Drug Mech: Antibiotics 2

The bacterial cell wall, a rigid outer layer, is not found in animal cells. It completely surrounds the cytoplasmic membrane, maintaining the shape of the cell and preventing cell lysis.

• Beta Lactams (Penicillins, Cephalosporins, etc.)
• Vancomycin
• Cycloserine
• Bacitracin
• Fosfomycin
• Dalbavancin
• Telavancin

• Gram-Positive Bacterial Cell Wall:
• Less developed biosynthetically.
• Simple cell wall.
• High internal osmolality.
• Very thick peptidoglycan layer.
• Antibiotic agents mostly active in gram positive!

• Gram-Negative Bacterial Cell Wall:
• Highly developed biosynthetically.
• Highly adaptive (more sophisticated cell wall).
• Complex; penetration of antibiotics is often restricted.
• Low internal osmolality.
• Very thin peptidoglycan layer.

The peptidoglycan polymer, also known as murein or mucopeptide, is a complex, cross-linked (rigid) polymer consisting of polysaccharides and polypeptides.

• The linear peptido-polysaccharide chains of the
• polymer contain alternating aminosugars, N-acetylglucosamine and N-acetylmuramic
• acid.

• A five-amino-acid peptide is linked to the N-acetylmuramic acid sugar. This peptide
• terminates in D-alanyl-D-alanine. The peptide side chains in the polymer are cross-linked, which gives the bacterial cell wall its structural rigidity.

The exact amino acid composition of the peptide side chains in the polymer varies among bacterial species. In Staph. aureus, the pentapeptide side chains are linked to each other by pentaglycine bridges (see below).



The linear polymeric chains in peptidoglycan must be cross-linked by transpeptidation of the peptide side chains in order to achieve the necessary strength and rigidity of the peptidoglycan polymer for cell viability.

• Gram Positive vs. Gram Negative Cell Wall
• Recall that for a gram positive bacteria, the peptidoglycan layer is very thick, and therefore there are many peptidoglycan layers sandwiched together in a gram positive cell wall.

• Recall that for a gram negative bacteria, the peptidoglycan layer is very thin, so there may only be one layer of peptidoglycan in a gram negative cell wall.
• Antibiotic Inhibitors of Gram Positive Cell WallLet's assume we are talking about a gram positive cell with a thick peptidoglycan layer because antibiotics that inhibit cell wall synthesis work on gram positive bacteria.

• What is Peptidoglycan?
• Recall that the bacterial cell wall in the domain Bacteria is composed of a rigid, tight-knit molecular complex called peptidoglycan.

Peptidoglycan, also known as murein, is a vast polymer consisting of interlocking chains of identical peptidoglycan monomers. It functions to prevent bacterial osmotic lysis.

• Peptidoglycan Synthesis: The Complete Process
• In order for bacteria to divide by binary fission and increase their size following division, links in the peptidoglycan must be broken, new peptidoglycan monomers must be inserted, and the peptide cross links must be resealed.
• A group of bacterial enzymes called autolysins break the glycosidic bonds between the peptidoglycan monomers at the point of growth along the existing peptidoglycan.
• Autolysins also break the peptide cross-bridges that link the rows of sugar together. In this way, new peptidoglycan monomers can be inserted to enable bacterial growth.

Peptidoglycan monomers are synthesized in the cytosol of the bacterium where they attach to a membrane carrier molecule called bactoprenol. The bactoprenols, which are similar to a "lipid anchors," transport the peptidoglycan monomers across the cytoplasmic membrane and helps insert them into the growing peptidoglycan chains.

• 1) Make peptidoglycan monomers
• First, N-acetylglucosamine (NAG) links up with uridine diphosphate (UDP) to form UDP-NAG. Some of the NAG is enzymatically converted to N-acetylmuramic acid (NAM) forming UDP-NAM.

• 2) Alanine Racemase Step
• Five amino acids are sequentially added to the UDP- NAM forming a pentapeptide. The last two are D-alanine molecules enzymatically produced from L-alanine, the usual form of the amino acid.

The NAM-pentapeptide is attached to the bactoprenol carrier molecule in the cytoplasmic membrane, the energy being supplied by one of the high-energy phosphate groups of the UDP.

The NAG is attached to the NAM-pentapeptide on the bactoprenol to complete the peptidoglycan monomer.

• 3) Bactoprenols help Transportation & Insertion
• Bactoprenols then insert the peptidoglycan monomers into the breaks in the peptidoglycan at the growing point of the cell wall.

• 4) Transglycosylation Step
• Transglycosidase enzymes catalize the formation of glycosidic bonds between the NAM and NAG of the peptidoglycan momomers and the NAG and NAM of the existing peptidoglycan.

• 5) Transpeptidation or Cross-Linking Step
• Finally, transpeptidase enzymes reform the peptide cross-links between the rows and layers of peptidoglycan to make the wall strong.

• Recall the Steps for Peptidoglycan Synthesis
• 1) Make peptidoglycan monomers
• First, N-acetylglucosamine (NAG) links up with uridine diphosphate (UDP) to form UDP-NAG. Some of the NAG is enzymatically converted to N-acetylmuramic acid (NAM) forming UDP-NAM.

• 2) Alanine Racemase Step
• Five amino acids are sequentially added to the UDP- NAM forming a pentapeptide. The last two are D-alanine molecules enzymatically produced from L-alanine, the usual form of the amino acid.

The NAM-pentapeptide is attached to the bactoprenol carrier molecule in the cytoplasmic membrane, the energy being supplied by one of the high-energy phosphate groups of the UDP.

The NAG is attached to the NAM-pentapeptide on the bactoprenol to complete the peptidoglycan monomer.

• 3) Bactoprenols help Transportation & Insertion
• Bactoprenols then insert the peptidoglycan monomers into the breaks in the peptidoglycan at the growing point of the cell wall.

• 4) Transglycosylation Step
• Transglycosidase enzymes catalize the formation of glycosidic bonds between the NAM and NAG of the peptidoglycan momomers and the NAG and NAM of the existing peptidoglycan.

• 5) Transpeptidation or Cross-Linking Step
• Finally, transpeptidase enzymes reform the peptide cross-links between the rows and layers of peptidoglycan to make the wall strong.

• What steps do Antibiotics Inhibit?
• 1) Inhibit the synthesis of peptidoglycan monomers: Fosfomycin

2) Inhibit the Alanine Rasinase Step: Cycloserine

3) Inhibit the Bactoprenols from Transportation & Insertion: Bacitracin

4) Inhibit the Transglycosylation Step: Vancomycin, Dalbavancin, Telavancin

5) Inhibit the Transpeptidation or Cross-Linking Step: b-Lactams, Vancomycin, Dalbavancin, Telavancin

Transpeptidase and Glycosyltranferase

• The Peptidoglycan Layer
• · The peptidoglycan polymer consists of a backbone chain of repeating two-sugar units (NAG and NAM) and a pentapeptide side chain bound to each NAM residue.

· The [NAG-NAM-pentapeptide] core (called ‘Lipid II’) is synthesized in the cell and tethered to the cell membrane by a lipid linker (called ‘Bactoprenol’). Lipid II is then transferred from the inside of the cell to the outside.

• Penicillin Binding Proteins (PBPs)
• · Once outside of the cell, Lipid II is grafted onto the pre-existing cell wall (ie, the pre-existing peptidoglycan polymer) with the help of membrane associated enzymes known as ‘Penicillin-Binding Proteins’ (PBPs) (e.g., PBP1b from E. coli and PBP2 from Staph. aureus). PBPs are found in the cell membrane of all bacteria.

· PBPs are bifunctional enzymes. They contain two separate domains that are connected by a linker. One of the domains is a glycosyltransferase domain; the other domain is a transpeptidase domain.

• Transpepdtidation Step
• · The transpeptidase domain of PBP catalyzes the
• cross-linking (transpeptidation) step in the biosynthesis of peptidoglycan. Only the transpeptidase domain is inhibited by the b-lactam antibiotics. The transpeptidase (C-terminal) domain is positioned on the outside of the cell, where it can find its substrate (the pentapeptide side chains). This is also where the hydrophilic b-lactam antibiotics can interact with the enzyme and block its function.

• Transglycosylation Step
• · The glycosyltransferase (or ‘transglycosylase’)
• domain of PBP catalyzes the transglycosylation step in the biosynthesis of peptidoglycan. The transglycosylation step involves attaching the NAM residue in Lipid II to the NAG residue of the pre existing linear peptidoglycan chain via a glycosidic linkage (a ‘carbon-oxygen-carbon’ linkage). The glycosyltransferase (N-terminal) domain is membrane-bound, where it interacts with its substrate (the aminosugar residues in Lipid II).

Fosfomycin inhibits the conversion of UDP-Nacetylglucosamine to UDP-Nacetylmuramic acid in the cytoplasm by inhibiting enolpyruvate transferase enzyme. It binds covalently to the active site of the enzyme and blocks the addition of phosphoenolpyruvate to UDP-N-acetylglucosamine (which is the first step in the formation of UDP-N-acetylmuramic acid).

• Cycloserine is a structural analog of D-alanine (meaning it is structurally related and has similar chemistry to D-alanine). As a result, it inhibits the incorporation of D-alanine into the peptidoglycan pentapeptide by inhibiting alanine racemase (which converts L-alanine to D-alanine) in the cytoplasm. It also inhibits the cytoplasmic enzyme D-alanyl-D-alanine ligase which is
• responsible for the formation of the dipeptide D-Ala–D-Ala.

• Bacitracin inhibits the dephosphorylation step in the cycling of the lipid carrier (bactoprenol). As a
• result, it inhibits the attachment of N-acetylmuramic acid (and the linear peptidoglycan chain) to the cell membrane.

• Vancomycin binds firmly to the D-Ala–D-Ala
• terminus of nascent peptidoglycan pentapeptide. As a result, vancomycin is able to sterically inhibit both the transglycosylation and transpeptidation steps in bacterial cell wall synthesis. (It is able to do this because it is such a large molecule in size).

• Resistance to vancomycin in enterococci and VRSA is due to modification of the D-Ala–D-Ala binding site of the peptidoglycan building block in which the terminal D-Ala is replaced by D-lactate.
• This results in the loss of a critical H-bond that facilitates high-affinity binding of vancomycin to its target and loss of activity.

• Same as vancomycin. Dalbavancin (Zeven®), a
• lipoglycopeptide, is structurally similar to vancomycin. However, unlike vancomycin, dalbavancin has a long lipophilic side chain attached to its glycopeptides backbone. It has been proposed that the lipophilic side chain serves as a membrane anchor, allowing for enhanced binding and improved antibacterial activity.

• Telavancin (Vibativ®), a lipoglycopeptide, has two mechanisms of action:
• 1) The first mechanism is the same as vancomycin. 2) The second mechanism involves depolarization of the bacterial cell membrane, resulting in disruption of the functional integrity of the membrane. Depolarization of the cell membrane is attributed to the interaction of a long lipophilic side chain in telavancin with the lipid bilayer of the cell membrane.

Beta Lactams inhibit the cross-linking of peptidoglycan polymer chains by inhibiting the transpeptidation reaction.

· PBPs catalyze the transpeptidation reaction (cross-linking) in the biosynthesis of peptidoglycan by removing the terminal D-alanine residue from a peptide side chain to form a crosslink with another nearby peptide side chain. This cross-linking reaction is inhibited by b-lactam antibiotics.





· b-lactam antibiotics are structural analogs of the natural [D-Ala-D-Ala] substrate for PBPs. b lactams bind covalently to the active site of the transpeptidase domain of PBPs, inhibiting the transpeptidation reaction. Consequently, peptidoglycan synthesis is inhibited and the bacterial cell dies. The exact mechanism responsible for cell death is not completely understood, but autolysins (bacterial enzymes that remodel and break down the cell wall) are involved.

• · b-lactam antibiotics and the other inhibitors of bacterial cell wall synthesis are bactericidal only if bacterial cells are actively growing (multiplying/dividing) and synthesizing cell wall.
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