Action of penicillins 1. mode of action 2. family of β lactams 3. route of traverse across cell envelope

1. Antibacterial agents Cell wall Cell membrane Intracellular metabolism Nucleic acid biosynthesis Protein synthesis New targets: genomics

Selective toxicity Ability of a biological agent to inhibit or arrest a pathogen without injury to the host unique targets in procaryotes Bactericidal kills Bacteriostatic inhibitory; host defences

Antibiotic Chemical substance produced by one spp Inhibits in small amounts growth of another micro-organism Discovery of penicillin –A. Fleming –benzylpenicillin –Penicillium notatum

2. β lactams Constrained 4-membered structure = β lactam ring Thiazolidine ring sulphur-containing 5- or 6-membered ring Substututions: –free amino group: β lactam ring –free carboxyl group: thiazolidine ring

β lactams Penicillins ampicillin, amoxicillin, methicillin Cephalosporins 1: cephalothin, cephaloridine, cephalexin 2: cephamandole, cefuroxime, cefaclor 3: cefotaxime, ceftriaxone, ceftazidine

β lactams Different properties to inhibit different m-o Diffuse across OM / porins Cleavage of β lactam ring β lactamase  inactivated antibiotic Targets: –enzymes of peptidoglycan biosynthesis –PBPs

PBPs Single target  multiple targets Expt #1: –Growing E. coli + 14 C-penicillin –Pen binds to target(s) –Collect cells; lysate; identify labelled material –SDS-PAGE / autoradiography –Multiple proteins – [ 14 C-penicillin] –All 14 C-proteins are in CM

PBPs Isolation: affinity chromatography Inert matrix –[penicillin] Expt #2: –Lysate of E. coli applied to matrix—[penicillin] –Some proteins bind to penicillin –Elute, assay in vitro activities –PBPs = penicillin-sensitive enzymes

Penicillin-sensitive enzymes PBP 1a, 1b transglycosylase transpeptidase p/g synthesis: cell elongation PBP2transpeptidasegrowth in rod shape; elongation PBP3transglycosylase transpeptidase p/g synthesis: cell septation PBP4DD-endopeptidase DD-carboxypep’ase X-link hydrolysis: elongation PBP5, 6DD-carboxypep’aseUnused pentapeptide

3. Route across cell envelope OM: porins Periplasm: degrading enzymes CM: enzymes of p/g assembly = PBPs

Desirable properties Penetrates OM via porins Free diffusion Hydrophilic compounds Resist β lactamase Bind with high affinity to targets = PBPs

Infectious antibiotic resistance β lactamase Plasmid-encoded Promiscuous inter species transfer; intra species transfer Proliferation of resistance elements Selective advantage

keywords Micro-organisms Streptomyces jumojeniensis, actinomycete bacteria: Proteus vulgaris, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Bacteriodes fragilis

keywords Anti-microbials –Penicillins: ampicillin –Cephalosporins: cephamycin C, cefmetazole, cefalothin, cefazolin –Nitrocefin –Aminoglycosides

keywords Terms: Soil samples, β lactamase, antibiotic activity, OM, cell wall, peptidoglycan, Gram-negative and Gram- positive, barrier layer, periplasm, MICs (minimum inhibitory concentrations: 3-13 and 100 μg/ml, vacuoles, blebs, spheroplasts, bacteriolysis, ghosts, snake-forms, indole-positive, opportunistic pathogen, septicemia, 7 PBPs in CM, PBP2- = spheres, PBP3- = filaments, t.s. phenotype, affinity for PBPs, autoradiography