Oxazolidinone resistance mutations in 23S rRNA of Escherichia coli reveal the central region of domain V as the primary site of drug action.

Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction, we selected Escherichia coli oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant isolates. Engineering of this mutation by site-directed mutagenesis in the wild-type rRNA operon produced an oxazolidinone resistance phenotype, establishing that the G2032A substitution was the determinant of resistance. Engineered U and C substitutions at G2032, as well as a G2447-to-U mutation, also conferred resistance to oxazolidinone. All the characterized resistance mutations were clustered in the vicinity of the central loop of domain V of 23S rRNA, suggesting that this rRNA region plays a major role in the interaction of the drug with the ribosome. Although the central loop of domain V is an essential integral component of the ribosomal peptidyl transferase, oxazolidinones do not inhibit peptide bond formation, and thus these drugs presumably interfere with another activity associated with the peptidyl transferase center.

Resistance mutations in 23 S rRNA identify the site of action of the protein synthesis inhibitor linezolid in the ribosomal peptidyl transferase center.

Oxazolidinones represent a novel class of antibiotics that inhibit protein synthesis in sensitive bacteria. The mechanism of action and location of the binding site of these drugs is not clear. A new representative of oxazolidinone antibiotics, linezolid, was found to be active against bacteria and against the halophilic archaeon Halobacterium halobium. The use of H. halobium, which possess only one chromosomal copy of rRNA operon, allowed isolation of a number of linezolid-resistance mutations in rRNA. Four types of linezolid-resistant mutants were isolated by direct plating of H. halobium cells on agar medium containing antibiotic. In addition, three more linezolid-resistant mutants were identified among the previously isolated mutants of H. halobium containing mutations in either 16 S or 23 S rRNA genes. All the isolated mutants were found to contain single-point mutations in 23 S rRNA. Seven mutations affecting six different positions in the central loop of domain V of 23 S rRNA were found to confer resistance to linezolid. Domain V of 23 S rRNA is known to be a component of the ribosomal peptidyl transferase center. Clustering of linezolid-resistance mutations within this region strongly suggests that the binding site of the drug is located in the immediate vicinity of the peptidyl transferase center. However, the antibiotic failed to inhibit peptidyl transferase activity of the H. halobium ribosome, supporting the previous conclusion that linezolid inhibits translation at a step different from the catalysis of the peptide bond formation.

Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA.

Functionally active large ribosomal subunits of thermophilic bacterium Thermus aquaticus have been assembled in vitro from ribosomal proteins and either natural or in vitro-transcribed 23S rRNA and 5S rRNA. Sedimentation properties of reconstituted subunits were similar to those of native ribosomal 50S subunits. Subunits reconstituted with in vitro-transcribed rRNAs exhibited high activity in the peptidyl transferase assay and in a poly(U)-dependent cell-free translation system (22 and 30%, respectively, compared to that of native 50S subunits). Catalytic activity of reconstituted subunits critically depended on the presence of 5S rRNA. rRNA mutations known to affect functions of the native ribosome produced similar effects in reconstituted T. aquaticus 50S subunits. Subunits assembled with in vitro-transcribed T. aquaticus 23S rRNA containing the G2267A mutation (G2252A in Escherichia coli), which interferes with binding of peptidyl-tRNA in the ribosomal P-site, showed drastically reduced peptidyl transferase activity, whereas clindamycin resistance mutation A2084G (A2058G in E. coli) rendered assembled subunits tolerant to clindamycin inhibition. Thus, reconstitution of functional subunits with in vitro-transcribed rRNA makes possible the use of in vitro genetics for mutational analysis of 23S rRNA functions in translation. In addition, the ability to assemble catalytically active 50S subunits from the rRNA transcript lacking any posttranscriptional modifications clearly demonstrates that modified nucleotides in 23S rRNA are dispensable for the principal activities of the ribosome.

Inhibition of translation and cell growth by minigene expression.

A random five-codon gene library was used to isolate minigenes whose expression causes cell growth arrest. Eight different deleterious minigenes were isolated, five of which had in-frame stop codons; the predicted expressed peptides ranged in size from two to five amino acids. Mutational analysis demonstrated that translation of the inhibitory minigenes is essential for growth arrest. Pulse-labeling experiments showed that expression of at least some of the selected minigenes results in inhibition of cellular protein synthesis. Expression of the deleterious minigenes in cells deficient in peptidyl-tRNA hydrolase causes accumulation of families of peptidyl-tRNAs corresponding to the last minigene codon; the inhibitory action of minigene expression could be suppressed by overexpression of the tRNA corresponding to the last sense codon in the minigene. Experimental data are compatible with the model that the deleterious effect of minigene expression is mediated by depletion of corresponding pools of free tRNAs.

Ketolide resistance conferred by short peptides.

Clones expressing pentapeptides conferring resistance to a ketolide antibiotic, HMR3004, were selected from a random pentapeptide mini-gene library. The pentapeptide MRFFV conferred the highest level of resistance and was encoded in three different mini-genes. Comparison of amino acid sequences of peptides conferring resistance to a ketolide with those conferring resistance to erythromycin reveals a correspondence between the peptide sequence and the chemical structure of macrolide antibiotic, indicating possible interaction between the peptide and the drug on the ribosome. Based on these observations, a "bottle brush" model of action of macrolide resistance peptides is proposed, in which newly translated peptide interacts with the macrolide molecule on the ribosome and actively displaces it from its binding site. Temporal "cleaning" of the ribosome from the bound antibiotic may be sufficient to allow continuation of protein synthesis even despite the presence of the drug in the medium.

Erythromycin resistance peptides selected from random peptide libraries.

Translation of a 5-codon mini-gene encoded in Escherichia coli 23 S rRNA was previously shown to render cells resistant to erythromycin (Tenson, T., DeBlasio, A., and Mankin, A. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5641-5646). Erythromycin resistance was mediated by a specific interaction of the 23 S rRNA-encoded pentapeptide with the ribosome. In the present study, peptides conferring erythromycin resistance were selected from in vivo expressed random peptide libraries to study structural features important for peptide activity. Screening of a 21-codon mini-gene library (the general structure ATG (NNN)20 TAA) demonstrated that only short peptides (3-6 amino acids long) conferred erythromycin resistance. Sequence comparison of erythromycin resistance peptides isolated from the 5-codon library (ATG (NNN)4 TAA) revealed a strong preference for leucine or isoleucine as a third amino acid and a hydrophobic amino acid at the C terminus of the peptide. When tested against other antibiotics, erythromycin resistance peptides rendered cells resistant to other macrolides, oleandomycin and spiramycin, but not to chloramphenicol or clindamycin. Defining the consensus amino acid sequence of erythromycin resistance peptides provided insights into a possible mode of peptide action and the nature of the peptide binding site on the ribosome.

Studies on the toxic effects of crambe meal and two of its constituents, 1-cyano-2-hydroxy-3-butene (CHB) and epi-progoitrin, in broiler chick diets.

1. Studies were undertaken to determine a safe inclusion rate for crambe (Crambe abyssinica) meal in broiler chick diets, and to determine the mechanism for adverse effects by investigating its constituents; 1-cyano-2-hydroxy-3-butene (CHB) and 3-butenyl glucosinolate (epi-progoitrin, E-PG). 2. Crambe meals were prepared to differ in E-PG (19, 36 and 40 g/kg) and CHB contents (0.1, 0.7 and 1.9 g/kg), and with either active or inactive thioglucosidase. 3. Meals were fed to 7-d-old broiler chicks at 50 or 100 g/kg of the diet for 12 or 13 d. In separate studies, isolated E-PG or CHB were mixed into the diet or administered by gavage to 7-d-old broiler chicks in amounts equivalent to 50 or 100 g/kg crambe meal diets for 10 and 12 d, respectively. 4. Weight gain decreased (P < 0.05) in chicks fed on the high glucosinolate crambe diets or isolated E-PG. Food consumption decreased (P < 0.05) in chicks fed on the diet containing the high E-PG meal with active enzyme. 5. Mild liver lesions and increased serum aspartate aminotransferase were found in chicks fed on the diet containing the high glucosinolate meal with active enzyme. Other organs, including thyroids, were normal. 6. Commercially-processed crambe meal appeared safe at an inclusion rate of 50 or 100 g/kg diet, but could not be recommended at this point for long term feeding.

Efficacy of feeding glucosinolate-extracted crambe meal to broiler chicks.

Glucosinolates and their breakdown products (nitriles) have long been implicated as toxic factors when feeding rapeseed (Brassica napus) meals and crambe (Crambe abyssinica) meals to poultry. Accordingly, various methods have been developed to remove these compounds from the meals to enhance their value as feed supplements. Glucosinolates and nitriles were extracted from commercially processed, defatted crambe meal by washing with water or various solvent-water mixtures: 50% isopropanol, 50% acetone, or 50% ethanol. In addition, crambe seed was extruded and extracted in the laboratory with isopropanol or hexane. Water washing of commercially defatted meal proved to be the most effective method of extraction, removing 95% of the glucosinolates and nitriles. Meals were fed to 7-d-old broiler chicks at 10% of the diet for 14 d. Weight gain decreased (P < .05) in most groups; however a greater decrease (P < .01) was observed in birds fed meals with high glucosinolate content. Feed intake also decreased (P < .05) in most groups; consequently, feed efficiencies were similar for all groups. No changes in serum chemistries, triiodothyronine, thyroxine, or tissue lesions were associated with glucosinolate or nitrile intake. A relationship (P < .05, r = .74) was found between weight gain and glucosinolate intake. No correlation was found between feed intake and meal glucosinolate or nitrile concentrations.

Ultrastructural study of cathepsin B immunoreactivity in rat brain neurons: lysosomal and extralysosomal localizations of the antigen.

Cathepsin B was localized in multiple neurons of the rat central nervous system by means of the peroxidase-antiperoxidase technique and immunogold labeling using a polyclonal antiserum produced in rabbits against rat liver enzyme. The main intracellular locus of cathepsin B antigenic sites was in lysosomes. In some cases, however, immunoreactive material was also detected outside lysosomes (i.e. at the membranes of the rough endoplasmic reticulum). The findings are discussed with respect to the proposed role of the enzyme in the general protein metabolism of the brain and the potency of the antiserum to label the proform of cathepsin B.

Cathepsin B immunoreactive neurons in rat brain. A combined light and electron microscopic study.

The regional distribution and cellular localization of the lysosomal proteinase cathepsin B was studied by use of monospecific antiserum. The application of the peroxidase-antiperoxidase technique at the light microscopic level revealed cathepsin B immunoreactive neurons in many brain areas. A strong immunoreaction was found in pyramidal cells of the cortex, large neurocytes of the septal region, some hippocampal neurons and magnocellular nerve cells of the hypothalamus. Immunogold labeling on ultrathin cryosections of rat neocortex revealed the enzyme protein to be associated with lysosomes.

Cathepsin B immunoreactivity is widely distributed in the rat brain.

The cellular localization and regional distribution of cathepsin B within rat CNS was revealed by immunohistochemistry using a monospecific antiserum. Cathepsin B protein was found to be widely but unevenly distributed throughout rat brain. Neurons were always cathepsin B immunoreactive. Glial elements were only occasionally immunostained. The distribution of the enzyme resembles largely that of cathepsin D.