Dissecting virulence of E. coli O157:H7 using genome alignments

E. coli

• Escherichia coli is a normal resident of the mammalian gut – Most strains are non-pathogenic

– E. coli K12 – lab strain

• Some strains are pathogenic to human: E. coli O157:H7

– Cause diarrhea, cramps

– The most virulent strains of O157:H7 can result in death

• Disease symptoms caused in part by the bacterial production of Shiga toxin– Shiga toxins act to inhibit protein synthesis in the host

– Cytotoxic

• Pathogenic strains transmitted through consumption of contaminated foods, (undercooked ground meat products/vegetables watered with contaminated water)

Pathogenesis

• Microbes living in or on a host AND• Producing negative consequences to host

– Killing host cells– Inducing an immune response in host (toxic shock)– Reducing the “fitness” of the host

• Virulence factors are proteins that facilitate the microbes ability to exploit the host

Virulence factors

• Virulence = severity of damage to the host– Things produced by the bacterial that make the host sicker– Example: Shiga toxin in enterohemorraghic E. coli

• Why would a pathogen “want” to harm the host?– Virulence is sometime a byproduct of the bacterial

efforts to live and propagate in a host• Proteins that adhere to host cells• Proteins injected into the host to manipulate the host cell• Stx encodes a protein that facilitates attachment to the host

mucosa, but it also inhibits protein synthesis in the host cell

Pathogenic bacteria and virulence factors

-Previously we showed a comparison of genomes of good E. coli (K-12) and bad E. coli (O157:H7)

-The genome of E. coli O157:H7 contains >1,000 extra genes in comparison to good E. coli (K-12)

-Many of these extra genes enable this strain of E. coli to cause disease in humans.

-A survey of all of the published literature has identified 394 genes that are believed to be involved in the ability of E. coli O157:H7 to cause disease, and are called virulence factors.

-These genes compose >7.5% of the genome of E. coli O157:H7 strains

14 categories of virulence factors for E. coli O157:H7

-Adhesion

-Antigen

-Clinical isolate (up-regulated gene)

-Effacing

-Effector

-Hemolysin

-Host barriers

-Iron acquisition

-Genomic island

-Regulator

-Secretion

-Toxins

-Epithelial (up-regulated gene)

-Norepinephrine (up-regulated gene)

adhesion23%

antigen3%

effector14%

iron acquisition3%

pathogen island4%

toxins1%

upregulated with norepinephrine

9%

hemolysin2%

defense from host barriers8%

secretion systems14%

upregulated with epithelial cells

12%

upregulated in human

clinical isolates5%

regulator1%

effacing1%

Known or putative virulence factors for E. coli O157:H7 separated into categories (n=394 genes)

Many pathogenic strains of E. coli O157:H7

• Differ in degree of virulence

• Most virulence factors are not yet known

• How can we begin to make hypotheses about which genes

play roles in the virulence of different strains?

-Determination of the complete E. coli sequence required almost 6 years.

-E. coli K-12 is a normal inhabitant of our intestinal tract. The presence of these good bacteria helps to prevent bad bacteria from colonizing there.

-E. coli is the preferred model in biochemical genetics, molecular biology, and biotechnology and its genomic characterization will undoubtedly further research toward a more complete understanding of this important experimental, medical, and industrial organism.

(Blattner et al. Science 1997)

The first E. coli genome sequenced: the non-pathogenic E. coli K-12 genome strain MG1655

(Perna et al. Nature 2001)

-In 1982, Escherichia coli O157:H7 recognized as a pathogen for human disease

-Also known as EDL933 from the 1982 Michigan outbreak from ground beef

-Shiga toxin producing (STEC)

Epidemiological statistics

-47 sickened individuals, no deaths

The first pathogenic E. coli genome sequence: enterohaemorrhagic (EHEC) Escherichia coli O157:H7

strain 933 EDL

E. coli K-12:

4,140 genes

4,639,675 bp

E. coli O157:H7

5,142 genes

5,528,423 bp

~1,000 extra genes in the genome of E. coli

O157:H7

Where are the differences?

Comparison of E. coli K-12 vs. E. coli O157:H7

EDL933 islands

Backbone

-Areas in red are only found in E. coli K12

-Areas in orange are E. coli O157:H7-specific

-Areas in blue are conserved in both

-Regions that are unique to one genome are called “islands”

Genome Comparison of E. coli O157:H7 EDL933 (pathogenic) vs. E. coli K-12 MG1655 (non-pathogenic)

K-12 islands

(unpublished image provided courtesy of Nicole T. Perna)

Pathogenicity islands

• Comparison of the genome structire of pathogenic vs. non-pathogenic strains indicate that pathogenesis is associated with multi-gene “islands”

• The functions of some of the genes could be inferred using BLAST– genes encoding type III secretion system,

adhesins, and phage- and plasmid-encoded genes

(Hayashi et al. DNA Res 2001)

-In July 1996, an outbreak of E. coli O157:H7 infection occurred among schoolchildren in Sakai City, Osaka, Japan.

Epidemiological statistics

-8,938 schoolchildren sickened, 3 deaths

- We are starting to ask: What genomic differences determine differences in virulence, epidemiology, and fatality?

The completion of the 2nd E. coli O157:H7 (EHEC) genome sequence (strain Sakai)

2006 E. coli O157:H7 outbreak from bagged spinach

-multi-state outbreak

Epidemiological Statistics (from CDC)

205 people sickened, 3 deaths

-Produce-associated outbreak strains caused higher incidence of hemolytic-uremic syndrome (HUS)

(Manning et al. PNAS 2008)

-Genome alignments can be used to find variations

Background information regarding epidemiology of E. coli O157:H7 outbreaks

(Figures addressing these topics can be found in the work of Rangel et al. 2005)

-E. coli O157:H7 outbreaks by year 1982-2002

-Median size of E. coli O157:H7 outbreaks by year

-Transmission routes of E. coli O157:H7 outbreaks by year

-Vehicles of foodborne E. coli O157:H7 outbreaks by year

-Hemolytic uremic syndrome (HUS) and case-fatality rate per 100 outbreak-related illnesses

Do genomes change over time?

-Yes, a genome sequence represents a “snapshot” at the time the strain was sequenced. Genomes may vary ~15% or more over time

-For example, E. coli O157:H7 strain EDL933 originally had two copies of the tellurite resistance- and adherence-conferring island (TAI) pathogenicity island.

-In the lab, it has lost one of the TAI pathogenicity islands (Nicole T Perna unpublished data)

-Do the other two genomes of E. coli O157:H7 have one or both of these islands?

-Comparative genomics allow us to start understanding differences that have occurred over time and geographical differences, and to investigate variations in the genomes that may have implications for virulence differences

As of 2010, there are 15 E. coli O157:H7 genomes sequenced. We will have you focus on three that are all in the ASAP database

(http://asap.ahabs.wisc.edu/asap/home.php)

The three strains you will focus on are:

Escherichia coli EDL933 (EHEC) [ground beef 1982 outbreak]

Escherichia coli Sakai (EHEC) [radish sprouts 1996 outbreak]

Escherichia coli EC4042 (EHEC) [fresh bagged spinach 2006 outbreak]

Comparative genomics using the tool Mauve: Multiple Genome Aligner

• Able to identify conserved regions of multiple genomes even in the presence of rearrangements

(Darling et al. 2004)

Student individual projects

Investigate a known or putative virulence factor to determine if it is conserved, and if it occurs in the same place in the genomes of the three E. coli O157:H7 strains.

Also using the Mauve alignment, look for islands unique to 1 out of 3, and 2 out of the 3 E. coli O157:H7 strains

-Things to consider when analyzing genome comparisons:

#1) Are there virulence factors that have been lost or gained in the different genomic islands?

#2) Could these genes present in these genomic islands play a role in one of the virulence mechanisms?

Here is a list of a few virulence factors

ABH-0027064 stx1B

ABH-0027065 stx1A

ABH-0025275 stx2A

ABH-0025276 stx2B

ABH-0028752 eae

ABH-0028754 tir

ABH-0024965 ureD

ABH-0026806 tccP

ABH-0025423 iha

ABH-0024997 terC

ABH-0028614 lpfC

ABH-0026805 espJ

For more putative and known virulence factors (394 identified) see attached excel spreadsheet called:

Supplemental data 1 Virulence factor list for E coli O157:H7 strain EDL933