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

2. 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)

3. 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

4. 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

5. 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

6. 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)

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

8. 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?

9. -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

10. (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

11. 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

12. 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)

13. 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

14. (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 2 nd E. coli O157:H7 (EHEC) genome sequence (strain Sakai)

15. 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

16. 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

17. 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

18. 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]

19. 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)

20. 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?

21. 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