Week 5

• Bacterial Tyrosine Kinase (BY-Kinases) are kinases unique to only bacteria. Note that they are membrane-embedded proteins with a catalytic kinase domain facing the cytosol

  
• BY-Kinases play a role in various physiological processes but often regulate capsule and polysaccharide production. Since they are kinases they autophosphorylate and phosphorylate other proteins within the cell

  
• BY-Kinases often have an associated protein tyrosine phosphatase (PTP) that can modulate the kinase activity

  
• Lipid A is unique to gram-negative bacteria. It is a key component for creating lipopolysaccharide

  
• Lipopolysaccharide (LPS) is made up of 4 subunits: Lipid A, Inner core, Outer core and O antigen.

  
• Bacterial capsule is made up of 3 different subunits: O antigen, Core, and phosphatidylglycerol.

  
• Bacterial capsules are much longer than LPS. different antigens are needed to mark different types of capsules.

  
• gfc A-E, etp and etk are the group 4 genes required for making group 4 capsules. gfc A is the regulator gene, gfcB is the assembly gene, gfc C and D are the transport gene, gfceE is the outer membrane exporter, etp is the phosphatase gene, and etk is the BY-kinase.

  
• The 5 steps of group 4 capsule biosynthesis.
• The 5 steps of capsule 4 Biosynthesis.

  1. the process starts with WbaP as the priming for glycosyltransferase which takes und-PP
  2. the und-PP is then flipped by the Wzx flippase into the periplasm.
  3. Wzy is a ligase that creates the repeat of saccharide unit by ligation
  4. transport of the polysaccharide is done by Etk,Etp, and the GfcE exporter.
  5. the capsule unit is then ligated to the extracellular leaflet by GfcD.

  
• Etp and Etk proteins are needed for Capsule 4 production.

  
• Etk regulates capsule assembly and export by modulating the activity of Wzy (polymerase) and GfcE (outer membrane transporter).

  
• Etk is activated in its dephosphorylated octameric form, while deacticated in its phosphorylated monomeric form. Capsule production and export require continuous Etk cycling between phosphorylation and dephosphorylation states with the help of Etp.

  
• Etk can phosphorylate Etp to modulate function. Phosphorylated Etp can dephosphorylate Etp to restore its activated function. Etk in its monomeric form has increased kinase activity and it phosphorylates Etp.

  
• Entropathogenic and enterohemorrhagic E.coli (EPEC and EHEC) cause acute gastroenteritis. To do this EPEC and EHEc have to endure 3 distinct environments during infection

  1. Stomach - acid stress
  2. Small intestine - antimicrobial peptides (where the capsule is most useful)
  3. Large Intestine - lower oxygen, lower iron levels, microbiome.

  
• Paneth cells in the small intestine secrete anti-microbial peptides (AMP). Panteth cells lie at the bottom of the intestinal crypts. The need for AMPs is to protect the stem cells from good and bad bacteria.

  
• The intestinal barrier is a highly organized mucosal surface that prevents the entry of microbes into the lamina propria. The epithelium is constructed of a single layer of intestinal epithelial cells (IECs). A mucus layer covers the IEC layer and is mainly devoid of microbes. The inner mucus layer contains IgA and AMPs.

  
• AMPs can be alpha helices or beta sheets in nature. AMPs are typically cationic and contain fewer than 50 amino acids. AMPs are the first line of defence against pathogens in the gastrointestinal tract. AMPs work through different mechanisms but often target the disruption of the bacterial membrane.

  
• Human Defensins (HDs) are well characterized beta sheet AMPs

  
• LL-37 is a well-characterized alpha-helical AMP.

  
• Deletion of gfcA (capsule 4 regulator gene) results in a significant increase in EHEC killing by HD5 (human defensins 5), in a concentration-dependent manner.

  
• EHEC has a type 3 secretion system that is required for infection of host enterocytes. The type 3 secretion system allows of intimate attachment to host cells, and host cell effacement of microvilli. Group 4 capsule must be turned off for EHEC to associate with the host cell

  
• BY-kinases play a role in various biological processes in a transcription factor independent manner

  
• BY-kinases are often associated with the production (or regulation) of surface-layer polysaccharides.

  
• Group 4 capsule production in EHEC is regulated by Etk and Etp.

  
• Group 4 capsule allows EHEC to withstand killing from AMPs, such as HD5 produced by Paneth cells.

  
• Group 4 capsule needs to be turned off in order for EHEC to infect the cecum of the host using the type 3 secretion system.