Adaptive Evolution in Microbial Genomes · Type 1 Fimbrial Adhesin of E. coli oropharynx stomach large intestine urinary tract * NON-PATHOGENIC (COMMENSAL) HABITAT PATHOGENIC (VIRULENCE)

Adaptive Evolution

in

Microbial Genomes

Sujay Chattopadhyay Department of Microbiology

University of Washington

Seattle WA

Gene

acquisition

NON-PATHOGENIC (COMMENSAL) HABITAT

PATHOGENIC (VIRULENCE) HABITAT

Gene

mutation

Gene

loss

low

fit

Mechanisms of Virulence Evolution

Patho-adaptive mutation *

*

*

[Welch et al. PNAS, 2002]

40.2% 7.8%

2.6%

21.8%

2.7%

18.0%

6.9%

MG1655 (K-12)

non-pathogenic

EDL933

enterohemorrhagic

CFT073

uropathogenic

Mosaic vs. Common Genes

3 Escherichia coli genomes

fimH – A Common Gene Encoding

Type 1 Fimbrial Adhesin of E. coli

oropharynx

stomach

large intestine

urinary tract

*

NON-PATHOGENIC

(COMMENSAL) HABITAT

PATHOGENIC

(VIRULENCE) HABITAT

- type 1 fimbriae are critical for colonization

FimH

*

*

g259a (G87R)

fimH

from a uropathogenic

E.coli

fimH

from a commensal

E.coli

Patho-adaptive Mutations in

E. coli FimH Adhesin

Weissman et al. Infect Immun, 2007

FimH Protein Tree

R187H

G160R

G87S

V4G

G87R

G87C

A48V

T6N

V4G

V184I:

V251A

T6P

S99N

V4E

T6Y

V184A

A48V

1 amino acid change

T95I A127V

Q290K

A48V

A149M

R248H G87C

G216F

A263V

A140V

A10I:

V77A

V4F G294A

N6T T6N

A279C G83A

A127T

R3Q:

A127V

A48T

G87R

T95I:

V184A

T95A

N91S

Sokurenko et al. Mol Biol Evol, 2004

I. Detecting adaptive evolution via

mutations in coding genes

synonymous

(or silent)

change

non-synonymous

(or amino acid)

change

Two Types of Gene Mutation

*

*

Comparing Rates of Gene Mutation

(dN/dS)

synonymous

(or silent)

change

non-synonymous

(or amino acid)

change

* Rate of synonymous change

(dS)

Rate of non-synonymous change

(dN)

dN/dS

<1 – Purifying Selection (against amino acid changes)

>1 – Positive Selection (for amino acid changes)

)3

41ln(

4

3Sp)

3

41ln(

4

3Np

[where pN and pS denote proportions of non-synonymous and synonymous changes]

= =

dN/dS Shows Purifying Selection in fimH

R187H

G160R

G87S

V4G

G87R

G87C

A48V

T6N

V4G

V184I:

V251A

T6P

S99N

V4E

T6Y

V184A

A48V

1 amino acid change

T95I A127V

Q290K

A48V

A149M

R248H G87C

G216F

A263V

A140V

A10I:

V77A

V4F G294A

N6T T6N

A279C G83A

A127T

R3Q:

A127V

A48T

G87R

T95I:

V184A

T95A

N91S

dN/dS = 0.08


Need for a

more sensitive approach

than dN/dS

Detecting

convergent evolution

at molecular level

Morphological Convergence

VERTEBRATES

TETRAPODS

AMNIOTES

Fish Turtles

Amphibians

Snakes and

Lizards

Crocodiles

and Birds

Mammals

develop fins

develop flippers

to adapt to the aquatic habitat

Ala 27 Val

* Ala 27 Val

Adaptive Mutations Tend to Repeat

non-synonymous

(or amino acid)

change

synonymous

(or silent)

change

non-synonymous

(or amino acid)

change

*

Ala 27 Thr

Ala 27 Val

Ala 27 Val

repeated phylogenetically-unlinked mutations

in the same amino acid position

Convergent Evolution via Mutations

Hotspot Mutations

G87S

G87S

G87C

A48V

A48V

1 amino acid change

A127V

A48V

G87C

A127T

A127V

A48T

G87S

Convergent Evolution of FimH


Chattopadhyay et al. J Mol Evol, 2007

Adaptive evolution of FimH

occurs via accumulation of

hotspot mutations

What about other genes

in the genome?

Genome-level study

Detect genes undergoing

adaptive convergent evolution

via HOTSPOT mutations

using TimeZone software [Chattopadhyay et al. Nature Protocols, 2013]

LT2 D23580

14028S

SL476

SL254

RKS4594

SPB7

ATCC 9150

AKU_12601

SC-B67

SL483

P125109

287/91 CT_02021853

CVM19633

CT18 Ty2

Paratyphi C

Typhimurium

Heidelberg

Newport

Choleraesuis Paratyphi B

Agona

Paratyphi A

Enteritidis Gallinarum Dublin

Schwarzengrund

Typhi

serovar

10 nucleotide changes

Phylogenetic tree of concatenated sequences of 7 housekeeping genes

used in multilocus sequence typing (MLST)

17 Salmonella enterica subsp. I Genomes

Shigella

EAEC (Enteroaggregative) ExPEC (Extraintestinal)

EHEC (Enterohemorrhagic)

ExPEC (Extraintestinal)

Fecal

Fecal

ETEC (Enterotoxigenic)

EAEC (Enteroaggregative)

Fecal


EPEC (Enteropathogenic)

Environmental

042

UMN026

EDL933

Sd197

Sb227

Ss046

Sf5str8401

SE11

55989

E24377A

IAI1

ATCC 8739

HS

MG1655

S88

UTI89

IAI39

536

CFT073

ED1a

E2348/69

SECEC SMS-3-5

pathotype

21 Escherichia coli Genomes


Phylogenetic tree of concatenated sequences of 7 housekeeping genes

used in multilocus sequence typing (MLST)

1488

common

5370 genes

E. coli

2797

common

4450 genes

S. enterica ss. I

Common Genes Analyzed

Based on >95% sequence-identity and >95% length-coverage

0

10

20

30

40

50

S.enterica E. coli

High Frequency of Genes with Hotspot Changes

% of genes

with hotspots

Ala 27 Val

Ala 27 Val

Ala 27 Thr

COINCIDENTAL

(same position,

different mutations)

PARALLEL

(same position,

same mutations)

% of genes

with hotspots

Predominance of Parallel Hotspot Changes

0

10

20

30

40

50

S. enterica E. coli

all hotspots

parallel

coincidentalAla 27 Val

Ala 27 Val

Ala 27 Thr

COINCIDENTAL

(same position,

different mutations)

PARALLEL

(same position,

same mutations)

Parallel Hotspots

Can Result from Recombination

These changes would appear as

hotspots in phylogenetic tree

Recombination Detection Analysis

Method P-value calculation References

Pairwise

Homoplasy Index

permutation Bruen et al.,

2006

Maximum 2

(MAXCHI)

2, permutation Maynard Smith,

1992

Neighbor Similarity

Score (NSS)

Contingency test for clustering

in compatibility matrix

Jacobsen &

Easteal, 1996

(in collaboration with Vladimir Minin, Dept. of Statistics, UW)

0

10

20

30

40

50

S. enterica E. coli S. enterica E. coli

Frequency of Hotspots Remains High

in Non-recombinant Genes

% of genes

with hotspots

all

genes

non-recombinant

genes

[Chattopadhyay et al. PNAS, 2009; J Bacteriol, 2012]

0

10

20

30

40

50

S. enterica E. coli S. enterica E. coli

all hotspots

parallel

coincidental

% of genes

with hotspots

all

genes non-recombinant

genes

Parallel Hotspots Predominant

Also in Non-recombinants


Can neutral (random) mutations

explain:

overall high frequency of

hotspot mutations?

predominance of parallel

hotspots?

S. enterica E. coli

non-recombinant

genes

Simulation of Mutations under Neutrality

using EvolveAGene3 software [Hall BG, Mol Biol Evol, 2008]

One allele from real dataset

as reference

Generating new alleles under the constraints of:

1. gene-length identical to real dataset

2. Number of alleles identical to real dataset

3. Equal probability of mutations in generating new alleles

4. No indels or stop codon

5. Branch-lengths of simulated phylogenetic tree ranging within

the limits of average branch-length of the real dataset tree

10 iterations to yield

10 simulated datasets

for each gene

0

5

10

15

20

real simulated real simulated

parallel

coincidental

% of genes

with hotspots

S. enterica E. coli

In simulated sequences:

Hotspot frequency significantly lower

Coincidental hotspots more frequent than parallel ones


Hotspot mutations are

results of adaptive evolution



II. Assessing functional bias of proteins

with adaptive mutations

23% Categories Enriched

for Hotspot Mutations in S. enterica ss. I

18

enriched

categories

60

non-enriched

categories

Enriched Functional Categories in S. enterica ss. I

Two-component

system

Amino acid

biosynthesis

DNA

repair

Purine

metabolism

Vitamin

metabolic

process

RNA

modification

Ubiquinone & other

terpenoid-quinone

biosynthesis

Homologous

recombinantion Carbohydrate

biosynthesis

Transition

metal

ion

transport

Bacterial

secretion

system

Pentose

phosphate

pathway

Glycolysis/

gluconeogenesis

Propanoate

metabolism

Pyrimidine

metabolism

Citrate (/TCA)

cycle

Nicotinate &

nicotinamide

metabolism

Bacterial

chemotaxis

18

enriched

categories

13

enriched

categories 35

non-enriched

categories

27% Categories Enriched

for Hotspot Mutations in E. coli

Two-

component

system

Amino acid

biosynthesis

DNA

repair

Purine

metabolism

Vitamin

metabolic

process

RNA

modification Ubiquinone & other

terpenoid-quinone

biosynthesis

Homologous

recombinantion

Pyruvate

metabolism

Lipid

synthesis

Cofactor

biosynthesis

Cellular

respiration

Carbohydrate

biosynthesis

Enriched Functional Categories in E. coli

13

enriched

categories

S. enterica ss. I E. coli

Common Enriched Functional Categories

significant overlap (P<0.001)

[Chattopadhyay et al. J Bacteriol, 2012]

Two-component

system

Amino acid

biosynthesis

DNA

repair

Purine

metabolism

Vitamin

metabolic

process

RNA

modification

Ubiquinone & other

terpenoid-quinone

biosynthesis

Homologous

recombinantion

Carbohydrate

biosynthesis

Gene Product Functional

Category

aer Aerotaxis sensor regulator Two component

system creB Response regulator

ruvA Holliday junction helicase subunit A DNA repair

trmA tRNA (uracil-5-)-methyltransferase RNA modification

thrC Threonine synthase Amino acid

biosynthesis

mrcA Transpeptidase of penicillin-binding protein 1a Peptidoglycan

synthesis murB UDP-N-acetylenolpyruvoylglucosamine reductase

murF D-alanine:D-alanine-adding enzyme

holB DNA polymerase III, subunit Purine metabolism

yfbB Putative metabolic enzyme Vitamin

metabolism

Gene-level Adaptive Convergence Between Species




II. Predicting functional role of adaptive

mutations

III. Identifying pathotype-specific adaptive

evolution

Shigella

EAEC (Enteroaggregative) ExPEC (Extraintestinal)

EHEC (Enterohemorrhagic)


Fecal

Fecal

ETEC (Enterotoxigenic)

EAEC (Enteroaggregative)

Fecal


EPEC (Enteropathogenic)

Environmental

042

UMN026

EDL933

Sd197

Sb227

Ss046

Sf5str8401

SE11

55989

E24377A

IAI1

ATCC 8739

HS

MG1655

S88

UTI89

IAI39

536

CFT073

ED1a

E2348/69

SECEC SMS-3-5

pathotype

Pathotypes: Shigella, ExPEC, Fecal

Highest rate of

accumulation

in Extra-intestinal

pathogens

(p<0.0001)

Rates of Accumulation of Genes with Hotspots

No. of genes with hotspot mutations

Evolutionary divergence of the strains

E. coli pathotypes

rate

of

ac

cu

mu

lati

on

of

ge

ne

s w

ith

ho

tsp

ot

mu

tati

on

s

Extra-

intestinal

Shigella

2000

0

Fecal

1000

3000

4000

5000

6000

[Chattopadhyay et al. PNAS, 2009]

E. coli require large number of convergent

mutations to adapt to extra-intestinal habitats

Systemically Invasive Serovars

LT2 D23580

14028S

SL476

SL254

RKS4594

SPB7

ATCC 9150

AKU_12601

SC-B67

SL483

P125109

287/91 CT_02021853

CVM19633

CT18 Ty2

Systemically

invasive

Paratyphi C

Typhimurium

Heidelberg

Newport


Agona

Paratyphi A


Schwarzengrund

Typhi

serovar

Systemically

invasive

Systemically

invasive

Systemically

invasive

Ch

ole

rae

su

is

Du

bli

n

Pa

raty

ph

i B

Gallin

aru

m

Typ

hi

Para

typ

hi A

Dublin 3

Paratyphi B 0 6

Gallinarum 5 1 0

Typhi 2 12 14 3

Paratyphi A 8 8 15 5 37

Paratyphi C 2 1 2 2 1 2

High Level of Hotspot-sharing

Between Typhi and Paratyphi A


Convergent Evolution

in Typhi and Paratyphi A

LT2 D23580

14028S

SL476

SL254

RKS4594

SPB7

ATCC 9150

AKU_12601

SC-B67

SL483

P125109

287/91 CT_02021853

CVM19633

CT18 Ty2

Paratyphi C

Typhimurium

Heidelberg

Newport


Agona

Paratyphi A


Schwarzengrund

Typhi

serovar

Genome Research 2007 Jan;17(1):61-8

yla

B e

ntD

yjjV

STM3549

yjjV

Genomic Distribution of Genes Sharing

Hotspot Mutations in Typhi and Paratyphi A


Convergent evolution

in Typhi and Paratyphi A

via mutations

Conclusions

Convergent molecular evolution is a major

mode of microbial adaptive evolution

Molecular convergence targets specific functional

categories and pathotypes

TimeZone offers a suite of tools to detect genes

targeted by adaptive mutations

B. bacilliformis KC583

B. bacilliformis INS (contigs)

B. clarridgeiae 73

B. grahamii as4aup

B. tribocorum CIP 105476

B. vinsonii (subsp. berkhoffii) Winnie

B. quintana RM-11

B. quintana Toulouse

B. henselae Houston-1

B. australis Aust/NH1 Kangaroo

Human

Cat

Mouse

Rat

Dog

Rhesus macaque

Human

Cat


10 Bartonella spp. Genomes

Phylogenetic tree of concatenated sequences of 4 housekeeping genes (rpoB,

groEL, ribC and gltA) used in multilocus sequence typing (MLST)

B. australis

Aust/NH1

43

112

87

38

31

22

88

1057

CORE

MOSAIC

1556

B. quintana

RM-11,

Toulouse

B. vinsonii

(subsp. berkhoffii)

Winnie

B. tribocorum

CIP 105476

B. bacilliformis

KC583

B. clarridgeiae

73

B. grahamii

as4aup

B. henselae

Houston-1

19

Pan-genomic profile of Bartonella species

Two human pathogens at two extreme ends

of the distribution of species-specific genes

Distribution of species-specific (i.e. unique) genes (in blue),

core genes with truncation mutations (in red) in Bartonella spp.

0

20

40

60

80

100

120 no. of species-specific (or unique) genes

no. of core genes with truncation mutations

Two human pathogens at two extreme ends

of the distribution of genes with truncation mutations

gene product

protein

length

(AA)

BARBAKC583_0311 Brp family immunodominant surface

antigen 551

BARBAKC583_0314 Brp family immunodominant surface

antigen 1235

BARBAKC583_0513 putative adhesin/invasin 800

BARBAKC583_0912 adhesin/hemagglutinin 1259

BARBAKC583_1109 outer membrane autotransporter 1193

BARBAKC583_1133 outer membrane autotransporter 1058

Outer membrane proteins

as sole overrepresented functional category

in Bartonella bacilliformis specific genes

gene product AA length non-syn / syn

BARBAKC583_0061 hypothetical protein 202 1 / 0

rimM (BARBAKC583_0085) 16S rRNA-processing protein 191 1 / 0

rpoH2

(BARBAKC583_0126) RNA polymerase factor sigma-32 303 1* / 0

BARBAKC583_0440 uracil-DNA glycosylase 275 1 / 0

BARBAKC583_0617 lipid A biosynthesis lauroyl acyltransferase 301 1 / 0

ribH (BARBAKC583_0633) 6,7-dimethyl-8-ribityllumazine synthase 152 0 / 1

pcs (BARBAKC583_0817) phosphatidylcholine synthase 253 1 / 0

BARBAKC583_0880 integral membrane protein 527 2 / 0

BARBAKC583_1011 acetyltransferase, GNAT family protein 185 1 / 0

flaA (BARBAKC583_1040) flagellin A 380 1 / 0



BARBAKC583_1133 outer membrane autotransporter 1058 1 / 0

BARBAKC583_1223 cell wall hydrolase family protein 284 1 / 0

BARBAKC583_1262 putative L-asparaginase 329 1 / 0

acnA (BARBAKC583_1282) aconitate hydratase 895 0 / 1

BARBAKC583_1342 ABC transporter, permease/ATP-binding protein 588 0 / 1

Polymorphic genes in B. bacilliformis

rimM (BARBAKC583_0085) 16S rRNA-processing protein 191 1 / 0

pcs (BARBAKC583_0817) phosphatidylcholine synthase 253 1 / 0

BARBAKC583_1011 acetyltransferase, GNAT family protein 185 1 / 0

gene product AA length non-syn / syn

10

nucleotide

changes

rimM

(16S rRNA-processing protein)

B. grahamii as4aup

B. tribocorum CIP 105476


B. quintana RM-11

B. quintana Toulouse

B. henselae Houston-1

B. clarridgeiae 73

B. australis Aust/NH1


B. bacilliformis INS

K11R

K11R



B. clarridgeiae 73

B. australis Aust/NH1

W85L

W85R

BARBAKC583_1011

(acetyltransferase,

GNAT family protein)

B. grahamii as4aup

B. tribocorum CIP 105476 B. quintana RM-11

B. quintana Toulouse B. bacilliformis KC583


B. clarridgeiae 73

B. australis Aust/NH1 B. henselae Houston-1


G116E

G116S

pcs

(phosphatidylcholine synthase)

Cross-species convergent mutations

Conclusions

Preliminary results indicate footprints of within-

clonal adaptive evolution in B. bacilliformis

Comparative genomics of strains within and

between Bartonella species representing strains from

diverse clonal groups, clinical outcomes and regions

of isolation can offer important clues to the

pathoadaptive significance of the genetic variations

Acknowledgments

Cal Poly State University, CA

Peter Chi

SUNY Stony Brook, NY

Daniel Dykhuizen

Univ of Minnesota, MN

James Johnson

Univ of Montana, MT

Michael Minnick

UPCH, Peru

Arturo Centurion

UW Microbiology

Evgeni Sokurenko

Sandip Paul

Veronika Tchesnokova

Dagmara Kisiela

Steve Moseley

UW Statistics

Vladimir Minin

Seattle Children’s Hospital

Scott Weissman

Documents

Adaptive Evolution in Microbial Genomes · Type 1 Fimbrial Adhesin of E. coli oropharynx stomach large intestine urinary tract * NON-PATHOGENIC (COMMENSAL) HABITAT PATHOGENIC (VIRULENCE)