Differential binding of penicillin by membrane fractions from penicillin-susceptible and -resistant gonococci

Area of Science:

• Microbiology
• Pharmacology
• Biochemistry

Background:

• Penicillin resistance in Neisseria gonorrhoeae is a growing public health concern.
• Previous studies indicated an inverse relationship between penicillin binding to whole cells and resistance.

Purpose of the Study:

• To investigate the relationship between penicillin binding to cytoplasmic membranes and penicillin minimal inhibitory concentration in Neisseria gonorrhoeae.
• To characterize penicillin binding sites within gonococcal cell membranes.

Main Methods:

• Isolation of cytoplasmic membranes from 10 Neisseria gonorrhoeae strains using Braun homogenization and differential centrifugation.
• Quantification of [(14)C]penicillin binding to isolated membranes.
• Competition assays with unlabeled penicillin and cephalothin.
• Analysis of membrane proteins by slab gel electrophoresis.

Main Results:

• Cytoplasmic membranes from penicillin-susceptible strains bound significantly more [(14)C]penicillin (140-200 nmol/g) than those from less susceptible strains (18-59 nmol/g).
• Cell wall binding was minimal, suggesting membrane localization of binding sites.
• Pre-exposure to unlabeled penicillin or cephalothin reduced [(14)C]penicillin binding, indicating specific binding interactions.
• Slab gel electrophoresis identified 13 protein bands in membranes, with 8 being labeled by penicillin.

Conclusions:

• A strong inverse correlation exists between penicillin binding to Neisseria gonorrhoeae cytoplasmic membranes and penicillin resistance.
• Penicillin binding primarily occurs in the cytoplasmic membrane, not the cell wall.
• These findings suggest specific penicillin-binding proteins (PBPs) in the gonococcal membrane are key determinants of susceptibility.