β-Lactams-penicillins, cephalosporins, carbapenems and monobactams

β-LACTAMS—PENICILLINS, CEPHALOSPORINS, CARBAPENEMS AND MONOBACTAMS

The final stage of peptidoglycan assembly is the cross-linking of the linear glycan strands assembled by trans-glycosylation to the existing peptidoglycan in the cell wall. This reaction is catalysed by transpeptidase enzymes, which are also located on the outer face of the cell membrane. They first remove the terminal d-alanine residue from each stem peptide on the newly synthesized glycan chain. The energy released from breaking the peptide bond between the two alanines is used in the formation of a new peptide bond between the remaining D-alanine on the stem peptide and a free amino group present on the third amino acid of the stem peptides in the existing cross-linked peptidoglycan. In many organisms, including E. coli, this acceptor amino group is supplied by the amino acid diaminopimelic acid. In other organisms, e.g. Staph. aureus, the acceptor amino group is supplied by the amino acid l-lysine. Although there is considerable variation in the composition of the peptide cross-link among different species of bacteria, the essential transpeptidation mechanism is the same. Therefore, virtually all bacteria can be inhibited by interference with this group of enzymes.

The β-lactam antibiotics inhibit transpeptidases by acting as alternative substrates. They mimic the d-alanyl-d-alanine residues and react covalently with the transpeptidases (Figure 12.3). The β-lactam bond (common to all members of the β-lactam antibiotics) is broken but the remaining portion of the antibiotic is not released immediately. The half-life for the transpeptidase-antibiotic complex is of the order of 10 minutes; during this time the enzyme cannot participate in further rounds of peptidoglycan assembly by reaction with its true sub-strate. The vital cross-linking of the peptidoglycan is therefore blocked while other aspects of cell growth continue. The cells become deformed in shape and eventually burst through the combined action of a weakened cell wall, high internal osmotic pressure and the uncontrolled activity of autolytic enzymes in the cell wall. Penicillins, cephalosporins, carbapenems and monobactams all inhibit peptidoglycan cross-linking through interaction of the common β-lactam ring with the transpeptidase enzymes. However, there is considerable variation in the morphological effects of different β-lactams owing to the existence of several types of transpeptidase. The transpeptidase enzymes are usually referred to as penicillin binding proteins (PBPs) because they can be separated and studied after reaction with 14C-labelled penicillin. This step is necessary because there are very few copies of each enzyme present in a cell. They are usually separated according to their size by electrophoresis and are numbered PBP1, PBP2, etc., starting from the highest molecular weight species. In Gram-negative bacteria most of the high molecular weight transpeptidases also possess trans-glycosylase activity, i.e. they have a dual function in the final stages of peptidoglycan synthesis with the trans-glycosylase and transpeptidase activities located in separate regions of the protein structures. Furthermore, the different transpeptidases have specialized functions in the cell; all cross-link peptidoglycan but some are involved with maintenance of cell integrity, some regulate cell shape and others produce new cross wall between elongating cells, securing chromosome segregation prior to cell division. The varying sensitivity of the PBPs towards different β-lactams helps to explain the range of morphological effects observed in treated bacteria. For example, penicillin G (benzylpenicillin), ampicillin and cephaloridine are particularly effective in causing rapid lysis of Gram-negative bacteria such as E. coli. These antibiotics act primarily upon PBP1B, the major transpeptidase of the organism. Other β-lactams have little activity against this PBP, e.g. mecillinam binds preferentially to PBP2 and it produces a pronounced change in the cells from a rod shape to an oval form. Many of the cephalosporins, e.g. cephalexin, cefotaxime and ceftazidime, bind to PBP3 resulting in the formation of elongated, filamentous cells. The lower molecular weight PBPs, 4, 5 and 6, do not possess transpeptidase activity. These are carboxypeptidases, which remove the terminal D-alanine from the pentapeptides on the linear glycans in the cell wall but do not catalyse the cross-linkage. Their role in the cells is to regulate the degree of cross-linking by denying the d-alanyl-d-alanine substrate to the transpeptidases but they are not essential for cell growth. Up to 90% of the amount of antibiotic reacting with the cells may be consumed in inhibiting the carboxypeptidases, with no lethal consequences to the cells.

Gram-positive bacteria also have multiple transpeptidases, but fewer than Gram-negatives. Shape changes are less evident than with Gram-negative rod-shaped organisms. Cell death follows lysis of the cells mediated by the action of endogenous autolytic enzymes (autolysins) present in the cell wall which are activated following β-lactam action. Autolytic enzymes able to hydrolyse peptidoglycan are present in most bacterial walls; they are needed to re-shape the wall during growth and to aid cell separation during division. Their activity is regulated by binding to wall components such as the wall and membrane teichoic acids. When peptidoglycan assembly is disrupted through β-lactam action, some of the teichoic acids are released from the cells, which are then susceptible to attack by their own autolysins.