Antimicrobial Chemotherapy

Peptidoglycan is an exclusively bacterial polymer and so potentially should provide an excellent target for selective chemotherapy. Unfortunately, not all the intermediate steps in peptidoglycan biosynthesis involve processes that are confined to bacteria and some antimicrobials that inhibit such reactions may be very toxic to humans as well as to bacteria. Peptidoglycan is unique among biological polymers because it contains both L- and D-isomers of its constituent amino acids. Antibiotics may act at several stages during peptidoglycan synthesis. Some are valuable chemotherapeutic agents; others are too toxic for human use.

Structure of peptidoglycan

The b-lactam group of antibiotics includes an enormous diversity of natural and semi-synthetic compounds that inhibit several enzymes associated with the final step of peptidoglycan synthesis. All of this enormous family are derived from a b-lactam structure: a four-membered ring in which the b-lactam bond resembles a peptide bond. The multitude of chemical modifications based on this four-membered ring permits the astonishing array of antibacterial and pharmacological properties within this valuable family of antibiotics.

Clinically useful families of b-lactam compounds include the penicillins, cephalosporins, monobactams and carbapenems. Many new variants on the b-lactam theme are currently being explored. Certain b-lactams have limited use directly as therapeutic agents, but may be used in combination with other b-lactams to act as b-lactamase inhibitors. Co-amoxyclav, for example is a combination of amoxycillin and the b-lactamase inhibitor clavulanic acid. During cross-linking of the peptidoglycan polymer, one D-alanine residue is cleaved from the peptidoglycan precursor and this reaction is prevented by b-lactam drugs. More recent studies have shown that the activity of this class of drugs is more complicated and involves other processes as well as preventing cross-linking of peptidoglycan.

Penicillin nucleus

Cephalosporin nucleus

Monobactam nucleus

Carbapenem nucleus

Clavulanic acid

The targets for b-lactam drugs are the penicillin binding proteins (PBP's), so called because they bind radioactive penicillin and can be detected by autoradiography of gels on which bacterial proteins have been separated electrophoretically. The penicillin binding proteins have transpeptidase or carboxypeptidase activity and they act to regulate cell size and shape. They are also involved in septum formation and cell division. Bacteria have several individual penicillin binding proteins, each with a separate function. Conventionally these are numbered according to size, with PBP 1 as the largest protein. The PBP 1 of one bacterium will not necessarily have the same function as the PBP 1 of a different organism.

The b-lactam antibiotics may bind preferentially to different penicillin binding proteins, and at sublethal concentrations may cause alterations in cell morphology. For example, mecillinam binds preferentially to Escherichia coli PBP 2 and causes spherical cells to form, whereas cephalexin causes Escherichia coli to grow as filaments as a result of its preferential binding to PBP 3. This indicates that PBP 2 in Escherichia coli is involved in cell elongation whereas its PBP 3 is has a role in the cell division of this bacterium.

The b-lactam antibiotics also stimulate the activity of autolysins. These are enzymes that are responsible for the natural turnover of cell wall polymers to permit growth of the cells. Under normal conditions, these enzymes produce controlled weak points within the peptidoglycan structure to allow for expansion of the cell wall structure. This activity is stimulated by b-lactams, causing a breakdown of peptidoglycan and leading to osmotic fragility of the cell and ultimately to cell lysis.

Some b-lactam antibiotics

Benzyl penicillin

Ampicillin

Cephalosporin C

Ceftriaxone

Aztreonam

Imipenem

The molecule of vancomycin is relatively large. The drug acts to prevent peptidoglycan subunits from being added to the growing cell wall polymer. This is accomplished by vancomycin binding to the D-alanyl D-alanine residue of the lipid-bound precursor. Its primary activity is against Gram-positive bacteria. It is particularly useful in the treatment of serious staphylococcal infections. In these cases, it is given either intramuscularly or intravenously since it is not absorbed from the gut. It is also used for the treatment of pseudomembranous colitis caused by Clostridium difficile when it is administered orally.

Chemical structure of vancomycin

The condensation reaction between UDP-N-acetyl glucosamine and phosphoenol pyruvate in the early stages of peptidoglycan synthesis is the target for fosfomycin. There is a rapid selection of resistance to fosfomycin, rendering it unsuitable for most clinical purposes.

Chemical structure of fosfomycin

The simple, cyclic molecule cycloserine is an analogue of alanine that interferes with two steps in peptidoglycan synthesis. It is a competitive inhibitor of the racemase that converts L-alanine to D-alanine and it also prevents the action of the D-alanyl D-alanine synthetase. The stable ring structure of cycloserine holds the molecule in a sterically favourable position, permitting preferential binding of this compound both to the racemase and to the synthetase, rather than their natural substrates. This results in competitive inhibition of these enzymes. Cycloserine is a neurotoxin and is not used clinically except for the treatment of drug-resistant Mycobacterium tuberculosis, or in other life-threatening infections where alternative therapies have failed.

Chemical structure of cycloserine and its analogue, D-alanine

The polypeptide antibiotic bacitracin is too toxic for human clinical use. It is, however, widely used in diagnostic laboratories to distinguish bactracin-sensitive Streptococcus pyogenes from other b-haemolytic streptococci. Its activity depends upon its ability to bind to the undecaprenyl pyrophosphate lipid carrier that transports the peptidoglycan monomers across the bacterial membrane. This impedes the dephosphorylation of the carrier, which, in turn, obstructs regeneration of the undecaprenyl phosphate and thus prevents recycling of the transport mechanism. Bacitracin also interferes with sterol synthesis in mammalian cells by binding to pyrophosphate intermediates, accounting for its human toxicity.

Chemical structure of bacitracin