Pathogenic E. coli

• Microbial diseases continue to present a major threat to human health
• Virulence factors

  Factors that contribute to the ability of a microorganism to cause disease
• Examples of bacterial virulence factors

  • Membrane proteins (adhesins, invasins)
  • Polysaccharide capsules
  • Secreted proteins (toxins)
  • Injected proteins (effectors, T3SS, T4SS, T6SS)
  • Outer membrane components (LPS)
• Koch's Postulates

  Traditional method to test if a bacteria causes a disease
• Molecular Koch's Postulates

  Modern approach to identify virulence genes and study pathogenesis using molecular biology techniques
• Molecular Koch's Postulates

  1. Virulence gene must be present in pathogenic bacteria but absent in non-pathogenic

  2. Disruption of virulence gene should lead to loss of pathogenicity

  3. Virulence gene should be expressed during infection

  4. Complementation should restore pathogenicity
• Approaches to study pathogenesis using molecular Koch's postulates

  1. Whole genome not known - screen for virulence factors by random mutagenesis

  2. Whole genome known - identify virulence factors by bioinformatics and target mutagenesis
• EPEC (Enteropathogenic Escherichia coli) can induce a cytopathic effect (CPE) in infected cells, blocking the cell cycle
• Investigating EPEC pathogenesis

  1. Random mutagenesis of EPEC to identify mutants defective in CPE

  2. Identify the gene disrupted in CPE-defective mutants as the Cycle Inhibiting Factor (Cif)

  3. Cif is encoded by a prophage, not the LEE pathogenicity island

  4. Complementation of cif mutant restores the CPE phenotype
• Cif is a novel virulence factor that contributes to the pathogenesis of EPEC and EHEC by blocking the cell cycle of infected cells
• orf1, orf3 and rorf1 are similar to lambda-phages genes present in EHEC O157:H7 strains EDL933 and Sakai (now identified as type III effectors)
• Genes ybhB and bioA are part of the core genome (common to all E coli)
• Molecular Koch's Postulates

  • Virulence gene must be present in the bacteria that cause disease but absent in bacteria that don't cause disease
  • Virulence gene should be expressed during infection (Human or animal models)
  • complementation or allelic replacement of the disrupted/mutated gene should lead to restoration of pathogenicity
• pBRCif

  bacterial plasmid expressing Cif protein
• Cloned cif gene restores the CPE in cif mutant and CPE negatives EPEC and EHEC strains
• Cif is expressed during cell infection and is targeted to the nucleus of infected cells
• Cif is a new effector molecule injected in the host cell through the Type 3 secretion system
• Cif is a member of the new family of virulence factors called Cyclomodulins
• EPEC and EHEC induce inflammatory response in the initial stage of infection
• Stimuli that activate NF-kB

  • pathogens
  • stress signals
  • cytokines (TNFa…)
• NF-kB inflammatory pathway


  1. Activation of IKK complex (IkB kinases)

  2. Phosphorylation of IkB

  3. Ubiquitination and degradation of IkB

  4. Translocation of free NF-kB to the nucleus

  5. Transcriptional activation of NF-kB dependent genes
• EPEC downregulate NF-kB in J774 in a T3SS dependant process
• EPEC uses its type 3 secretion system to suppress cytokine secretion in monocyte derived dendritic cells
• Effectors encoded on Island 4 are required for inhibition of IL-8 secretion in infected dendritic cells
• NleE effector inhibits pro-inflammatory response in EPEC infected dentritic cells
• EPEC and EHEC T3SS effectors that inhibit NF-kB

  • NleE: Blocks translocation of activated NF-kB to the nucleus. Inactivates TAB2/3 adaptor proteins by methylation.
  • NleC: Protease that cleaves and inactives NF-kB p65 subunit.
  • NleH: By binding to RPS3,regulator of NF-kB dependent transcription, NleH1 reduces its nuclear abundance and activation of NF-kB. NleH2 induce RPS3 translocation.
  • Tir: enhance association of inhibitors SHP1/2 with TRAF6
  • NleB: Inhibits NF-kB activation by inhibition of cell death receptor pathway (TNF-receptor).
• T3SS essential for virulence – substrates of T3SS are called effectors
• T3SS EPEC-EHEC – Essential for attaching-effacing (A/E) lesions
• Genomics and genome sequencing allow to address a whole range of questions including the study of pathogenicity but also outbreak analysis or antimicrobial resistance to determine what drugs can be effective