Host specificity of pathogenic Escherichia coli Eliora Z. Ron, Tel-Aviv University

1. Host specificity of pathogenic Escherichia coliEliora Z. Ron, Tel-Aviv University תודה

3. References Ideses, D., U. Gophna, et al. ( 2005). A Degenerate Type-III Secretion System from Septicemic Escherichia coli Contributes to Pathogenesis. J Bacteriolin press. Mokady, D., U. Gophna, et al. (2005). Virulence factor of septicemic E. coli strains. IJMMin press. Mokady, D., U. Gophna, et al. (2005). Extensive gene diversity in septicemic Escherichia coli strains. J Clin Microbiol43: 66-73. Ideses, D., D. Biran, et al. (2005). The lpf operon of invasive Escherichia coli. Int J Med Microbiol295:227-236. Gophna, U., D. Ideses, et al. (2004). OmpA of a septicemic Escherichia coli O78--secretion and convergent evolution. Int J Med Microbiol294: 373-381. Gophna, U., E. Z. Ron, et al. (2003). Bacterial type III secretion systems are ancient and evolved by multiple horizontal-transfer events. Gene312: 151-163. Gophna, U., A. Parket, et al. (2003). A novel ColV plasmid encoding type IV pili. Microbiology149:177-84. Gophna, U. and E. Z. Ron (2003). Virulence and the heat shock response. Int J Med Microbiol292:453-61. Adiri, R. S., U. Gophna, et al. (2003). Multilocus sequence typing (MLST) of Escherichia coli O78 strains. FEMS Microbiol Lett222: 199-203. Gophna, U., T. A. Oelschlaeger, et al. (2002). Role of fibronectin in curli-mediated internalization. FEMS MICROBIOLOGY LETTERS212: 55-58. Gophna, U., T. A. Oelschlaeger, et al. (2001). Yersinia HPI in septicemic Escherichia coli strains isolated from diverse hosts. FEMS Microbiol. Let196: 57-60. Gophna, U., M. Barlev, et al. (2001). Curli Fibers Mediate Internalization of Escherichia coli by Eukaryotic Cells. Infect Immun69: 2659-65. Dobrindt, U., G. Blum-Oehler, et al. (2001). S-Fimbria-Encoding Determinant sfaI Is Located on Pathogenicity Island III536 of Uropathogenic Escherichia coli Strain 536.Infect. Immun.69: 4248-4256. Babai, R., B. E. Stern, et al. (2000). New Fimbrial Gene Cluster of S-Fimbrial Adhesin Family. Infect Immun68: 5901-5907. Babai, R., G. Blum-Oehler, et al. (1997). Virulence patterns from septicemic Escherichia coli O78 strains. FEMS Microbiol Lett149: 99-105. Yerushalmi, Z., N. I. Smorodinsky, et al. (1990). Adherence pili of avian strains of Escherichia coli O78." Infect Immun58: 1129-31.

4. 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)

5. 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

6. 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

7. Colisepticemia in farm animals • A lethal disease in newborn lambs – bacteria carry K99 fimbriae and colV plasmid. Colonize lambs on birth, death within two weeks. • Avian colisepticemia - serious disease of chickens and turkeys. Heavy direct losses due to high morbidity and mortality, as well as indirect losses due to intensification of other respiratory diseases caused by viruses or mycoplasma • Losses of several million dollars a year reported by DelMarVa industries alone

8. Avian Colisepticemia • Bacteria enter the host by adherence and colonization of upper respiratory tract • Later, the bacteria cross epithelial barriers and invade deeper tissues • Finally the bacteria enter bloodstream and proceed to the vital organs of the host • A good model system for ExPEC infections • 80% of the cases of colisepticemia, world –wide, are caused by E. coli serotypes O2 and O78

9. ExPEC strains • Known virulence factors include genes for efficient iron uptake, serum resistance and adherence to host tissues. Do not produce toxins • What makes them virulent? • Are the virulence factors host dependent ? • Is there a clonal relationship? (host dependent?) • Can we predict an outbreak of ExPEC (early warning) ?

10. Goals: • Define virulence-essential ExPEC-specific genes • Profile strains involved in UTI, NBM and sepsis using these ExPEC-specific genes • Look for virulence genes which determine host specificity • Use the data to define potential targets for development of vaccines and/or antibacterial drugs.

11. What is a virulence factor? • Encoded by a gene present only in pathogenic strains • Without this factor virulence is decreased without a decrease in growth rate • Example: toxins • Virulence factors can determine host specificity • Example: adherence fimbriae (pili)

12. E. coli O78 – septicemic in humans and birds AC/I pili – contributes to specific adherence only in avian septicemic strains

13. minor subunits major subunits S-fimbrial adhesin family • Found in human pathogenic E. coli strains • Composed of around 1000 protein units, major and minor subunits

14. Degree of identity between AC/I (Fac) orfs to SfaI, SfaII and Foc Fac orfs Sfa I Sfa II Foc (UTI) (NBM) (UTI) FacA (major) 66 100 69 FacD 98 ? ? FacE 99 ? 99 FacF 98 ? 97 FacG (minor) 100 99 98 FacS (minor) 72 71 60 FacH (minor) 80 82 96

15. Adherence of strain 781 to avian epithelial cells - adherence of strain 781 producing AC/I compared with - 781 strain not expressing AC/I (grown in 180C) - Unpiliated strain

16. Preferential adherence of strain 781 to avian epithelial cells Unpiliated strain CFA/I expressing strain AC/I expressing strain AC/I fimbriae appear to be a virulence factor, probably avian specific

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

21. 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 • Over 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

23. 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 • The virulence related genes identified by SSH include iron uptake systems, adhesins autotransporters and secretion genes, including a new T3SS

24. T3SS • Type three secretion systems (T3SS) of E. coli O157 and other invasive bacteria deliver effectors into the cytosol of the host cells • A novel T3SS was discovered in genomic studies of E. coli O157 and others – called ETT2 (E. coli type-three secretion system 2) • So far if is not clear if ETT2 has a role of in pathogenesis • Our results of SSH indicated that septicemic strains have the ETT2 type of TTSS – first demonstration in septicemic strains

25. However - • The cluster contains a large deletion and several stop codons and appears to be “dead”

27. ETT2sepsis • ETT2sepsis has several premature stop codons and a large (five Kb) deletion • These genetic modifications result in an inability to produce the “needle” • The 5 kb deletion is conserved in elevenE. coli strains from septicemia and newborn meningitis.

28. Is ETT2sepsis really dead? • Although the ETT2sepsis is degenerate, the gene cluster is transcribed • A null mutantdeleted for ETT2sepsis was constructed and grows as well as the wild type • However, the deletion of ETT2sepsis results in modification of bacterial surface properties which could affect interaction with host cells and immune system

29. Growth of strain 789 & the ETT2 deletion

30. 250C 250C 370C 370C Null mutation in ETT2sepsis affects surface properties but only above 370C (host conditions?)

31. ETT2sepsis null mutants are not virulent

32. T3SS • SSH indicated that septicemic E. coli strains have the ETT2 type of TTSS – ETT2sepsis • ETT2sepsis is degenerate but important for virulence • These results are the first demonstration of the importance of ETT2 in pathogenesis • The biological role of ETT2sepsis probably does not involve classical secretion of effectors

33. Some virulence factors are missed by the genomic approaches • Genomics and proteomics give information about sequences of proteins which are unique to virulent strains • By the classical definition, a virulence factor is encoded by a gene present only in pathogenic strains • This is not always so – example: curli fibers

34. E. coli O78 are internalized and replicate within cellshuman (Hela, T24) and avian (T24)

35. Confocal laser scanning microscopy of internalized bacteria Cells are visualized using anti-actin or anti-tobulin antibodies and the bacteria express GFP

36. Transmission electron microscopy of internalized bacteria

37. A clone from E. coli O78 cosmid library mediates internalization The clone carries the csg gene cluster encoding curli

38. Curli fibers Bian, Z., Branuer, A., Li, Y. and Normark, S., 2000. JID 181: 602

39. Curli - thin coiled fiberswith high affinity binding for several host proteins: • Plasminogen and plasminogen activator • MHC Class I molecules • Laminin • Fibronectin

40. However... • Non pathogenic E. coli strains also contain the csg cluster encoding curli... • Is curli of O78 different than this of K-12? • Is Curli a virulence factor?

41. A high level of curli expression promotes internalization by eukaryotic cells When expressed in multicopy, the presence of the csg gene cluster of pathogenic and non pathogenic strains promotes internalization.The csg clone from E. coli O78 is more effective than that of the E. coli K-12 cloneIs it a better curli or more curli?

42. E. coli O78 expresses high levels of curli at host conditions Curli expression at 42°C

43. Expression of O78 curli is differnet from K-12 curli • Curli of O78 are expressed at higher levels • Curli of O78 are expressed under host conditions (high temperature, high osmolarity) • Sequencing indicated that the CsgD - activator required for transcription curli operons – of O78 is different from K-12 and similar to that of Salmonella. Could explain the differences in expression of curli.

44. Recent support for the role of curli in virulence • Isolates from human sepsis constitutively express high levels of curli. • O157:H7 isolates with increased curli expression are more invasive to cells and more virulent in mice. • Curli mutants of avian septicemic E. coli serotype O78 are attenuated in vivo

45. Virulence factors are encoded by a gene present only in pathogenic strains • Other virulence factors encoded by genes which are presnet in virulent and non virulent strains, but have a different expression or activity in virulent strains • These virulence factors are missed in genomic studies

46. Analysis of the unique “septicemic” sequences • Using subtractive hybridization (SSH) of septicemic strains and K-12, we identified over 80 sequences unique to strain O78-9 and over 110 sequences unique to another septicemic strain, O2-1772 • Are the unique sequences of O78 similar to the unique sequences of O2?

47. Screening of additional septicemic strains of E. coli O78 and O2 serotypes • Comparison of the two subtractive hybridiation libraries indicated a large diversity between the O2 and the O78 strains • Is this diversity serotype specific? • To determine this we screened additional septicemic strains of the same serotypes the presence of each of the unique sequences

48. Kb Kb Kb 5 5 5 3 3 3 2 2 2 O O 78 78 - - 18 18 ( ( 1369 1369 bp bp ) ) 1 1 1 O O 78 78 - - 55 55 ( ( 570 570 bp bp ) ) 0 0 0 . . . 5 5 5 O O 2 2 - - 210 210 ( ( 419 419 bp bp ) ) O O 2 2 - - 334 334 ( ( 264 264 bp bp ) ) PCR of septicemic E. coli O2 and O78 strains with virulence specific sequences Large variability, not serotype related

49. Screening of additional septicemic strains of serogroups O2 and O78 for presence of specific sequences

50. Comparison of unique sequences of septicemic strains of serotype O2 and O78 • high level of genome plasticity • there is a high diversity between the SSH libraries of O2 and O78 strains, with only a few shared genes coding for virulence factors • unexpected for two 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