Pathogenomics in Israel Eliora Z. Ron <eliora@post.tau.ac.il>

1. Pathogenomics in IsraelEliora Z. Ron <eliora@post.tau.ac.il> • Metagenomics • LGT = lateral gene transfer • Typing of bacterial strains and drug resistance • Identification of virulence factors • Whole genome analysis

2. 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

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

4. 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@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

5. LGT – lateral gene transfer in relation to pathogenesis • Uri Gophna ugophna@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

6. LGT – lateral gene transfer in relation to pathogenesis • Yair Aharonowitz, Ilya Borovok and Gerald Cohen from Tel Aviv University study gluthathione synthesis yaira@tauex.tau.ac.il • Identified GshF orthologs, consisting of a γ-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

7. 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 Bacillus subtilis Anabaena cylindrica 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

8. Typing of bacterial strains and drug resistance • The group of Chezi Kashi in the Technion kashi@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@tx.technion.ac.il

9. 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 libraryof plasmids with GFP as a reporter • The group of Gil Segal (Tel Aviv U.) GilS@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

10. Identification of fungal virulence factors • The group of Nir Asherov (Tel Aviv U.) nosherov@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 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

11. 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@iibr.gov.il • Septicemic Escherichia coli – strain of serotype O78(Ron, Tel Aviv U.)eliora@post.tau.ac.il

12. 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)

13. 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

14. 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 casesincommunity acquired infections

15. 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.

16. Welch et al. 2002,PNAS

17. Identification of virulence related sequences in septicemic strains • Whole genome sequencing • Subtractive hybridization

18. 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

19. Pathogen Non-Pathogen Pathogen Specific Subtractive hybridization A way to study comparative genomics with organisms which have not been sequenced Library of pathogen specific genes

20. 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

21. 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

22. O78 * * * O2 * * *

23. 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

24. 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

25. 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)

27. 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

28. 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

29. 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

30. 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

31. 80 sequences specific to the pathogenic strain and absent from the driver strain K-12.

32. 117 sequences specific to the pathogenic strain and absent from the driver strain K-12.

33. 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

34. 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 Buchnera aphidicola Escherichia coli Escherichia coli DdlA Vibrio cholerae 0.1 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) GshFs ATP-grasp GshBs