Week 5

• Bacterial Tyrosine Kinases: these are a unique class of protein kinases that are only found in bacteria. They are membrane-embedded proteins with a catalytic kinase domain in the cytosol.

  
• Bacterial Tyrosine Kinases play a role in various physiological processes but often regulate capsule and polysaccharide production.
• Bacterial Tyrosine Kinases autophosphorylate and phosphorylate other proteins within the cell. They often have an associated protein tyrosine phosphatase (PTP) that can modulate the kinase activity.

  
• Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS). LPS is made up of Lipid A, inner core, outer core, and a repeated sequence of O antigen.

  
• LPS is the signature of bacterial strain.

  
• Lipid A has its specific antigen depending on what type of capsule it is.

  
• group 4 capsule genes: gfc A-E, etp, and etk

  
• group 4 capsule: gfc A-D genes are for regulation, assembly, and transport. gfcE is the gene for outer membrane exporters. etp is the gene that makes the phosphatase while etk makes the BY-kinase (bacterial tyrosine kinase).
• group four capsule (GFC) biosynthetic complex in E.coli: [1] Repeat saccharide unit synthesis (WbaP is the priming glycosyltransferase) [2] Flipping across the membrane (Wzx is the flippase) [3] Repeat saccharide unit ligation (Wzy is the repeat unit ligase [4] Transport and secretion (Etk/Etp/GfcE) [5] Ligation to extracellular leaflet of membrane (GfcD?)

  
• Etk and Etp are required for group 4 capsule biosynthesis. An SDS-Page + Western blot and dot blot was done to etp and etk genes. the deletion of either etk or etp shows that no capsule was made. Wt and compliment provided a capsule although the compliment capsules did not look the same as the Wt.

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

  
• multi-phosphorylated Etk is found as a monomer while dephosphorylated Etk is found as an octamer

  
• Capsule production and export requires continuous Etk cycling between phosphorylated and dephosphorylated states

  
• Etk can phosphorylate Etp to modulate function. Etk is turned on by dephosphorylation of Etp, and Etp is turned on by phosphorylation of Etk. Note that Etk is OFF when phosphorylated.

  
• EPEC and EHEC have to endure 3 distinct environments during infection. [1] Stomach - acid stress [2] Small intestine - antimicrobial peptides [3] Large intestine - lower oxygen, lower iron levels, microbiome

  
• Paneth cells in the small intestine secrete anti-microbial peptides (AMPs)

  
• The intestinal barrier is a highly organized mucosal surface that entry of microbes into the lamina propria.

  
• The small intestine 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

  
• A mucus layer covers the IEC layer and is mainly devoid of microbes. The inner mucus layer contains immunoglobulin A (IgA) and antimicrobial peptides (AMPs)

  
• Anti-microbial peptides (AMPs) made by the host prevent bacterial colonization in IEC crypts.

  
• AMPs can be a-helical or B-sheet in nature. LL-37 is a well-characterized a-helical AMP. Human Defensins (HDs) are well-characterized B-sheets AMPs.

  
• AMPs are typically cationic and contain fewer than 50 amino acids.

  
• AMPs are the first line of defence against pathogens in the gastrointestinal tract. They work through different mechanisms, but often target the disruption of the bacterial membrane

  
• Group 4 capsule production protects E.coli from host-derived antimicrobial peptides. Human defensin 5 (HD-5) is a host-derived antimicrobial peptide (AMP). Deletion of gfcA results in a significant increase in EHEC killing by HD5, in a concentration-dependent manner

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