Inhibition Cell wall syntheses and division in E.coli Flashcards
Inhibitors cell wall
Penicillin Cephalosporins Carbapenem Monobactam Glycopeptides Lipopeptides
Inhibitors RNA / Protein synthesis
Rifamycin Aminoglycoside Tetracyclines Macrolides Chloramphenicol Lincosamides Mupirocin Streptogramin Oxazolidinones Pleuromutilins
Cell division E.coli
Elongation»_space; Z-ring formation»_space; Divisome assembly»_space; Constriction»_space; Dell division
All steps can be targeted
Function peptidoglycan layer
And target
Gives strengt to bacterial wall
Mesh network of sugar and peptides
» Peptidoglycan down > Not a good shape and bacteria will die
Trans glycosylation
Bind sugars in long strand
PBP catalyse this reaction
Transpeptidation
Bind neighbour sugars with peptide bonds
PBP catalyse this reaction
Penicillin
Competition reaction: With D-ALA:
Binds in pocket region of transpeptidation reaction takes place > Breaks bond > Inhibit elongation
Most important targets in periplasm because:
Membrane enzymes are interesting targets. They’re easy to reach
Derogation
Afbraak peptidoglycan
Because it’s a dynamic proces
Elefheria terrae and its target
- It’s a gramnegative bacterium against gram positive bacteria. Tested for antimicrobial activity:
- Soil bacteria with Lipid II target peptidoglycan
- Antibiotic can’t pass outer membrane
- Couldn’t find resistance mechanism in bacteria
Peptidoglycan synthesis
2 models:
1.Takes place during elongation: Inserted in cylindrial wall
2. During division: Complete new synthesis of the cell poles
Different PBPs and different enzymes during this processes
MreB
Peripheral membrane protein at membrane in helical way:
- Binds to enzymes that are important for peptidoglycan
- Helps localising the elongasome
- Without MreB: Structure will not form > Bacteria grows in spheres and die
FTS-Z and Z-ring
Indispensible cellular entity (onmisbaar voor de cel)
Formation of Z-ring in middle of elongated cell
- The ring binds to the membrane via Zap A and FtsA
- Polymerizes to form a contractile ring
- GTP hydrolyzes : GTP couples to FtsZ monomers head to tail and forms a ring
> The ring recruit division proteins (ZipA , FtsK)
> The diameter decreases in this proces
»_space; After cell division you get disassembly (demontage)
Membrane protein FTS-Q in biogenesis E.coli:
Onderzocht by site specific photo crosslinking.
In a study of interactions of proteins.
- Membrane protein with only one transmembrane segment.
Involved in cell division:
- Best protein in biogenesis
- Connects all major players: Alle major players must work together otherwise it won’t work.
- Recruits all proteins to division site
- Interacts with FTS-b and FTS-L «_space;Drug targets
FTS-Z Advantages as target (5)
- Target (Z-ring) is essential for bacteria
- Broad range antibiotics
- Catalytic activity when binding of ligands
- Developing resistance is not easy
- Structure is known
FTS-Z Disadvantages as target
- Has a human homologue (Tubulin)
- In cytosol (location isn’t great)
- You need lots of this protein > So also need lots of antibiotics «_space;It’s abundant (Z-ring used a-lot)
FTS-Z inhibitors
Structure based design: Look to structure to inhibit
None has made market yet.
FTS-Z dimer interface:
- Analogue of GTP (morpholino-8GTP) replaces real GTP > Little bigger and inhibits polymerisation
- No normal Z-ring
How FTS-Z knows were to form Z-ring
2 mechanisms
- Min system: Minc is polymerisation inhibitor and oscillates between poles.
Low midcell [MinC] allows FTS-Z ring formation and inhibits FtsZ polymerisation
2 Nucleoid occlusion (opstopping): SlmA bound chromosome and inhibits FTS-Z.
Normal role of nucleoid occlusion prevent the genetic material in E.coli & SlimA also plays temporal role in organisation of division
Binary fission process
Prokaryotic Cell division
- Divide Cell in two equal parts
- Also happens in our mitochondrien
PBP3:
Is an transpeptidase crosslinks peptidoglycan polymers.
Essential for cell division
Divisome and cell division
Recruitment of division proteins is hierarchical
- FTS-Z is the first protein to localize the division site
- FTS-Q : Connects all divisome proteins but function unknown.
FTS-Q as target
When mutations in FTS-Q > Typical filamentous growth of cells > Cells stop dividing and grow as filaments
FTS-Q as target Advantages
- Target essential for survival
- Catalytic activity / binding ligands
- Broad range antibiotics
- Hasn’t a close human homologue
- Developing resistant isn’t easy
- Toegankelijk for drugs: Subcellular location (Key interactions of FtsQ/FtsB/FtsL take place in periplasm)
- Structure is known (crystal structure available)
- Not abundant so low [drug] is enough
FTS-Q as target Disadvantages
- Catalytic activity / Binding of ligands
Finding new antibiotics in soil
- Culture soil bacteria in soil (mimic their environment)
- Plate with chip: Sealed in platcid that is permeable to small nutrients > Nutrients can go soil in
- But antibiotics that are produced can’t pass the soil and remain in the welsh
- Test extracts for antimicrobial activity
Elongasome
Complex that catalyzes peptidoglycan biosynthesis
«_space;MreB helps localizing the elongasome
Development FtsZ inhibitors:
In vitro assay/ In vivo screening:
- In vitro: Screen for polymerisation & GTPase activity
- In vivo screening: Compounds that affect membrane potential (indirect effect through decreased membrane association of FtsA)
Screen for interaction ZipA, FtsA
Look to effects on host > Toxicity and permeability of cel enveloppe
FTSQ interactions
Upstream interactions take place in alpha domain (FtsK)
Downstream interactions take place in beta domain (FtsB/FtsL)
Medicinal chemistry to find inhibitors for FtsQ
- Screen small molecules that bind FtsQ
- Test hits for inhibition
- Optimize hits for inhibition cell division
- Test efficacy of the molecules
