The penicillin binding proteins and beta-lactamases of Neisseria gonorrhoeae

Several strains of Neisseria gonorrhoeae have been characterised and minimum inhibitory concentrations of benzyl penicillin and methicillin against each strain were determined. Three penicillin binding proteins (PBPs) labelled with 3H-penicillin were separated and visualised using traditional fluorography. Antisera against beta-lactam conjugates were prepared and subsequently characterised using ELISA. The antisera raised against the methicillin conjugate were used in Western blots to probe for methicillin bound to PBPs from sensitive and resistant strains of gonococci. PBPs 1 and 2 bound methicillin and were visualised using the anti-methicillin sera. The reduced affinity for methicillin of PBPs 1 and 2 in chromosomally resistant strains was demonstrated by Western blotting. This correlates with a reduced level of binding of radio labelled penicillin to PBPs of these strains as detected by fluorography. Affinity chromatography with beta-lactam ligands was used to isolate and purify PBPs from detergent solubilised membranes of both a sensitive and resistant strain of Neisseria gonorrhoeae. PBP3 proved difficult to solubilise compared with the other two PBPs. All 3 PBPs bound avidly to the ligands: 6-amino penicillanic acid and 7-amino cephalosporanic acid. The bound PBPs were difficult to elute, PBPS was removed from both ligands in small amounts using rigorous conditions. A beta-lactamase was purified from Neisseria gonorrhoeae and partially characterised. Antibodies were prepared against this protein using material cut from polyacrylamide gels. The antibodies recognised the beta-lactamase from Neisseria gonorrhoeae in Western blots and also cross reacted with beta- lactamase from Escherichia coli (R-TEM) and Bacillus cereus type I. This suggests that proteins have regions of amino acid homology which can be recognised by the antibodies. The potential use of the anti-methicillin and anti gonococcal beta-lactamase antibodies to detect chromosomally mediated resistance and beta- lactamase mediated resistance respectively, is discussed