Week 4/5: Prokaryotic Tyrosine Phosphorylation (BY Kinases)

• bacterial tyrosine kinases (BY kinases) are membrane bound proteins with a catalytic kinase domain in the cytosol only in bacteria, play role in regulating capsule and polysaccharide production (making surface layer polysaccharides)

  
• BY kinases can auto phosphorylate and phosphorylate other proteins in the cell, have an associated Protein Tyrosine Phosphatase (PTP) that modulates kinase activity

  
• gram negative cell wall layers:

  inner membrane - periplasm - outer membrane - LPS (glycolipid) - capsular polysaccharide (CPS)

  • BY kinases regulate the CPS layer

  
• lipopolysaccharides (LPS) have 3 components which are essential for structural integrity of gram negative membranes

  • lipid A -> embeds in outer membrane
  • inner/outer core oligosaccharide
  • O antigen -> repeated units of sugars (molecular signature for every bacterial strain)

  
• capsule CPS have 3 components directly linked to membrane

  • phosphatidylglycerol (PG)
  • core
  • O/K antigen depending on capsule type
  • O antigen is group 4 capsule which uses the same sugar as O antigen but differs in membrane anchoring
  • K antigen is group 1 capsule which differs from the LPS repeat unit

  
• the genes for Group 4 Capsule (GFC) are

  • gfcABCD (regulator, assembly, transport)
  • gfcE (outer membrane exporter)
  • etp (PTP)
  • etk (BY kinase)

  
• 5 general steps for GFC biosynthetic complex in E. coli

  1. repeat saccharide unit synthesis
  2. WbaP -> priming glycosyltransferase in the inner membrane
  3. cytosolic domain binds lipid (Undecaprenyl phosphate/UndPP)
  4. flipping across membrane to periplasm
  5. Wzx -> flippase
  6. repeat saccharide unit ligation
  7. Wzy -> repeat unit ligase -> attaches sugars together and recycles lipid
  8. transport and secretion
  9. Etk/Etp/GfcE (kinase/phosphatase/exporter)
  10. ligation to extracellular leaflet of membrane
  11. GfcD

  
• Etk and Etp are required for GFC biosynthesis in E. coli not LPS synthesis

  
• model for role of Etp/Etk in GFC formation

  • Etk regulates capsule assembly and export by modulating Wzy polymerase and GfcE OM transporter (its adjacents)
  • GFC production and export needs continuous Etk cycling between its states
  • phosphorylated (multi) Etk -> monomer, closed
  • dephosphorylated Etk -> octamer to form open pore with GfcE exporter to allow GFC out of cell
  • as such: monomeric phos Etk activates kinase activity and phosphorylates Etp to dephosphorylate Etk and make it octameric
  • Etk can phosphorylate Etp to modulate function

  
• enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli cause acute gastroenteriritis, have to endure 3 distinct environments during infection of humans

  • stomach -> acid stress
  • small intestine -> antimicrobial peptides (AMP)
  • large intestine -> lower oxygen and iron levels, microbiome

  
• intestinal epithelial cells (IEC) form the epithelium in a single layer which is covered by a mucus layer and is devoid of microbes whose inner mucus layer contains IgA and AMP (secreted by paneth cells)

  
• AMP made by the host prevent bacterial colonization in IEC crypts

  • can be alpha helical (LL-37) or beta sheet (human defensins)
  • usually cationic and less than 50 AA
  • are the first line of defense against pathogens in the GI tract
  • work through different mechanisms but usually target the disruption of bacterial membrane

  
• the deletion of gfcA gene results in significant increase of killing by HD5 AMP in concentration dependent manner, the presence of GFC on bacterial membranes prevent HD5 from disrupting these membranes

  
• bacteria don't need to produce GFC throughout the GI tract for ex

  • EHEC have type 3 secretion system for infecting host cells by allowing intimate attachment to cells and host cell effacement of microvilli -> GFC must be off for this to happen
  • *LPS is still present but no GFC
• BY kinases play roles in TF independent manner
• Etk and Etp regulate GFC production by its octameric model
• BY kinases have a distinct active site for auto phosphorylation, they are monomeric/dissociate from octameric form when phosphorylated on their allosteric C terminal tyrosine rich tail site