Antibiotics & Mechanisms of Action

Stages of Cell Wall Synthesis

The synthesis of the bacterial cell wall, specifically peptidoglycan (PG), is a complex, multi-stage process involving numerous enzymes, precursor molecules, and regulatory mechanisms. This process is essential for bacterial survival and is a key target for many antibiotics. It can be broadly divided into three main phases:

1. Cytoplasmic Synthesis: This initial stage involves the creation of peptidoglycan precursors within the cytoplasm. It includes the synthesis of UDP-N-acetylglucosamine (UDP-NAG) and UDP-N-acetylmuramic acid (UDP-NAM), as well as the assembly of the pentapeptide chain that is attached to NAM. These precursors are then linked to form UDP-NAM-pentapeptide, a crucial building block for peptidoglycan.

2. Membrane-Associated Synthesis: This stage involves the transfer of UDP-NAM-pentapeptide to the lipid carrier bactoprenol, which is located in the cytoplasmic membrane. The resulting Lipid I intermediate is then glycosylated with NAG to form Lipid II, the final peptidoglycan precursor. Lipid II is then flipped across the membrane by flippases, such as MurJ, to the extracellular side.

3. Extracellular Polymerization: This final stage involves the polymerization and cross-linking of peptidoglycan monomers outside the cell membrane to form the mature cell wall. Transglycosylases polymerize the NAG-NAM subunits to form glycan strands, while transpeptidases (penicillin-binding proteins or PBPs) catalyze the cross-linking of the pentapeptide chains between adjacent glycan strands, providing strength and rigidity to the cell wall.

Antibiotics Targeting Cell Wall Synthesis

Acting Inside the Cell (Cytoplasm)

Several antibiotics interfere with the early stages of peptidoglycan synthesis, targeting enzymes within the cytoplasm. These antibiotics prevent the formation of essential precursors, thereby halting cell wall synthesis.

• Fosfomycin: This is a phosphoenol pyruvate (PEP) analog that inhibits the enzyme pyruvate transferase (MurA). This enzyme is crucial for catalyzing the conversion of N-acetylglucosamine (NAG) to N-acetylmuramic acid (NAM), an essential precursor for peptidoglycan synthesis.

  $NAG + PEP \rightarrow NAM$

  Fosfomycin covalently binds to the active site of MurA, blocking the addition of PEP to UDP-NAG and preventing the formation of UDP-NAM.

• Cycloserine: This is an alanine analog that inhibits two key enzymes: alanine racemase (Alr) and D-alanyl-D-alanine ligase (Ddl). Alr converts L-alanine to D-alanine, while Ddl ligates two D-alanine molecules to form the D-ala-D-ala dipeptide. This dipeptide is necessary for peptidoglycan cross-linking. By inhibiting these enzymes, cycloserine prevents the formation of the D-ala-D-ala dipeptide, disrupting peptidoglycan synthesis.

Acting at the Cell Membrane

Some antibiotics target the lipid carriers and membrane proteins involved in transporting peptidoglycan precursors across the cell membrane. These antibiotics disrupt the translocation of essential building blocks, leading to cell wall defects.

• Bacitracin: This cyclic polypeptide antibiotic inhibits the dephosphorylation of bactoprenol pyrophosphate ($C<em>{55}-PP$) to bactoprenol phosphate ($C</em>{55}-P$). Bactoprenol is a lipid carrier responsible for transporting peptidoglycan subunits (NAM and NAG) across the cytoplasmic membrane. Inhibition of its dephosphorylation prevents the recycling of bactoprenol, halting peptidoglycan synthesis. Without the dephosphorylation, bactoprenol cannot accept new peptidoglycan precursors, effectively stopping cell wall assembly.

  $C<em>{55}-PP + H</em>2O \rightarrow C<em>{55}-P + P</em>i$

• Muraymycin, Tunicamycin, Mureidomycin, Capuramycin, Liposidomycin, Phage QX174 protein E, Humimycin, Phage M lysis protein, DMPI, CDFI, Anthranilic acid analogs: These antibiotics inhibit MraY and MurG, which are essential enzymes involved in the synthesis of Lipid I and II. MraY catalyzes the transfer of phosphoenolpyruvate from UDP-MurNAc to undecaprenyl phosphate, forming Lipid I. MurG then catalyzes the addition of GlcNAc to Lipid I, forming Lipid II. By inhibiting these enzymes, these antibiotics prevent the formation of the Lipid II precursor, disrupting peptidoglycan synthesis.

• Vancomycin, Ramoplanin, Nisin: These antibiotics inhibit transglycosylation, which is the process of adding the NAG-NAM subunit to the growing peptidoglycan chain. Vancomycin binds to the D-alanyl-D-alanine terminus of the NAM/NAG-precursor, preventing the addition of the subunit. Ramoplanin and Nisin work similarly to prevent transglycosylation.

Acting Outside the Cell Membrane

Other antibiotics target enzymes involved in the polymerization and cross-linking of peptidoglycan outside the cell membrane. These antibiotics interfere with the final stages of cell wall assembly, leading to weakened cell walls and cell death.

• β-Lactams (Penicillins, Cephalosporins, Carbapenems, Monobactams): These antibiotics inhibit transpeptidases, also known as penicillin-binding proteins (PBPs), which are essential for cross-linking the peptidoglycan strands. β-lactams mimic the D-ala-D-ala structure, binding to the active site of PBPs and preventing cross-linking. This weakens the cell wall, leading to cell lysis and bacterial cell death.

Antibiotics Targeting the Cell Membrane

Certain antibiotics directly target the bacterial cell membrane, disrupting its integrity and function. These antibiotics cause leakage of cellular contents, leading to cell death.

Polymyxins and Colistin

• These are cationic antibiotics that disrupt the structure of the bacterial cell membrane by interacting with phospholipids, similar to a detergent. They are active against Gram-negative bacteria but have serious side effects, limiting their use to skin and eye infections or as a last resort for multi-drug resistant (MDR) Gram-negative infections. Polymyxins bind to the lipid A component of lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria, disrupting membrane integrity and increasing permeability.

Daptomycin

• This lipopeptide antibiotic binds to the bacterial cell membrane, causing rapid depolarization and loss of membrane potential. This leads to the inhibition of protein, DNA, and RNA synthesis, resulting in bacterial cell death. Daptomycin is used in the treatment of Gram-positive infections. It inserts into the cell membrane in a calcium-dependent manner, forming channels that disrupt ion gradients and lead to cell death.

Bacterial Cell Envelope

The bacterial cell envelope differs between Gram-positive and Gram-negative bacteria. This difference affects the ability of some antibiotics to reach their target.

• Gram-positive bacteria have a thick peptidoglycan layer outside of the cell membrane, which is readily accessible to many antibiotics.

• Gram-negative bacteria have a thin peptidoglycan layer between the inner cytoplasmic membrane and an outer membrane containing lipopolysaccharide (LPS). This outer membrane acts as a barrier, limiting the entry of many antibiotics.

Some antibiotics cannot cross the outer membrane of Gram-negative bacteria to reach the peptidoglycan layer. Porins, which are protein channels in the outer membrane, allow some small, hydrophilic antibiotics to enter the cell. However, larger or hydrophobic antibiotics are often excluded.

Lipid II Flippases

Lipid II is a crucial precursor in peptidoglycan synthesis, and its translocation across the inner membrane is facilitated by flippases, such as MurJ. These flippases are essential for moving Lipid II from the cytoplasm to the extracellular side of the membrane, where it can be incorporated into the growing peptidoglycan.

Mechanism of MurJ Flipping:

1. Entry: Lipid II enters the MurJ central cavity through a lateral portal.

2. Binding: The cavity binds the diphosphate of lipid II, while the undecaprenyl tail fits into a hydrophobic groove.

3. Occlusion: Lipid II is occluded from the cytosol by a thin gate.

4. Transition: Upon sodium binding, MurJ transits to an outward-facing state.

5. Release: Lipid II is released due to the narrow outward-facing cleft.

6. Reset: Chloride binding resets MurJ to an inward-facing apo state.

$Net Reaction: Export \ Lipid II + Uptake \ Na^+ + CL^-$

Non-Ribosomal Peptide Synthesis (NRPS)

Many antimicrobial peptides, including bacitracin, vancomycin and daptomycin, are synthesized by NRPS, which are large enzyme complexes that use a nucleic-acid-independent mechanism. These systems contain multiple modules, each responsible for adding a single amino acid to the growing peptide chain.

Bacitracin Synthesis:

• Bacitracin, a cyclic polypeptide antibiotic, is synthesized by three NRPSs (BacA, BacB, and BacC) organized into 12 modules.

• Each module incorporates one amino acid into the growing peptide chain.

• The peptide chain remains covalently tethered to the NRPSs via thioesters.

• After reaching the final module, the linear peptide is released by macrocyclization. This cyclization step is essential for the antimicrobial activity of bacitracin.

Penicillin-Binding Proteins (PBPs)

PBPs are bacterial enzymes involved in the transpeptidation and transglycosylation steps of peptidoglycan synthesis. These proteins are essential for cell wall assembly and are the primary targets of beta-lactam antibiotics. Different PBPs have different functions and contribute to different aspects of cell wall synthesis.

Beta-Lactam Structures

Beta-lactam antibiotics share a core beta-lactam structure and include:

• Penicillins

• Cephalosporins

• Carbapenems

• Monobactams

All beta-lactams bind to PBPs and inhibit transpeptidation, preventing cross-linking of peptidoglycan. The beta-lactam ring is essential for their activity, as it binds to the active site of PBPs in a mechanism-based inhibition process.

Cephalosporins

Cephalosporins are structurally similar to penicillins and are classified into generations based on their spectrum of activity and resistance to beta-lactamases. Each generation has improved activity against Gram-negative bacteria and increased resistance to enzymatic degradation.

• First-generation: Primarily active against Gram-positive bacteria; examples include cefazolin and cephalexin.

• Second-generation: Extended Gram-negative activity and increased resistance to beta-lactamases; examples include cefuroxime and cefaclor.

• Third-generation: Broad spectrum and can penetrate the central nervous system (CNS); examples include ceftriaxone and ceftazidime.

• Fourth-generation: Extended spectrum, greater resistance to beta-lactamases, and can cross the blood-brain barrier; example include cefepime.

Carbapenems

Carbapenems are a class of beta-lactams with a broad spectrum of antibacterial activity and high resistance to beta-lactamases. They are typically administered intravenously for serious infections and are often reserved for infections caused by multi-drug resistant bacteria.

• Imipenem is co-administered with cilastatin to prevent degradation by renal enzymes. Cilastatin inhibits dehydropeptidase I, an enzyme found in the kidneys that can break down imipenem.

• Meropenem is stable to dehydropeptidase I and can be given without cilastatin, providing a more convenient administration option.

Exam Questions

Several past exam questions focused on cell wall inhibiting antibiotics, their mechanisms of action, and specific targets such as PBPs and glycopeptide binding sites. These questions are designed to test understanding of the key concepts related to cell wall synthesis and antibiotic mechanisms.

1. Name three structurally different groups of beta-lactam antibiotics used in the clinic.

2. What does the term PBP mean?

3. Which function of PBP’s, when inhibited by beta-lactams, results in bacterial cell death?

4. What is the specific binding target of glycopeptide antibiotics, such as vancomycin?
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