Transcript Pathogenomics in Israel

Pathogenomics in Israel
Eliora Z. Ron <[email protected]>
• Metagenomics
• LGT = lateral gene transfer
• Typing of bacterial strains and drug
resistance
• Identification of virulence factors
• Whole genome analysis
Facilities for pathogenomic studies
• Good facilities for genomics,
transcriptomics and proteomics in
several universities and research
institutes
• High level bioinformatics
• All facilities are also available as service
Metagenomics
• Metagenomics of culturable and non-culturable
microorganism populations present in biofilms of
Acute Otitis Media (AOM, Middle Ear Infection)
– This infection involves a variety of bacterial
species, found in the form of biofilms, which are
inherently resistant to antibiotic treatment
– Detection of microbial biodiversity of AOM
biofilm is limited due to current cultivation
methods
– The group of Fauzi Silbaq (ArabQual) and Racheli
Kreisberg-Zakarin (IBEX) [email protected]
in involved in a study the biodiversity of AOM
using a metagenomics approach, including
microbiology, functional genomics and
bioinformatics methodologies
Metagenomics
• Metagenomics of intestinal microflora in health
and disease and the effect of the TLR
mutations
– Toll-Like Receptors (TLRs) recognize
pathogen specific patterns of microorganisms.
Mutations in TLRs are associated with
inflammatory bowel diseases (Crohn's disease
and ulcerative colitis). Patients with those
diseases have altered intestinal microflora
• Uri Gophna, currently with Ford Doolitle in
Halifax [email protected] performs cultureindependent profiling of intestinal microflora of
TLR knockout mice under normal conditions and
after a challenge which models inflammatory
bowel disease
LGT – lateral gene transfer in
relation to pathogenesis
• Uri Gophna [email protected] studies the
role of lateral gene transfer (LGT) in
the evolution of pathogens
• Uses bioinformatics for the
identification of laterally acquired
genes and pathways in pathogens
LGT – lateral gene transfer in
relation to pathogenesis
• Yair Aharonowitz, Ilya Borovok and Gerald Cohen
from Tel Aviv University study gluthathione
synthesis [email protected]
• Identified GshF orthologs, consisting of a γglutamylcysteine ligase (GshA) domain fused to
an ATP-grasp domain, in 20 gram-positive and
gram-negative bacteria.
• Remarkably, 95% of these bacteria are
mammalian pathogens. Presumably, this fusion
gene, once formed, spread between mammalian
hosts most likely by horizontal gene transfer
Distribution of fused glutathione biosynthetic genes mapped onto a universal
tree of bacterial 16S rRNA (Minimum Evolution, ME)
Chlamydia trachomatis
Anabaena cylindrica
Lactobacillus delbrueckii
Lactobacillus plantarum
Clostridium acetobutylicum
Staphylococcus aureus
Bacillus subtilis
Clostridium perfringens Enterococcus faecium
Listeria monocytogenes
Enterococcus faecalis
Listeria innocua
Streptococcus uberus
Streptococcus pyogenes
Streptococcus agalactiae
Streptococcus suis
Streptococcus gordonii
Streptococcus pneumoniae
Streptococcus sobrinus
Streptococcus mutans
Rhodospirillum rubrum
Pseudomonas aeruginosa
Actinobacillus pleuropneumonia
Lactococcus lactis
Haemophilus somnus
Actinobacillus actinomycetemco
Haemophilus influenzae
Pasteurella multocida
Vibrio cholerae
Salmonella typhimurium
Escherichia coli
Streptomyces coelicolor
Corinebacterium glutamicum
Mycoplasma pneumoniae
Mycobacterium tuberculosis Leptospira interrogans
Borrelia japonica
Thermotoga maritima
Thermus aqaticus
Chloroflexus aurantiacus
0.05
Typing of bacterial strains and
drug resistance
• The group of Chezi Kashi in the Technion
[email protected]
• uses novel methods for molecular typing
ofVibrio cholera in order to study emerging
new pathogenic strains
• The group of Sima Yaron (Technion) is involved
in typing the serovars of Salmonella enterica in
respect to drug resistance and virulence
[email protected]
Identification of bacterial virulence
factors
• The group of Sima Yaron has developed a
rapid, simple screen for real-time
quantification of promoter - activity in S.
enterica using a library of plasmids with GFP
as a reporter
• The group of Gil Segal (Tel Aviv U.)
[email protected] is involved in the study of
hyper variable genes are found in the
Legionella icm/dot pathogenesis region. They
have demonstrated the role of these genes in
virulence
Identification of fungal virulence
factors
• The group of Nir Asherov (Tel Aviv U.)
[email protected] studies Aspergillus
fumigatus, which causes serious disease (around
60% mortality) in immunocompromised people.
They concentrate on studying novel cell wall
proteins and identified 68 such cwp’s. They are
studying these CWPs in vitro and in infected
animals by the use of deletion mutants.
• They are currently in the process of preparing a
Cwp-specific microarrays to study Cwp gene
expression patterns
Whole genome analysis
• Two genomes of bacterial pathogens are
being sequenced and analyzed in Israel
– Bacillus anthracis (Shaefferman et al,
Biological Institute, Nes Ziona)
[email protected]
– Septicemic Escherichia coli – strain of
serotype O78 (Ron, Tel Aviv U.)
[email protected]
Virulent E. coli strains
• Most of the E. coli strains are commensal, but
a small number are pathogenic
• Pathogenic E. coli strains are divided into two
groups:
– Intestinal strains. These produce
enterotoxins and constitute a major
problem, especially in young children and
travellers (Montesumu’s revenge)
– Extraintestinal strains – ExPEC
(Extraintestinal Pathogenic E. coli)
Extraintestinal diseases caused by E. coli
• Urinary
tract infections (UTI) (pyeolonephritis, kidney
failure, productivity loss)
• UTIs are responsible for > seven million patient visits
and one million hospital admissions (due to
complications) per year in the United States only. 80 90% of the cases are caused by E. coli
• Neonatal meningitis: bacterial meningitis
•0.25 per 1000 live births in industrialized countries
(2.66 per 1000 in developing countries). ~30% caused
by E. coli , ~10% mortality
•Intra-abdominal infections, Respiratory tract infections,
Wound and surgical infections
•Septicemia
Septicemia (colibacillosis)
• Colisepticemia is the major causes of
mortality from community and hospitalacquired infections (more than 80%)
• Main cause of mortality in immunosupressed patients (HIV, chemotherapy,
old age)
• Colisepticemia is an emerging disease –
83% increase 1980 – 1992, over 40% of
the bacteremia cases in community
acquired infections
Goals:
• Define virulence-essential ExPEC-specific
genes
• Profile strains involved in UTI, NBM and
sepsis using these ExPEC-specific genes
• Use the data to define potential targets
for development of vaccines and/or
antibacterial drugs.
Welch et al. 2002, PNAS
Identification of virulence related
sequences in septicemic strains
• Whole genome sequencing
• Subtractive hybridization
Subtractive hybridization
• Obtain pathogen specific sequences,
absent from non-pathogenic K12
strain
• Excellent chance of “hitting”
pathogenicity islands which are
pathogen specific and very large
• Faster (and much cheaper) than whole
genome sequencing
Subtractive hybridization
A way to study comparative genomics
with organisms which have not been
sequenced
Pathogen
NonPathogen
Pathogen
Specific
Library of
pathogen specific
genes
Search for unique “septicemic”
sequences
• Using suppression subtractive hybridization
(SSH) we identified sequences unique to strain
O78-9 and absent from the non-pathogenic
strain K-12
• Oover 80 O78-specific open reading frames
were found (91 to 1473 bp in length)
• The same experiment was repeated with
another septicemic strain O2-1772
• 117 unique O2 sequences were identified
Comparison of unique sequences of
O2 and O78
• Although the two strains cause the
same disease, there is a high diversity
between the SSH libraries of O2 and
O78 strains, with only a few shared
genes coding for virulence factors.
• Is this diversity serotype specific? To
determine this we profiled additional
septicemic strains of the same
serotypes the presence of each of the
unique sequences
% of O78 strains with sequence
120
100
O78
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*
*
80
60
40
20
2D Graph 2
0
% of O2 strains with sequence
120
100
O2
*
*
80
60
40
20
0
Sequence
*
Comparison of unique sequences of two
septicemic strains - O2 and O78
• high level of genome plasticity
• there is a high diversity between the
septicemic strainsunexpected for
strains causing the same disease
• Septicemic strains of serogroups O2
and O78 contain a large pool of virulence
genes which are used in a “mix and
match” fashion
Current-future research
• Molecular-physiological studies of the
newly-identified virulence genes
• Bioinformatic studies to determine the
evolution of these virulence genes (many
in PAIs with evidence of LGT)
• Whole genome sequencing – joint
project with Prof. Joerg Hacker and
Prof. Gerhard Gottschalk
Thank you!!
• TAU group
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Uri Gophna
Diana Ideses
Daphna Mokady
Dr. Dvora Biran
• Collaborations
– Wuerzburg University (Prof. Joerg Hacker)
– Greifswald University (Prof. Michael Hecker)
– Goetingen University (Prof. Gerhard Gottschalk)
Metagenomics
• Uri Gophna, currently with Ford Doolitle in
Halifax studies the effect of TLR mutations on
micrflora in health and disease by culture-
independent profiling of intestinal
microflora of TLR knockout mice under
normal conditions and after a challenge
which models inflammatory bowel disease
MLST of O78 strains
• Multi Locus Sequence Typing
• 450 – 500 bp of 7 “housekeeping”
genes
• Criteria for chosing genes:
– 97-98% homology to E. coli K-12 (from
blast data)
– appear in pathogenic and non pathogenic
strains
– map at considerable distance from each
other
– several allels in the population
• There is a positive correlation between
virulence, invasiveness and clonal origin
• Clonal division in E. coli O78 strains is
host independent - closely related
clones reside in different hosts
• The MLST results are compatible
with the results from subtractive
hybridization and sequencing
• The profile of virulence factors in
ExPEC strains is independent of the
host and independent of the serotype
• There is a high diversity of virulence
genes in the various E. coli septicemic
strains and each strain has its own
profile of virulence genes
O78-9 sequences
known
functions
putative virulence-associated
putative and known
virulence factors
unknown
functions
phage related
mobility-related
80 sequences specific to the pathogenic strain and absent from
the driver strain K-12.
O2-1772 sequences
unknown
functions
known functions
putative
virulence-associated
putative and known
virulence factors
phage associated
mobility-related
117 sequences specific to the pathogenic strain and absent
from the driver strain K-12.
• Both libraries contain many sequences
associated with genomic plasticity evolution by horizontal gene transfer
• Many sequences of O2 and O78 are
homologous to virulence related
sequences of human ExPEC strains
Distribution of fused GshFs based of the C-terminal domain sequences and
mapped onto a tree of bacterial GSHB and ATP-Grasp proteins (ME analysis)
Streptococcus agalactiae Streptococcus mutans
Enterococcus faecalis
Streptococcus suis
Enterococcus faecium
Leptospira interrogans
Actinobacillus pleuropneumonia
Haemophilus somnus
Clostridium perfringens
Pasteurella multocida
Desulfotalea psychrophila
Mannheimia succiniciproducens
Listeria monocytogenes
Listeria innocua
Bordetella parapertussis MurC
Ralstonia eutropha MurC
Clostridium acetobutylicum
Acinetobacter sp. MurC
Gloeobacter violaceus CphA
Anabaena variabilis CphA
Bordetella pertussis
Anabaena variabilis DdlA
Neisseria meningitidis
Nitrosomonas europaea
Listeria monocytogenes DdlA
Acinetobacter sp.
Bacillus subtilis DdlA
Pseudomonas syringae
Escherichia coli
Vibrio cholerae
Buchnera aphidicola
Escherichia coli DdlA
0.1
GshFs
ATP-grasp
GshBs