Cell Wall Synthesis Inhibitors: Examples, Inhibition, Resistance

Generally, the bacterial cell consists of a cell wall, cell membrane, and nucleus. The cell wall is the outer covering of the bacteria-containing peptidoglycan layer which is made up of cross-linked polymers. Peptidoglycan is mainly responsible for all the mechanisms including resistivity, virulence factors including- the shape of the bacteria.

Gram positive and gram negative cell wall structure — Gram-positive and gram-negative cell wall structure.

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What are Cell wall synthesis inhibitors?

Cell wall synthesis inhibitors include antibiotics of class β- lactams and Glycopeptides. Β- lactam antibiotics consist of Penicillins, Cephalosporins, Monobactams, and Carbapenem. Glycopeptides include Vancomycin and Teicoplanin which are the most commonly used antibiotics of this class. Penicillin includes Ampicillin, Oxacillin, Cephalosporins include Cefpodoxime, Cefepime, Monobactams include Aztreonam which is the only commercially available antimicrobial of this class. Carbapenem consists of Imipenem, Meropenem and Ertapenem. Cell wall synthesis inhibitors are the most used antibiotics for treating Gram-negative as well as Gram-positive infections.

Table: Cell wall biosynthesis inhibitors and their targets. Table Source: https://doi.org/10.1039/C6MD00585C

Peptidoglycan is made up of a polymer of peptides and glycan. Mainly synthesis of peptidoglycan occurs in three stages which include; cytoplasm, membrane, and periplasm.
In the cytoplasmic stage, UDP-GLcNAc is catalyzed in four steps process. MurA and MurB catalyze forming UDP-MurNAc. Four enzymes Mur C, Mur D, Mur E, and Mur F catalyzes alanine, glutamic acid which are involved in important steps. Alanine ligase and Alanine racemase are two important enzymes involved in the formation of D-Ala-D-Ala.
The second membrane-associated stage includes the formation of lipid intermediates.
In the third stage, peptidoglycan chains were formed by glycosyltransferases which are then cross-linked by the enzyme transpeptidases. This enzyme transpeptidase consists of PBPs (Penicillin Binding Proteins) and Rod A
Mur enzymes, Transpeptidases, glycosylases, and lipid components are the main components involved in cell wall synthesis.

**Figure: Cell Wall Biosynthesis.** The bacterial cell wall consists of strands of repeating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) subunits. The NAM subunits have short peptide chains attached to them. The composition of the peptide chain varies between bacteria, but the proximal alanine is usually L-Ala and the distal two are usually D-Ala. Enzymes with transpeptidase activity that also bind penicillin (Penicillin Binding Proteins: PBPs) form bonds between the peptide side chains with the expulsion of a terminal D-Alanine from one of the peptide side chains. The PBP dissociates from the wall once the cross-link has been formed. Separate enzymatic domains with glycosyltransferase activity (GT) form linkages between NAM & NAG residues. Some high molecular mass PBPs (e.g. PBP2) are enzymatic complexes containing both transpeptidase and glycosyltransferase domains. Teichoic acid fibers are found in the cell wall of gram-positive bacteria and are composed of polymers of either glycerol phosphate or ribitol phosphate. They are involved in the attachment of bacteria to mucosal cells and can induce septic shock, similar to LPS (endotoxin) released by gram-negative bacteria. Image Source: Tulane University.

Examples of Cell wall synthesis inhibitors

Beta-lactam antibiotics

a. Penicillins- Ampicillin, Amoxicillin, Piperacillin, Oxacillin

b. Cephalosporins:

First-generation: Cefazolin, Cefalexin
Second-generation: Cefoxitin, Cefuroxime
Third-generation: Cefixime, Ceftazidime, Ceftriaxione
Fourth-generation: Cefepime

c. Monobactams: Aztreonam, the only commercially available monobactam.

d. Carbapenems: Imipenem, Meropenem

Glycopeptides

Vancomycin, Oritavancin, Teicoplanin, Telavancin, Bleomycin, Ramoplanin, Decaplanin.

**Cell wall synthesis inhibitors.** Created with BioRender.com. Image Source: Tulane University.

Process/Steps of Inhibition

Beta-lactams interfere in synthesis by acting as a component of D-Alanine- D- Alanine with the help of transpeptidase enzyme in transpeptidase reaction. Glycine residues cross-link amino acid portion of peptide-chain in the presence of penicillin-binding proteins (PBPs). New peptidoglycan chains are formed resulting in cell rupture due to osmotic lysis.
Bacterial cells contain autolytic enzymes which are known for cell growth and division and beta-lactam antibiotics affect the autolytic activity resulting in bacterial cell death.
Glycopeptide antibiotics bind to lipid II component that prevents the recycling of central lipid transporter that plays important role in the mode of action. They inhibit peptidoglycan synthesis by linking with the pentapeptide chains and prevent the addition of new peptidoglycan units. Transglycosylation and transpeptidation are inhibited by this interaction resulting in bacterial cell death.
Vancomycin, a last resort drug of glycopeptides, prevents the binding of D-alanyl with penicillin-binding proteins which inhibits cell wall synthesis causing bacterial lysis.
Permeabilization and depolarization of bacterial cell membrane resulting in inhibition of cell wall synthesis by some of the glycopeptides.

**Figure: Mechanism of action of beta-lactam antibiotics. Top:** In the absence of drug, transpeptidase enzymes (PBPs) in the cell wall catalyze cross-links between adjacent glycan chains, which involves the removal of a terminal D-alanine residue from one of the peptidoglycan precursors. Glycosyltransferases (GT), which exist as either separate subunits, or tightly associated with transpeptidases (e.g. as is the case for PBP-2) create covalent bonds between adjacent sugar molecules NAM & NAG. The net result of covalent bonds between both the peptide and sugar chains creates a rigid cell wall that protects the bacterial cell from osmotic forces that would otherwise result in cell rupture. **Bottom:** Beta-lactam antibiotics, which include penicillins (Pen), cephalosporins (Ceph), monobactams (Mono), and carbapenems (Carba) **bear a structural resemblance to the natural D-Ala-D-Ala substrate** for the transpeptidase, and exert their inhibitory effects on cell wall synthesis by tightly **binding to the active site of the transpeptidase (PBP)**. NAG: N-acetylglucosamine; NAM: N-acetylmuramic acid. Image Source: Tulane University.

Mechanisms of Resistance

The affinity of the drug, its permeability, and stability determines the activity against bacteria. During the past two decades, resistant organisms towards antibiotics are increasing which is of great concern. Organisms develop resistance to beta-lactam antibiotics through a synthesis of PBP2a (penicillin-binding protein 2a). It has a low affinity allowing transpeptidase activity which results in bacterial colonization in patients. Chromosomal mutations also directly or indirectly increase the level of penicillin-binding protein 2a and transcription causes bacterial resistance. Β-lactams resistance is mainly due to the alteration of Penicillin-binding proteins and enzymatic degradation. Glycopeptides resistivity is due to the alteration of targets.

Both Gram-positive and Gram-negative organisms produce beta-lactamases. In Gram-positive organisms, penicillinase is the enzyme causing resistance towards antibiotics and can also cause a mutation by altering the enzymatic activity.
Gram-negative organisms can express both chromosomal as well as plasmid-mediated enzymes that have broad activity in causing resistivity.
Resistant to methicillin and oxacillin in S. aureus staphylococcal cassette chromosome mec which contains mec A gene. PBP2a is encoded by mec A gene which shows high resistance to beta-lactam antibiotics.
Different antibiotics are exported from cells by membrane proteins to maintain their concentration called efflux pumps. These pumps carry a wide range of antibiotics that contribute to forming MDR organisms.
D-alanyl-alanine is changed to D-alanyl-lactate which inhibits the cross-linking of glycopeptides hence causing resistance.
Seven van genes are responsible for causing vancomycin resistance. These genes encode dehydrogenases that form lactate which is important for the formation of unmodified peptidoglycan.
Characterization of different enzymes that are required for the transfer of plasmid results in vancomycin resistance.

Figure: Mechanism of MRSA (ORSA) resistance to β-lactam antibiotics. In antibiotic-sensitive strains of bacteria, β-lactam antibiotics permanently inactivate PBP enzymes, which are essential for bacterial life, by permanently binding to their active site. **Top Row:** Antibiotic Sensitive PBP. Once in the active site, the β-lactam ring springs open, permanently inactivating the sensitive enzyme. **Bottom Row:** Strains of Methicillin-Resistant *S. aureus* (MRSA/ORSA) express a PBP (PBP2a) that has an altered active site that will not allow β-lactam antibiotics to bind, resulting in resistance to this entire subclass of antibiotics. Image Source: Tulane University.

References

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Kang H and Park Y (2015). Glycopeptide Antibiotics : Structure and Mechanisms of Action. 45(2), 67–78.
Kapoor G, Saigal S and Elongavan A (2017). Action and resistance mechanisms of antibiotics: A guide for clinicians. Journal of Anaesthesiology and clinical pharmacology. 33: 300-305
Nikolaidis I and Dessen A (2014). Resistance to antibiotics targeted to the bacterial cell wall. 23, 243–259. https://doi.org/10.1002/pro.2414
Scheffers, D and Pinho M G. (2005). Bacterial Cell Wall Synthesis : New Insights from Localization Studies. 69(4). 585–607. https://doi.org/10.1128/MMBR.69.4.585
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