI Tract
Problems with rRNA PCR
• Doesn’t predict biology of organisms well
• Doesn’t work for viruses
• Not very quantitative
Genome-Scale Methods and Uncultured Species
• High throughput rDNA PCR (e.g., Sogin, Eisen)
• rDNA “phylochips” (e.g., Brown, Anderson)
• Virus chip (e.g., Derisi)
• Metagenomics– Large inserts (e.g., Delong)
– Environmental shotgun sequencing (Venter, Banfield, everyone doing because of power of random sampling)
• Single cell genomics
Environmental Shotgun Sequencing
shotgunshotgun
sequencesequenceWarner Brothers, Inc.Warner Brothers, Inc.
Using a rRNA anchor allowed the
identification of a new form of phototrophy:
Proteorhodopsin
Beja et al. 2000
rRNA Phylotypes
Venter et al., 2004
taxonomic content per SHOTGUN 16S
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
GS-02
GS-03
GS-04
GS-05
GS-06
GS-07
GS-08
GS-09
GS-10
GS-11
GS-12
GS-13
GS-14
GS-15
GS-16
GS-17
GS-18
GS-19
GS-20
GS-21
GS-22
GS-23
GS-25
GS-26
GS-27
GS-28
GS-29
GS-30
GS-31
GS-32
GS-33
GS-34
GS-35
GS-36
Station
Shotgun Sequencing Detects More Diversity than PCR-methods
What Next?
• Selected as new NIH Roadmap Initiative• Current state of knowledge incredibly
limited• 100s of body locations, and likely variation
across people, places, diets, ages, etc need to be surveyed
• New molecular and informatics methods needed
ABCDEFG
TUVWXYZ
Binning in More Complex Systems?
Metagenomic Challenges (Examples)
• Fragmentary data
• Sparse sampling
• Parasitizing methods from standard genome analysis
• Structure of communities unknown
• Analyses frequently cover multiple levels and multiple fields of methods