Pathogenomics in Israel Eliora Z' Ron eliorapost'tau'ac'il

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Pathogenomics in IsraelEliora Z. Ron
lteliora_at_post.tau.ac.ilgt

• Metagenomics
• LGT lateral gene transfer
• Typing of bacterial strains and drug resistance
• Identification of virulence factors
• Whole genome analysis

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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

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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) racheli_at_ibexperts.com in
  involved in a study the biodiversity of AOM using
  a metagenomics approach, including microbiology,
  functional genomics and bioinformatics
  methodologies

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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 ugophna_at_dal.ca performs
  culture-independent profiling of intestinal
  microflora of TLR knockout mice under normal
  conditions and after a challenge which models
  inflammatory bowel disease

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LGT lateral gene transfer in relation to
pathogenesis

• Uri Gophna ugophna_at_dal.ca 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

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LGT lateral gene transfer in relation to
pathogenesis

• Yair Aharonowitz, Ilya Borovok and Gerald Cohen
  from Tel Aviv University study gluthathione
  synthesis yaira_at_tauex.tau.ac.il
• Identified GshF orthologs, consisting of a
  ?-glutamylcysteine 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

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Distribution of fused glutathione biosynthetic
genes mapped onto a universal tree of bacterial
16S rRNA (Minimum Evolution, ME)
Chlamydia trachomatis
Lactobacillus delbrueckii
Lactobacillus plantarum
Clostridium acetobutylicum
Staphylococcus aureus
Anabaena cylindrica
Bacillus subtilis
Enterococcus faecium
Clostridium perfringens
Listeria monocytogenes
Enterococcus faecalis
Listeria innocua
Streptococcus uberus
Rhodospirillum rubrum
Streptococcus pyogenes
Streptococcus agalactiae
Streptococcus suis
Streptococcus gordonii
Pseudomonas aeruginosa
Streptococcus pneumoniae
Streptococcus sobrinus
Streptococcus mutans
Actinobacillus pleuropneumonia
Lactococcus lactis
Haemophilus somnus
Actinobacillus actinomycetemco
Haemophilus influenzae
Pasteurella multocida
Vibrio cholerae
Salmonella typhimurium
Escherichia coli
Streptomyces coelicolor
Corinebacterium glutamicum
Mycoplasma pneumoniae
Leptospira interrogans
Mycobacterium tuberculosis
Borrelia japonica
Thermotoga maritima
Thermus aqaticus
Chloroflexus aurantiacus
0.05
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Typing of bacterial strains and drug resistance

• The group of Chezi Kashi in the Technion
  kashi_at_techunix.technion.ac.il
• 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
  simay_at_tx.technion.ac.il

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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.)
  GilS_at_tauex.tau.ac.il 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

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Identification of fungal virulence factors

• The group of Nir Asherov (Tel Aviv U.)
  nosherov_at_post.tau.ac.il 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 cwps. 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

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Whole genome analysis

• Two genomes of bacterial pathogens are being
  sequenced and analyzed in Israel
• Bacillus anthracis (Shaefferman et al, Biological
  Institute, Nes Ziona) ashaefferman_at_iibr.gov.il
• Septicemic Escherichia coli strain of serotype
  O78 (Ron, Tel Aviv U.) eliora_at_post.tau.ac.il

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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 (Montesumus
  revenge)
• Extraintestinal strains ExPEC (Extraintestinal
  Pathogenic E. coli)

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Extraintestinal diseases caused by E. coli

• Urinary tract infections (UTI) (pyeolonephritis,
  kidney failure, productivity loss)
• UTIs are responsible for gt 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

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Septicemia (colibacillosis)

• Colisepticemia is the major causes of mortality
  from community and hospital-acquired infections
  (more than 80)
• Main cause of mortality in immuno-supressed
  patients (HIV, chemotherapy, old age)
• Colisepticemia is an emerging disease 83
  increase 1980 1992, over 40 of the bacteremia
  cases in community acquired infections

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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.

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Welch et al. 2002, PNAS
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Identification of virulence related sequences in
septicemic strains

• Whole genome sequencing
• Subtractive hybridization

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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

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Subtractive hybridization

• A way to study comparative genomics with
  organisms which have not been sequenced

Library of pathogen specific genes
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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

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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

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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

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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

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Thank you!!

• TAU group
• 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)

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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

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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

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• 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

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• 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

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80 sequences specific to the pathogenic strain
and absent from the driver strain K-12.
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117 sequences specific to the pathogenic strain
and absent from the driver strain K-12.
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• 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

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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)
GshBs
GshFs
ATP-grasp