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21 Φεβ 2013 (πριν από 4 χρόνια και 3 μήνες)

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Pathogenomics

Goal:

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


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.


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


Eukaryotic
-
like pathogen genes

-

YopH, a protein
-
tyrosine phosphatase, of
Yersinia pestis


-

Enoyl
-
acyl carrier
protein reductase
(involved in lipid
metabolism) of
Chlamydia trachomatis

0.1

Aquifex aeolicus

Haemophilus influenza

Escherichia coli

Anabaena

Synechocystis

Chlamydia trachomatis

Petunia x hybrida

Nicotiana tabacum

Brassica napus

Arabidopsis thaliana

Oryza sativa

100

100

100

96

63

64

52

83

99

Prioritize for biological study.

-

Previously studied biologically?

-

Can UBC microbiologists study it?


-

C. elegans

homolog?


Screen for candidate genes.

Search pathogen genes against
sequence databases. Identify those
with eukaryotic similarity/motifs

Rank candidates.

-

how much like host protein?

-

info available about protein?

Modify
screening
method
/algorithm

Approach

Evolutionary significance.


-

Horizontal transfer? Similar by
chance?

Pathogens

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

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 similar gene
in model host,
C. elegans.


Study function
of gene.


Investigate role
of bacterial
gene in disease:
Infection study
in model host

C. elegans

DATABASE

Contact other
groups for
possible
collaborations.

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


Interdisciplinary team unique ideas and collaborations


Automated approach continually updated


Better understanding: pathogen gene
and

similar host
gene


Insight into horizontal gene transfer events and the
evolution of pathogen
-
host interactions.


Public database



other researchers may capitalize on
the findings


promote further collaboration

Power of the Approach

Database front end

PhyloBLAST


a tool for the analysis

Bacterium Eukaryote Horizontal 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.

N
-
acetylneuraminate lyase (sialic acid lyase, NanA)

Intracellular enzyme
involved in sialic acid
metabolism


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


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

N
-
acetylneuraminate lyase


role in pathogenicity?

Pasteurellaceae



Mucosal pathogens of the
respiratory tract



Intracellular NanA enzyme with
sialic acid transporter


T. vaginalis



Mucosal pathogen, causative
agent of the STD Trichomonas



Extracellular enzyme, so avoids
need for transporter?


Eukaryote Bacteria Horizontal Transfer?

0.1

Rat

Human

Escherichia coli

Caenorhabditis elegans

Pig roundworm

Methanococcus jannaschii

Methanobacterium thermoautotrophicum

Bacillus subtilis

Streptococcus pyogenes

Aquifex aeolicus

Acinetobacter calcoaceticus

Haemophilus influenzae

Chlorobium vibrioforme

Guanosine
monophosphate
reductase of
E. coli

is
81% similar to the
corresponding enzyme
studied in humans and
rats, and shares a
significant phylogenetic
relationship with
metazoans (left).


Its role in virulence has
not been investigated.

Eukaryote Bacteria Horizontal Transfer?

Ralstonia
solanacearum

cellulase (
ENDO
-
1,4
-
BETA
-
GLUCANASE
) is
56% similar to
endoglucanase
present in a number
of fungi.


Demonstrated
virulence factor for
plant bacterial wilt

Hypocrea jecorina
EGLII

Trichoderma viride
EGL2

Penicillium janthinellum
EGL2

Macrophomina phaseolina
EGL2

Cryptococcus flavus
CMC1

Ralstonia solanacearum egl

Humicola insolens
CMC3

Humicola grisea
CMC3

Aspergillus aculeatus
CMC2

Aspergillus nidulans
EGLA

Macrophomina phaseolina
egl1

Aspergillus aculeatus
CEL1

Aspergillus niger
EGLB

Vibrio
species

manA

Trends in the Analysis


Most cases of probable recent cross
-
domain gene
transfer involve movement of a bacterial gene to a
unicellular eukaryote


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


A control: The method identifies all previously
reported
Chlamydia trachomatis

eukaryotic
-
like
genes.


G+C Analysis: Identifying Pathogenicity Islands


%G+C S.D. Location Strand Gene Product




52.24 879443..880738
-

NMB0854 histidyl
-
tRNA synthetase


46.42 880832..881488
-

NMB0855 put. bacteriocin resist.


26.07
-
2 881770..882237
-

NMB0856 hypothetical protein


37.29
-
1 882294..882470
-

NMB0857 hypothetical protein


42.29
-
1 882474..882674
-

NMB0858 hypothetical protein


29.37
-
2 882677..883054
-

NMB0859 hypothetical protein


35.27
-
2 883112..883369
-

NMB0860 hypothetical protein


47.99 883459..884004
-

NMB0861 hypothetical protein


35.00
-
2 884001..884120
-

NMB0862 hypothetical protein


26.37
-
2 884167..884439
-

NMB0863 hypothetical protein


33.33
-
2 884705..884995
-

NMB0864 hypothetical protein


47.05 885001..885474
-

NMB0865 hypothetical protein


53.33 885517..886386
-

NMB0866 hypothetical protein


52.38 886550..887473 + NMB0867 YabO/YceC/SfhB fam. prot.


57.63 887551..888192
-

NMB0868 conserved hypothetical


54.42 888247..889038
-

NMB0869 hypothetical protein


55.56 889531..890322 + NMB0870 3
-
methyl
-
2
-
oxobutanoate


hydroxymethyltransferase

G+C of ORFs: Analysis of Variance


Low G+C variance correlates with an intracellular
lifestyle for the bacterium and a clonal nature (P
= 0.004)



This variance is similar within a given species



Useful marker for investigating the clonality of
bacteria? Relationship with intracellular lifestyle
may reflect the ecological isolation of intracellular
bacteria?

Future Developments


Incorporate unfinished genomes, plasmids into
analysis (including eukaryotic)



Motif
-
based and domain
-
based analyses



G+C analysis graphical viewer for identification of
pathogenicity islands



Functional tests




Peter Wall Foundation


Pathogenomics group


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

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











www.pathogenomics.bc.ca