Antibiotics & Mechanisms of Action

Stages of Cell Wall (Peptidoglycan) Synthesis

• The lecture outlines the stages of cell wall (peptidoglycan, PG) synthesis and discusses antibiotics that inhibit this process. It covers antibiotics acting inside the cell (in the cytoplasm), at the cell membrane, and outside the cell membrane. It also discusses antibiotics that target the cell membrane.

Bacterial Cell Envelope/Cell Wall Structures

• Gram-positive bacteria: Have a cytoplasmic membrane (CM) and a thick peptidoglycan (PG) layer.

• Gram-negative bacteria: Have a cytoplasmic membrane (CM), a thin peptidoglycan (PG) layer, an outer membrane (OM), and lipopolysaccharide (LPS).

• Some antibiotics cannot cross into the cytoplasm.

• Peptidoglycan provides shape and rigidity to bacteria.

Stages of Peptidoglycan Synthesis

• The stages of peptidoglycan synthesis involve multiple enzymatic steps and precursors.

• Undecaprenol is linked by a pyrophosphate to the PG building block, which is composed of a NAG-NAM disaccharide and a pentapeptide stem.

• This unit of peptidoglycan is flipped across the cytoplasmic membrane to build the cell wall.

Key Precursors and Intermediates

• UTP: Uridine triphosphate.

• PEP: Phosphoenol pyruvate.

• NADPH: Reduced nicotinamide adenine dinucleotide phosphate.

• A_2pm: Diaminopimelic acid.

• Ala: Alanine.

• Glu: Glutamic acid.

• GlcNAc: N-acetylglucosamine.

• MurNAc: N-acetylmuramic acid.

• Lipid I and Lipid II: Membrane-bound intermediates in peptidoglycan synthesis.

• DAP: Diaminopimelic acid (used in E. coli pentapeptide).

• Lys: Lysine (used in S. aureus pentapeptide).

Lipid Flippases and Peptidoglycan Biosynthesis

• Proposed bacterial flippase candidates: FtsW, MurJ, and AmJ.

• Precursors UDP-NAG and UDP-NAM-Pentapeptide are made in the cytoplasm.

• The latter precursor is linked to Undecaprenol-P by MraY to generate lipid I.

• MurG utilizes lipid I and UDP-NAG to synthesize lipid II.

• A lipid II flippase translocates lipid II across the inner membrane (IM) so that transglycosylases (TG) can polymerize the disaccharide-pentapeptide into glycan chains.

• TPs catalyze peptide bonds between stem peptides in adjacent glycan chains.

• CPs remove the terminal D-Ala residue of stem peptides.

Lipid II Flippase MurJ

• Lipid II enters the MurJ central cavity through a lateral portal.

• The cavity binds the diphosphate of lipid II, while the undecaprenyl tail fits into the hydrophobic groove formed by transmembrane domains (TMs) 13 and 14.

• Lipid II is occluded from the cytosol by a thin gate, poised for outward transition.

• Upon sodium binding to the C-lobe, MurJ transitions to the outward-facing state.

• Lipid II is released because the outward-facing cleft is too narrow to accommodate the lipid II headgroup.

• Chloride binding resets MurJ to an inward-facing apo state where the portal is closed. Membrane potential might also facilitate this inward reset.

• Release of sodium and chloride ions into the cytosol mediates reopening of the portal, completing the transport cycle.

• The net reaction is the export of lipid II, uptake of one sodium ion, and uptake of one chloride ion.

Lipid II Synthesis and Antibiotics Inhibiting the Process

• Lipid I is formed at the inner face of the membrane by coupling UDP-NAM-pentapeptide to the lipid carrier C55-P.

• Lipid II is produced through the linkage of UDP-GlcNAc to Lipid I.

• Lipid II translocates across the membrane mediated by the membrane protein FtsW (MurJ in E. coli).

• On the outside, the peptidoglycan unit is incorporated into the peptidoglycan network by the action of penicillin-binding proteins (PBPs) by transglycosylation (TG) and transpeptidation (TP) reactions.

• C55-PP is recycled through de-phosphorylation, and the retrieved C55-P is available for the next biosynthesis cycle.

• Equivalent terms for C55-P: Bactoprenol-P, Undecaprenyl phosphate, Lipid-P.

• Pentapeptide: DAP in E. coli, Lys in S. aureus, 5x Gly linker in S. aureus.

Antibiotics Inhibiting Peptidoglycan Synthesis

• Fosfomycin: Inhibits conversion of NAG to NAM; a phosphoenol pyruvate (PEP) analog that binds to pyruvate transferase.

• Cycloserine: Inhibits the conversion of L-ala to D-ala, thus inhibits the formation of D-ala-D-ala; it's an alanine analog.

Bactoprenol and Bacitracin

• The peptidoglycan subunit is passed across the cytoplasmic membrane attached to Bactoprenol (C55-isoprenyl pyrophosphate).

• The nascent peptidoglycan monomer leaves the carrier on reaching the cell wall, and Bactoprenol is dephosphorylated to its monophosphate form.

• Bacitracin inhibits the dephosphorylation of bactoprenol, which halts peptidoglycan subunit synthesis.

Bacitracin

• Bacitracin is a cyclic polypeptide antibiotic made by non-ribosomal peptide synthesis.

• Used in topical applications against Gram-positives.

• Often used in combination with other topical antibiotics (Polymixin B and neomycin) in ointment form for topical treatment of localized skin and eye infections and for the prevention of wound infections.

• Also used as an aftercare antibiotic on tattoos.

Non-Ribosomal Peptide Synthesis (NRPS)

• Bacterial NRPSs have a modular organization, organized in operons.

• One module is responsible for the incorporation of one amino acid into the final product.

• Modules are subdivided into domains, which catalyze the individual steps of nonribosomal peptide synthesis.

• Small peptide molecules represent a large subclass of bioactive natural products, containing unique structural features not found in normal proteins.

• They are made by nucleic-acid-independent synthesis using large enzyme complexes (NRPS).

Example of NRPS – Bacitracin

• Synthetic machinery of the branched cyclic dodecapeptide bacitracin A.

• 12 modules distributed over three NRPSs (BacA, BacB, and BacC) process the growing peptide chain along the protein template.

• Elongation intermediates remain covalently tethered as thioesters to the cofactors of the NRPSs.

• After the linear peptide reaches the final module, it is released by macrocyclization.

• Genetic alterations can reprogram the protein template for the synthesis of novel peptides.

Antibiotics That Do Not Need to Pass the Inner Membrane

• These antibiotics act on the cell wall of Gram-positive and Gram-negative bacteria.

• Examples include β-lactam antibiotics, which are susceptible to β-lactamase.

Glycopeptide Antibiotics

• Glycopeptide antibiotics (e.g., Vancomycin, Teicoplanin) bind D-Ala-D-Ala, inhibiting transglycosylase by steric hindrance, thus preventing polymerization.

Vancomycin

• Vancomycin is a glycopeptide made by non-ribosomal peptide synthesis.

• Used to treat Methicillin-resistant S. aureus (MRSA).

• Bacteriostatic, not bactericidal.

• Must be given intravenously.

• Can be nephrotoxic and ototoxic.

Transpeptidation and β-Lactams

• Transpeptidation is carried out by penicillin-binding proteins (PBPs).

• β-lactams resemble D-ala-D-ala, bind to transpeptidase (PBP) enzyme, and inhibit cross-linking, leading to cell lysis.

• Linear glycan strands are crosslinked by transpeptidases on the outer surface of the cytoplasmic membrane.

• They remove the terminal D-alanine, and a new peptide bond forms between the remaining D-ala and a free amino group on m-Dap.

Penicillin-Binding Proteins

• PBPs are identified and named by gel electrophoresis.

• They have varying molecular weights and functions, including transpeptidases involved in peptidoglycan synthesis during elongation and enzymes required for septum formation and maintenance of cell shape.

Core Beta-Lactam Structures

• Different groups of β-lactam antibiotics include penicillins, cephalosporins, carbapenems, and monobactams.

• All bind to penicillin-binding proteins (PBPs) and stop cross-linking (transpeptidation).

Variants of Penicillins

• Different penicillins (Penicillin G, Penicillin V, Methicillin, Dicloxacillin, etc.) have different side chains and properties, affecting their resistance to acid and penicillinases, their spectrum of activity, and their route of administration.

Cephalosporins

• Cephalosporins are structurally similar to penicillins.

• Absorption, distribution, and elimination are similar to penicillin.

• Hypersensitivity is similar to penicillins.

• Incidence of resistance is lower than penicillins; they are less susceptible to β-lactamases.

• Cefotaxime is a 3rd generation cephalosporin.

Cephalosporin Generations

• First-generation: Primarily Gram-positive coverage.

• Second-generation: Some Gram-negative activity and more resistant to β-lactamase.

• Third-generation: Broader spectrum, penetrate the CNS, anti-Pseudomonal (Ceftazidime).

• Fourth-generation: Extended spectrum, greater resistance to β-lactamases, can cross the blood-brain barrier, anti-Pseudomonal (Cefepime).

Monobactams

• β-lactams where the β-lactam ring is not fused to another ring.

• Work against Gram-negative bacteria only.

• Aztreonam inhibits peptidoglycan synthesis by blocking peptidoglycan crosslinking.

• Has strong activity against susceptible Pseudomonas aeruginosa.

• Also used for patients who are allergic to penicillin.

Carbapenems

• A class of β-lactams with a broad spectrum of antibacterial activity and high resistance to β-lactamases.

• Active against Gram-positive, Gram-negative bacteria, and anaerobes.

• Administered intravenously in hospital settings for serious infections due to their expanded spectra and poor oral bioavailability.

• Imipenem: Co-administered with cilastatin to prevent degradation by renal enzyme dehydropeptidase 1.

• Meropenem: Ultra-broad spectrum, injectable, used to treat a wide variety of infections, including meningitis and pneumonia. Stable to dehydropeptidase 1 and can be given without cilastatin.

Mechanism of Action of Imipenem and Aztreonam

• Imipenem targets transpeptidases involved in peptidoglycan synthesis during elongation.

• Aztreonam targets enzymes required for septum formation.

Antibiotics Targeting the Cell Membrane

• A cell with a damaged membrane dies from disruption in metabolism or lysis.

Polymyxins and Colistin

• Polymyxins and colistin (cationic) destroy bacterial cell membranes.

• Active against Gram-negatives.

• Have serious side effects.

• Used mostly for skin & eye infections.

• Colistin is a last-resort antibiotic for MDR Gram-negatives.

Daptomycin

• A lipopeptide used in the treatment of Gram-positive infections.

• Binds to the bacterial cell membrane, causes rapid depolarization, leading to inhibition of protein, DNA, and RNA synthesis, resulting in bacterial cell death.

• Synergistic with beta-lactam antibiotics.

• Made by non-ribosomal peptide synthesis (NRPS).

Relevant Exam Questions

• Examples of relevant exam questions from previous years focus on the identification of antibiotic groups, mechanisms of action, and specific targets in bacterial cell wall synthesis.