INF1 - F. BACTERIAL WALL SYNTHESIS AND TARGETING BY ANTIBIOTICS-COVERED

Question 1

what is the structure of peptidoglycan monomer

Answer 1

2 amino sugars
- NAG
- NAM
- NAG-NAM joined into polymer chains via a glycosidic bond

• 5 amino acids (pentane-tide) attached to NAM, including 2 terminal D-alanines but one D-alanine is cleaved during crosslinking assembly of cell wall
  (some gram +ve bacteria - 5 glycine amino acids added to NAM-pentapeptide aswell)
• polymer chains interlocked via the NAM-tetrapeptides either directly or via an extra sequence of approximately 5 glycine amino acids

Question 2

what is the process of cross-linking peptidoglycan chains called

Answer 2

transpeptidation
(amount of cross-linking varies)

Question 3

peptidoglycan biosynthesis in cytoplasm of cell

Answer 3

1. NAM synthesised from NAG by addition of lactic acid derived from phosphoenolpyruvate (PEP) and UDP used as an energy source, losing inorganic phosphate in process (attached at all times)
   Enolpyruvate transferase aids this
   Fosfomycin blocks this
2. 3 amino acids added to NAM:
   L-alanine
   D-glutamic acid and either
   L-lysine (gram +ve)
   mDap - diaminopimelic (gram -ve)
   Position 3 is a diamino for cross-linking
3. L-alanine converted to D-alanine by a racemes enzyme. 2 D-alanines added by a ligase enzyme to form the NAM-pentapeptide
   1 D-alanine cleaved off as an energy source to drive cross-linking between polymer chains
   D-cycloserine blocks D-alanine addition

Question 4

transport of peptidoglycan across cell membrane via lipid carrier

Answer 4

4. NAM pentapeptide passed to a lipid carrier bactoprenol in cell membrane
   Bacitracin blocks this - no longer licensed in UK
5. NAG added to NAM-pentapeptide forming NAG-NAM-pentapeptide
   (gram +ve: pentaglycine bridge may beg added to pentapeptide)
6. lipid carrier binds to NAG-NAM-pentapeptide and takes it across cell membrane
7. after delivering NAG-NAM-pentapeptide across cell membrane, carrier is recycled back to pick up more NAG-NAM monomers

Question 5

insertion and cross-linking of peptidoglycan in cell wall

Answer 5

8. NAG-NAM pentapeptide arrives at cell wall and linked to peptidoglycan polymer by enzyme peptidoglycan glycosyltransferases located on outer membrane of cytoplasmic membrane, building the polymer chains
   Vancomycin and Teicoplanin bind to D-alanyl-D-alanine dipeptide which blocks glycosidic bond formation
9. Transpeptidase (in outer membrane of cell membrane) cross-links pentapeptides. Terminal D-alanine is removed, energy generated used to cross-link new peptidoglycan monomer to third amino cid of existing cross-linked peptidoglycan. Gram +ve: pentaglycine sequence forms cross-bridge
   Beta-lactams (pencillins, cephalosporins, monobactams, carbapenems) block transpeptidase and hence cross-linking

Question 6

what activity do most of antibiotics that block cell wall display

Answer 6

Bacteriocidal - kill bacterium
Those that block translation may be bacteriostatic - stop bacteria growing but still alive effectively
Allows time for immune system to clear up the living bacteria

Question 7

how does fosfomycin work

Answer 7

• PEP analogue that inhibits enzymes that convert NAG to NAM
• needs to be taken into bacterial cytoplasm
• active uptake via transporters to acquire nutrients eg-glucose
• especially effective against gram -ve bacteria that infect urinary tract including E. coli as it is excreted unchanged in urine ie - not metabolised by body
• antibacterial synergy with beta-lactams, aminoglycosides (target protein synthesis) and fluoroquinolines (target DNA synthesis)
• resistance due to altered transporter activity/reduced uptake/mutation in enolpyruvate transferase

Question 8

how does D-cycloserine work

Answer 8

• acts as a structural homologue for D-alanine inhibiting:
  racemase that converts L-alanine to D form
  ligase that couples D-alanyl-D-alanine dipeptide
• used as a 2nd line antibiotic for treatment of TB
• resistance due to over expression of alanine racemase

Question 9

how do glycopeptides (vancomycin and teicoplanin) work

Answer 9

• affect polymerisation of peptidoglycan, bactericidal
• transglycosylase assembles linear glycol chains in cell wall by transferring growing glycan chain to disaccharide peptidoglycan precursor on lipid carrier as it crosses cell membrane
• glycopeptides block this by binding to D-alanyl-D-alanine dipeptide which prevents binding and function of TG-ase
• resistance due to DNA encoding enzymes that catalyse formation of D-Ala-D-lactacte instead of D-Ala-D-Ala so it no longer binds
• Vancomycin = complex tricyclic glycopeptide. Large so can’t penetrate outer membrane of gram -ve bacteria and not effect against them.
  Treats gram +ve infections - MRSA and C. difficile

Question 10

how do beta-lactams work

Answer 10

• bactericidal
• inhibit transpeptidase enzymes (PBP) involved in cross-linking
• mimic D-alanyl-D-alanine residues (where enzyme binds for cross-linking so antibiotic occupies active site and D…residues can’t bind)
• beta-lactam ring broken, enzyme inactivated
• destabilised cell wall causing cell wall to become leaky, osmotic burst

Question 11

what are the 4 subclasses of beta-lactams

Answer 11

• penicillins
• cephalosporins
• monobactams
• carbapenems
  all have different chemical substituents attached to ring
  main side effects are associated with allergic reactions (rashes to anaphylaxis, sickness, diarrhoea)

Question 12

what determines if an antibiotic is broad-spectrum for narrow-spectrum

Answer 12

properties of antibiotic and its uptake by bacteria

Question 13

what type of antibiotics pass through OM of gram -ve bacteria more easier

Answer 13

hydrophilic agents due to hydrophilic fatty acids

Question 14

what spectrum of activity do hydrophilic agents have

Answer 14

broad spectrum
ampicillin, amoxicillin, piperacillin

Question 15

what spectrum of activity do hydrophobic agents have

Answer 15

narrow spectrum
flucloxacillin, penicillin

Question 16

where do penicillins come from

Answer 16

naturally occurring from moulds penicillium notatum and penicillium chrysogenum

Question 17

what is the biggest cause of resistance to beta-lactams

Answer 17

beta-lactamases

Question 18

what structure do cephlasporins have

Answer 18

beta-lactam ring but more accessible and better targeted to actual site of infection via 2 side chains (R1 and R2) which alter stability, PKs, resistance breakdown

Question 19

what type of activity does cephlasporins display

Answer 19

broad-spectrum
treats septicaemia, meningitis, UTIs

Question 20

why were carbapenems developed

Answer 20

to avoid issues of developing reistance to penicillins and cephlasporins (4 drugs)

Question 21

what is the structure of carbapenems

Answer 21

derivatives of penicillin and cephalosporin where S atom is replaced with a C atom (R1R2 R3)

*stable against most beta-lactamases, not effective against MRSA

Question 22

what activity do cephlasporins display

Answer 22

broad spectrum
treat septicaemia and severe HCAI
reserved for severe infections

Question 23

what is the only drug in monobactams

Answer 23

aztreonam

Question 24

what are monobactams active against

Answer 24

gram-ve bacteria
useful for those with penicillin allergy

Question 25

what bacteria are resistant to aztreonam

Answer 25

gram -ve bacteria with extended-spectrum beta-lactamases (super-enzymes)

Question 26

what is antibiotic resistance encoded by

Answer 26

chromosomal or acquired (extrinsic) genes

Question 27

what type of resistance does gram +ve bacteria show

Answer 27

• chromosomal mutation in a transpeptidase encoding gene
• stops transpeptidases ability to bind to particular beta-lactam
• or by acquisition of a gene encoding a transpeptidase with low affinity for the beta-lactam

Question 28

where is resistance associated with beta-lactamases encoded

Answer 28

chromosome or plasmid

Question 29

where are beta-lactamases located

Answer 29

periplasmic space

Question 30

what decoy agents are used in antibiotics for beta-lactamases

Answer 30

clavulanic acid