Host specificity of pathogenic escherichia coli eliora z ron tel aviv university
This presentation is the property of its rightful owner.
Sponsored Links
1 / 64

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


  • 84 Views
  • Uploaded on
  • Presentation posted in: General

Host specificity of pathogenic Escherichia coli Eliora Z. Ron, Tel-Aviv University. תודה. References Ideses, D., U. Gophna, et al. ( 2005). A Degenerate Type-III Secretion System from Septicemic Escherichia coli Contributes to Pathogenesis. J Bacteriol in press .

Download Presentation

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

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

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

תודה


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

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.


Virulent e coli strains

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)


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

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

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


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

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


Avian colisepticemia

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


Expec strains

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) ?


Goals

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.


What is a virulence factor

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)


E coli o78 septicemic in humans and birds

E. coli O78 – septicemic in humans and birds

AC/I pili – contributes to specific adherence

only in avian septicemic strains


S fimbrial adhesin family

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


Degree of identity between ac i fac orfs to sfai sfaii and foc

Degree of identity between AC/I (Fac) orfs to SfaI, SfaII and Foc

Fac orfs Sfa ISfa IIFoc

(UTI)(NBM)(UTI)

FacA(major)6610069

FacD98??

FacE99?99

FacF98?97

FacG(minor)1009998

FacS(minor)727160

FacH(minor)808296


Adherence of strain 781 to avian epithelial cells

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


Preferential adherence of strain 781 to avian epithelial cells

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


Identification of virulence related sequences in septicemic strains

Identification of virulence related sequences in septicemic strains

  • Whole genome sequencing

  • Subtractive hybridization


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 hybridization1

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


Search for unique septicemic sequences

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


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

  • 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


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

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


However

However -

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


Ett2 sepsis

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.


Is ett2 sepsis really dead

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


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

Growth of strain 789 & the ETT2 deletion


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

250C

250C

370C

370C

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


Ett2 sepsis null mutants are not virulent

ETT2sepsis null mutants are not virulent


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

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


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

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


E coli o78 are internalized and replicate within cells human hela t24 and avian t24

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


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

Confocal laser scanning microscopy of internalized bacteria

Cells are visualized using anti-actin or anti-tobulin antibodies and the bacteria express GFP


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

Transmission electron microscopy of internalized bacteria


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

A clone from E. coli O78 cosmid library mediates internalization

The clone carries the csg gene cluster encoding curli


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

Curli fibers

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


Curli thin coiled fibers with high affinity binding for several host proteins

Curli - thin coiled fiberswith high affinity binding for several host proteins:

  • Plasminogen and plasminogen activator

  • MHC Class I molecules

  • Laminin

  • Fibronectin


However1

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?


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

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?


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

E. coli O78 expresses high levels of curli at host conditions

Curli expression at 42°C


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

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.


Recent support for the role of curli in virulence

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


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

  • 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


Analysis of the unique septicemic sequences

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?


Screening of additional septicemic strains of e coli o78 and o2 serotypes

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


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

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


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

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


Comparison of unique sequences of septicemic strains of serotype o2 and o78

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


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14

15 16

15 16

15 16

Kb

Kb

Kb

5

5

5

3

3

3

2

2

2

O

O

78

78

-

-

95

95

(

(

955

955

bp

bp

)

)

0

0

0

.

.

.

5

5

5

Byproduct:

- we found one sequence which is present in all O78 strains and in none of the O2 strains

- can be used for detecting O78, especially in food

O2 strains

O78 strains


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

  • There is large diversity in the profiles of virulence specific genes

    • this is in contrast to the results of O157

  • The profile of virulence specific genes is independent of the host

  • Is there a virulence-associated or host-dependent clonal relationship between the strains?

  • Clonal relationship was determined using MLST


Mlst of o78 strains

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


Genes chosen for use in mlst

Genes chosen for use in MLST


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

Genes selected for MLST


Bacteria used for mlst

Bacteria used for MLST

  • O78 strains, pathogens (ExPEC) and non pathogens (28 strains)

    • Human

    • Avian

    • Sheep - cattle


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

Neighbor-joining MLST phylogenetic tree of E. coli strains and virulence to 1-day-old chick.

H: Human; C: cattle; A: avian pathogen; -: no mortality; +: less than 25% mortality; ++: less than 25–49% mortality; +++: 50–74% mortality; ++++: 75–100% mortality


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

  • 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


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

  • 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

  • Is there host specificity in ExPEC strains??


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

  • E. coli strains isolated from avian septicemia are more virulent to chicks than strains isolated from NBM

  • This result was unexpected in view of the finding that the virulence genes and the clonal profile of virulence factors in ExPEC strains is independent of the host


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

Open Questions

  • Which genes are involved in the higher virulence to chicks?

  • Are there strains that are more virulent to mammals than to chicks (“human specific”)?

  • What is the zoonotic risk of human infection with avian colisepticemic strains?


Virulence factors and host specificity of expec strains beyond the omics

Virulence factors and host specificity of ExPEC strains – beyond the “omics”

  • It is possible to identify unique genetic sequences, present only in virulent strains

  • There is a high variability in these sequences, which is host independent

  • There are virulence factors which are encoded by genes present also in non virulent strains (i.e., curli)

  • “Dead” gene clusters can nevertheless be important virulence factors (ETT2)

  • There is a clear host specificity, but its basis is not obvious from examining “virulence genes”


Support

Support

  • European Community project COLIRISK

  • Center for Emerging Diseases, Israel

  • GIF – German Israeli Science Foundation

  • Manja and Morris Leigh Chair of Biophysics and Biotechnology


Host specificity of pathogenic escherichia coli eliora z ron tel aviv university

Tel Aviv University

Eliora Z. Ron

Reuven Babai

Uri Gophna

Diana Ideses

Daphna Mokady

Roni Segal-Adiri

Zohar Yerushalmi

Michael Naveh

Dvora Biran

Collaborations

U. Würzburg

Joerg Hacker

U. Helsinki –

Timo Korhonen

INRA - Centre de Tours

M. Moulin-Schouleur


  • Login