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20 Φεβ 2013 (πριν από 4 χρόνια και 5 μήνες)

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1.
Pathogenomics Project


2.
Cross
-
Domain Horizontal Gene
Transfer Analysis


3.
Horizontal Gene Transfer:
Identifying Pathogenicity Islands

Pathogenomics

Goal:

Identify previously unrecognized
mechanisms of microbial pathogenicity
using a combination of informatics,
evolutionary biology, microbiology and
genetics.



Explosion of data



2
6

of the 3
6

publicly available
bacterial

genome sequences are for pathogens



Approximately 2
4
,000 pathogen genes with no
known function!



~177

bacterial genome projects in progress …



Data as of June, 2001

Bacterial Pathogenicity

Processes of microbial pathogenicity at the molecular level
are still minimally understood



Pathogen proteins identified that manipulate
host cells by interacting with, or mimicking,
host proteins




Yersinia

Type III secretion system

Approach

Idea: Could we identify novel
virulence factors by identifying
bacterial pathogen genes more similar
to host genes than you would expect
based on phylogeny?

Prioritize for biological study.

-

Previously studied in the laboratory?

-

Can UBC microbiologists study it?


-

C. elegans

homolog?


Search pathogen genes against databases.
Identify those with eukaryotic similarity.

Evolutionary significance.


-

Horizontal transfer? Similar by chance?

Modify
screening
method
/algorithm

Approach

Genome
data for…

Anthrax


Necrotizing fasciitis

Cat scratch disease

Paratyphoid/enteric fever

Chancroid


Peptic ulcers and gastritis

Chlamydia


Periodontal disease

Cholera



Plague

Dental caries


Pneumonia

Diarrhea (E. coli etc.)

Salmonellosis

Diphtheria


Scarlet fever

Epidemic typhus

Shigellosis

Mediterranean fever

Strep throat

Gastroenteritis


Syphilis

Gonorrhea


Toxic shock syndrome

Legionnaires' disease

Tuberculosis

Leprosy



Tularemia

Leptospirosis


Typhoid fever

Listeriosis


Urethritis

Lyme disease


Urinary Tract Infections

Meliodosis


Whooping cough




Meningitis


+Hospital
-
acquired infections

Bacterial Pathogens

Chlamydophila psittaci


Respiratory disease, primarily in birds

Mycoplasma mycoides


Contagious bovine pleuropneumonia

Mycoplasma hyopneumoniae

Pneumonia in pigs

Pasteurella haemolytica


Cattle shipping fever

Pasteurella multicoda


Cattle septicemia, pig rhinitis

Ralstonia solanacearum


Plant bacterial wilt

Xanthomonas citri



Citrus canker

Xylella fastidiosa



Pierce’s Disease
-

grapevines


Bacterial wilt

World Research
Community

Approach

Prioritized candidates


Study function
of homolog in
model host

(
C. elegans)


Study function of
gene in
bacterium.


Infection of
mutant in model
host

C. elegans

DATABASE

Collaborations
with others

Informatics/Bioinformatics


BC Genome Sequence Centre


Centre for Molecular Medicine
and Therapeutics


Evolutionary Theory


Dept of Zoology


Dept of Botany


Canadian Institute for Advanced
Research

Pathogen Functions


Dept. Microbiology


Biotechnology Laboratory


Dept. Medicine


BC Centre for Disease Control

Host Functions


Dept. Medical Genetics


C. elegans Reverse Genetics
Facility


Dept. Biological Sciences SFU


Interdisciplinary group

Coordinator


For each complete bacterial and eukaryote genome: BLASTP
(and MSP Crunch) of all deduced proteins against non
-
redundant
SWALL database



Overlay NCBI taxonomy information


form ACEDB database



Query database for bacterial proteins who’s top scoring hit is
eukaryotic (and eukaryotic proteins who’s top hit is bacterial)



Perform similar query, but filtering different taxonomic groups
from the analysis


Development of first database:

Sequence similarity
-
based approach

BAE
-
watch Database: Bacterial proteins with unusual similarity
with Eukaryotic proteins

Problem: Proteins highly conserved in the three domains of life

Top hit to a protein
from another
domain may occur
by chance.


“StepRatio” score
helps detect these.



Example:

Glucose
-
6
-
Phosphate
Reductase

Example of a
case with a high
StepRatio:


Enoyl ACP
reductase

BAE
-
watch Database: Bacterial proteins with unusual similarity
with Eukaryotic proteins

Haemophilus influenzae

Rd
-
KW20

proteins
most strongly
matching eukaryotic proteins

PhyloBLAST


a tool for analysis

Brinkman et al. (2001)

Bioinformatics.
17:385
-
387.


Trends in this Sequence
-
based Analysis


Identifies the strongest cases of lateral gene transfer
between bacteria and eukaryotes



Most common “cross
-
domain” horizontal transfers:


Bacteria Unicellular Eukaryote



Identifies nuclear genes with potential organelle origins



A control: Method identifies all previously reported
Chlamydia trachomatis

“plant
-
like” genes.

First case: Bacterium Eukaryote Lateral Transfer

0.1

Bacillus subtilis

Escherichia coli

Salmonella typhimurium

Staphylococcua aureus

Clostridium perfringens

Clostridium difficile

Trichomonas vaginalis

Haemophilus influenzae

Acinetobacillus actinomycetemcomitans

Pasteurella multocida

N
-
acetylneuraminate
lyase (NanA) of the
protozoan
Trichomonas vaginalis

is 92
-
95% similar to
NanA of
Pasteurellaceae
bacteria.

de Koning et al. (2000) Mol. Biol. Evol.
17:1769
-
1773

N
-
acetylneuraminate lyase


role in pathogenicity?

Pasteurellaceae



Mucosal pathogens of the
respiratory tract



T. vaginalis



Mucosal pathogen, causative
agent of the STD Trichomonas



N
-
acetylneuraminate lyase (sialic acid lyase, NanA)

Involved in sialic acid
metabolism


Role in Bacteria: Proposed
to parasitize the mucous
membranes of animals for
nutritional purposes


Role in Trichomonas: ?

Hydrolysis of glycosidic
linkages of terminal sialic
residues in glycoproteins,
glycolipids


Sialidase

Free sialic acid




Transporter


Free sialic acid


NanA


N
-
acetyl
-
D
-
mannosamine


+ pyruvate

Another case: A Sensor Histidine Kinase for a

Two
-
component Regulation System

Signal Transduction


Histidine kinases common in bacteria


Ser/Thr/Tyr kinases common in eukaryotes



However, a histidine kinase was recently
identified in fungi, including pathogens
Fusarium solani

and
Candida albicans


How did it get there?


Candida

N
eurospora crassa NIK
-
1

F
usarium solani

FIK2

Streptomyces coelicolor

SC4G10.06c

Candida albicans

CaNIK
1

Escherichia coli

RcsC

Erwinia carotovora

RpfA / ExpS

Escherichia coli

BarA

Salmonella typhimurium
BarA

Pseudomonas aeruginosa

GacS

Pseudomonas
fluorescens

GacS / ApdA

Pseudomonas tolaasii

R
tp
A

/ PheN

Pseudomonas syringae

GacS / LemA

Pseudomonas viridiflava

Rep
A

Azotobacter vinelandii

GacS

0.1

Streptomyces coelicolor

SC7C7.03

Xanthomonas campestris

RpfC

Vibrio cholerae

TorS

Escherichia coli

TorS

Fusarium solani
FIK1

Fungi

Pseudomonas aeruginosa

PhoQ

100

100

51

100

100

100

100

100

100

100

100

100

100


86

54

39

100

100

Streptomyces

Histidine Kinase. The Missing Link?

virulence

factor

=

virulence factor ?

Brinkman et al. (2001)
Infection and Immunity
. In Press.

“Plant
-
like” genes in
Chlamydia



Chlamydiaceae: Obligate intracellular pathogens of humans


Proteins: Unusually high number most similar to plant
proteins


Previous proposal: Obtained genes from a plant
-
like amoebal
host? (a relative of Chlamydiaceae infects Acanthamoeba)



“Plant
-
like” genes in
Chlamydia

NCBI GI


Protein description


Subcellular localization in plants


4377270


Glycyl tRNA Synthetase


Chloroplast


4376626


c
ADP/ATP Translocase


Chloroplast


4376667


c
Glycogen Hydrolase


Chloroplast


4377189


GTP Cyclohydratase & DHBP Synthase


Chloroplast


4377237


c
Beta
-
Ketoacyl
-
ACP Synthase


Chloroplast


4376686


c
Enoy
-
Acyl
-
Carrier Reductase


Chloroplast


4376591


c
Thioredoxin Reductase


Chloroplast


4377185


Metal Transport P
-
type ATPase


Chloroplast


4377346


Similar to NA+/H+ Antiporter


Chloroplast


4376650


c
Phosphate Permease


Chloroplast


4376637


GcpE protein


Chloroplast


4376637


Tyrosyl tRNA Synthetase


Chloroplast


4377360


c
Malate Dehydrogenase


Chloroplast


4376763


GTP Binding protein


Chloroplast


4376911


c
ADP/ATP Translocase


Chloroplast


3329179


Phosphoglycerate Mutase


Chloroplast


4377281


c
Glycerol
-
3
-
Phosphate Acyltransferase


Chloroplast


4376993


ABC Transporter ATPase


Chloroplast


4376509


d
Deoxyoctulonosic Acid Synthetase


Chloroplast


4376872


e
Sugar Nucleotide Phosphorylase


Chloroplast


4377368


c
Shikimate 5
-
Dehydrogenase


Chloroplast


4377054


Geranyl Transferase


Chloroplast


3328465


1
-
Deoxyxylulose 5
-
Phosphate Reductoisomerase


Chloroplast


“Plant
-
like” genes in
Chlamydia

6578112


rRNA Methytransferase


Chloroplast


3329217


HSP60


Chloroplast


3328745


c
Phosphoribosylanthranilate Isomerase


Chloroplast


6578104


c
Aspartate Aminotransferase


Chloroplast
f



4377328


c
Polyribonucleotide Nucleotidyltransferase


Chloroplast
f


4377362


Putative D
-
Amino Acid Dehydrogenase


Chloroplast
g


4377331


Cytosine Deaminase


Chloroplast?
h


4376915


Lipoate
-
Protein Ligase A


Mitochondrial


4377272


Glycogen Synthase


N/A
i


4377065


c
Dihydropteroate Synthase


N/A
i


4377239


c
Inorganic Pyrophosphatase


N/A
i


4376904


Uridine 5’
-
Monophosphate Synthase


N/A
i


4377173


c
UDP
-
Glucose Pyrophosphorylase


N/A
i


4376815


GutQ/Kpsf Family Sugar
-
Phosphate Isomerase


Mitochondrial?
j


Chlamydiaceae share
an ancestral
relationship with
Cyanobacteria and
Chloroplast

0.1

Pyrococcus

furiosus

(Archaea)

Thermotoga

maritima

Aquifex

pyrophilus

Bacillus

subtilis

C
hlamydophila
pneumoniae

C
hlamydophila
psittaci

C
hlamydia
muridarum

C
hlamydia
trachomatis

1000

704

1000

Chlamydomonas

reinhardtii

Klebsormidium

flaccidum

Zea

mays

Nicotiana

tabacum

1000

988

998

Synechococcus

PCC6301

Synechocystis

PCC6803

Microcystis

viridis

1000

1000

1000

530

E
scherichia
coli

Zea

mays

mitochondrion

R
ickettsia
prowazekii

Caulobacter

crescentus

868

986

764

349

1000

538

C
hloro
plasts

Cyanobacteria

Chlamydiaceae

Chlamydiaceae share an ancestral relationship with
Cyanobacteria and Chloroplast

L3

L4

L23

L2

S19

L22

S3

L16

L29

S17

L14

L24

L5

S14

S8

L6

L18

S5

L30

L15

S10

Escherichia

Bacillus

Thermatoga

Synechocystis

Chlamydia

Unique shared
-
derived characters unite Chlamydiaceae and
Synechocystis

Chlamydiaceae “plant
-
like” genes reflect an ancestral
relationship with Cyanobacteria and Chloroplast



Chlamydia do not appear to be exchanging DNA with their
hosts




Existing knowledge of Cyanobacteria may stimulate ideas
about the function and control of pathogenic
Chlamydia?


Non
-
unique shared characters include a multistage
developmental lifecycle, storage of glucose primarily
as glycogen, and non
-
flagellar motility


Expanding the Cross
-
Domain Analysis


Identify cross
-
domain lateral gene transfer between
bacteria, archaea and eukaryotes


No obvious correlation seen with protein functional
classification


Most cases: no obvious correlation seen between
“organisms involved” in potential lateral transfer

Exceptions:


Unicellular eukaryotes


“Organelle
-
functioning” proteins in
Rickettsia
,
Synechocystis,
and Chlamydiaceae

Horizontal Gene Transfer and
Bacterial Pathogenicity

Transposons:


ST enterotoxin genes in
E. coli


Prophages:

Shiga
-
like toxins in EHEC

Diptheria toxin gene, Cholera toxin

Botulinum toxins


Plasmids:

Shigella, Salmonella, Yersinia

Pathogenicity Islands:


Uro
/
Entero
-
pathogenic
E. coli

Salmonella typhimurium

Yersinia spp.

Helicobacter pylori

Vibrio cholerae

Pathogenicity Islands

Associated with



Atypical %G+C


tRNA sequences


Transposases, Integrases and other mobility genes


Flanking repeats


IslandPath: Identifying Pathogenicity Islands


Yellow circle = high %G+C


Pink circle = low %G+C



tRNA gene lies between the two dots


rRNA gene lies between the two dots


Both tRNA and rRNA lie between the two dots


Dot is named a transposase


Dot is named an integrase


Neisseria meningitidis serogroup B strain MC58


Mean %G+C: 51.37 STD DEV: 7.57



%G+C SD Location Strand Product


39.95
-
1 1834676..1835113 + virulence associated pro. homolog


51.96 1835110..1835211
-

cryptic plasmid A
-
related


39.13
-
1 1835357..1835701 + hypothetical


40.00
-
1 1836009..1836203 + hypothetical


42.86
-
1 1836558..1836788 + hypothetical


34.74
-
2 1837037..1837249 + hypothetical


43.96 1837432..1838796 + conserved hypothetical


40.83
-
1 1839157..1839663 + conserved hypothetical


42.34
-
1 1839826..1841079 + conserved hypothetical


47.99 1841404..1843191
-

put. hemolysin activ. HecB


45.32 1843246..1843704
-

put. toxin
-
activating


37.14
-
1 1843870..1844184
-

hypothetical


31.67
-
2 1844196..1844495
-

hypothetical


37.57
-
1 1844476..1845489
-

hypothetical


20.38
-
2 1845558..1845974
-

hypothetical


45.69 1845978..1853522
-

hemagglutinin/hemolysin
-
rel.


51.35 1854101..1855066 + transposase, IS30 family

Variance of the Mean %G+C for all Genes in a Genome:

Correlation with bacteria’s clonal nature

non
-
clonal clonal

Pathogenomics Project: Future Developments



Identify eukaryotic motifs and domains in pathogen genes



Threader: Detect proteins with similar tertiary structure



Identify more motifs associated with


Pathogenicity islands


Virulence determinants



Functional tests for new predicted virulence factors



Expand analysis to include viral genomes





Jeff Blanchard (National
Centre for Genome
Resources, New Mexico)



Olof Emanuelsson
(Stockholm
Bioinformatics Center)



Genome Sequence
Centre, BC Cancer
Agency

Acknowledgements

Pathogenomics group


Ann M. Rose,
Yossef Av
-
Gay, David L. Baillie, Fiona S. L.
Brinkman, Robert Brunham, Artem Cherkasov, Rachel C.
Fernandez, B. Brett Finlay, Hans Greberg, Robert E.W.
Hancock, Steven J. Jones,

Patrick Keeling, Audrey de
Koning, Don G. Moerman, Sarah P. Otto, B. Francis
Ouellette, Nancy Price, Ivan Wan.








www.pathogenomics.bc.ca