Inhibition of exogenous 3-deoxy-D-manno-octulosonate incorporation into lipid A precursor of toluene-treated Salmonella typhimurium cells.
Analogs of 3-deoxy-D-manno-octulosonate (KDO) were designed to inhibit CTP:CMP-KDO cytidylyltransferase (CMP-KDO synthetase). Since these analogs lacked whole-cell antibacterial activity, a permeabilized-cell method was developed to measure intracellular compound activity directly. The method employed a mutant of Salmonella typhimurium defective in KDO-8-phosphate synthetase (kdsA), which accumulated lipid A precursor at 42 degrees C. Cells permeabilized with 1% toluene were used to evaluate inhibitor effect on [3H]KDO incorporation into preformed lipid A precursor. KDO incorporation proceeded through the enzymes CMP-KDO synthetase and CMP-KDO:lipid A KDO transferase. Optimum KDO incorporation occurred between pH 8 and 9 and required CTP, prior lipid A precursor accumulation, and a functional kdsB gene product, CMP-KDO synthetase. The apparent Km for KDO in this coupled system at pH 7.6 was 1.38 mM. The reaction products isolated and characterized contained 1 and 2 KDO residues per lipid A precursor molecule. Several KDO analogs produced concentration-related reductions of KDO incorporation in toluenized cells with 50% inhibitory concentrations comparable to those obtained in purified CMP-KDO synthetase systems. Two compounds, 8-amino-2-deoxy-KDO (A-60478) and 8-aminomethyl-2-deoxy-KDO (A-60821), competitively inhibited KDO incorporation, displaying Kis of 4.2 microM for A-60478 and 2.5 microM for A-60821. These data indicated that the inactivity of the KDO analogs on intact bacteria was the result of poor permeation into cells rather than intracellular inactivation.
D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002848:Chromatography, DEAE-Cellulose; D003570:Cytidine Triphosphate / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006017:Glycolipids; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008050:Lipid A / Q000031:analogs & derivatives* / Q000378:metabolism; D009154:Mutation; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013400:Sugar Acids / Q000378:metabolism*; D014050:Toluene / Q000494:pharmacology*
D006017:Glycolipids; D008050:Lipid A; D013400:Sugar Acids; C040142:lipid A precursors, bacterial; C002532:2-keto-3-deoxyoctonate; D014050:Toluene; D003570:Cytidine Triphosphate; D009713:Nucleotidyltransferases; C028984:3-deoxy-manno-octulosonate cytidyltransferase