Anti-CD3 recombinant diphtheria immunotoxin therapy of cutaneous T cell lymphoma.

The recombinant CD3 immunotoxin, A-dmDT(390)-bisFv(UCHT1), composed of the catalytic and translocation domains of diphtheria toxin fused to two single chain Fv fragments of an anti-CD3epsilon monoclonal antibody was administered to five patients with cutaneous T cell lymphoma (CTCL) by eight 15 min intravenous infusions over four days. Side effects were fever, chills, nausea, hypoalbuminemia, transaminasemia and reactivation of EBV and CMV. Half-life of drug was 40 min. Anti-immunotoxin antibodies developed in all patients after two weeks. Two patients had partial remissions lasting 1 and 6+ months. The agent is undergoing further dose escalation and shows promising results in this disease.

Ribosomal and non-ribosomal resistance to oxazolidinones: species-specific idiosyncrasy of ribosomal alterations.

A derivative of Mycobacterium smegmatis, which carries only one functional rRNA (rrn) operon, was used to isolate mutants resistant to the ribosome-targeted antibiotic linezolid. Isolation and characterization of linezolid-resistant clones revealed two classes of mutants. Ribosomes from class I mutants are resistant to oxazolidinones in an in vitro peptidyl transferase assay, indicating that resistance maps to the ribosome component. In contrast, ribosomes from class II mutants show wild-type susceptibility to a linezolid derivative in vitro, pointing to a non-ribosomal mechanism of resistance. Introduction of a wild-type ribosomal RNA operon into linezolid-resistant strains restored linezolid sensitivity in class I mutants, indicating that resistance (i) maps to the rRNA and (ii) is recessive. Sequencing of the entire rrn operon identified a single nucleotide alteration in 23S rRNA of class I mutant strains, 2447G --> T (Escherichia coli numbering). Introduction of mutant rrl2447T into M. smegmatis rrn- resulted in a linezolid-resistant phenotype, demonstrating a cause-effect relationship of the 2447G --> T alteration. The 2447G --> T mutation, which renders M. smegmatis linezolid resistant, confers lethality in E. coli. This finding is strong evidence of structural and pos-sibly functional differences between the ribosomes of Gram-positive and Gram-negative bacteria. In agreement with the results of the in vitro assay, class II mutants show a wild-type sequence of the complete rRNA operon. The lack of cross-resistance of the class II mutants to other antibiotics suggests a resistance mechanism other than activation of a broad-spectrum multidrug transporter.

Binding site of macrolide antibiotics on the ribosome: new resistance mutation identifies a specific interaction of ketolides with rRNA.

Macrolides represent a clinically important class of antibiotics that block protein synthesis by interacting with the large ribosomal subunit. The macrolide binding site is composed primarily of rRNA. However, the mode of interaction of macrolides with rRNA and the exact location of the drug binding site have yet to be described. A new class of macrolide antibiotics, known as ketolides, show improved activity against organisms that have developed resistance to previously used macrolides. The biochemical reasons for increased potency of ketolides remain unknown. Here we describe the first mutation that confers resistance to ketolide antibiotics while leaving cells sensitive to other types of macrolides. A transition of U to C at position 2609 of 23S rRNA rendered E. coli cells resistant to two different types of ketolides, telithromycin and ABT-773, but increased slightly the sensitivity to erythromycin, azithromycin, and a cladinose-containing derivative of telithromycin. Ribosomes isolated from the mutant cells had reduced affinity for ketolides, while their affinity for erythromycin was not diminished. Possible direct interaction of ketolides with position 2609 in 23S rRNA was further confirmed by RNA footprinting. The newly isolated ketolide-resistance mutation, as well as 23S rRNA positions shown previously to be involved in interaction with macrolide antibiotics, have been modeled in the crystallographic structure of the large ribosomal subunit. The location of the macrolide binding site in the nascent peptide exit tunnel at some distance from the peptidyl transferase center agrees with the proposed model of macrolide inhibitory action and explains the dominant nature of macrolide resistance mutations. Spatial separation of the rRNA residues involved in universal contacts with macrolides from those believed to participate in structure-specific interactions with ketolides provides the structural basis for the improved activity of the broader spectrum group of macrolide antibiotics.

EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance.

Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid-borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin-binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.

pKa of adenine 2451 in the ribosomal peptidyl transferase center remains elusive.

The universally conserved A2451 of 23S rRNA has been proposed to participate directly in the catalysis of peptide bond formation in the ribosomal peptidyl transferase center. An unusually high, near neutral, pKa of A2451 is a prerequisite for its action as a general acid-base catalyst. Increased reactivity of A2451 to dimethylsulfate (DMS) at pH 8.5 compared to pH 6.5 was taken as evidence that the pKa of this nucleotide falls within this pH range. Structural data suggested that the interaction between A2451 and G2447 in the ribosome is responsible for A2451 pKa perturbation. In contrast to expectation, our studies did not show pH dependence of A2451 dimethylsulfate modification in ribosomes of Thermus aquaticus and Mycobacterium smegmatis. Other rRNA regions, however, showed major alterations in DMS reactivity at pH 8.5 compared to pH 6.5, suggesting that conformational rearrangements in the structure of the large ribosomal subunit may occur upon the pH shift. The G2447U mutant of M. smegmatis was viable, indicating that the G2447-A2451 interaction is not critical for the ribosome function. We concluded that the proposed unusual pKa of A2451, if existing, may not be crucial for the ribosome activity and that the previously reported pH-dependent alterations in the DMS modification of A2451 do not necessarily reveal an unusual pKa of this nucleotide.

Short peptides conferring resistance to macrolide antibiotics.

Translation of specific short peptides can render the ribosome resistant to macrolide antibiotics such as erythromycin. Peptides act in cis upon the ribosome on which they have been translated. Amino acid sequence and size are critical for peptide activity. Pentapeptides with different consensus sequences confer resistance to structurally different macrolide antibiotics, suggesting direct interaction between the peptide and the drug on the ribosome. Translation of resistance peptides may result in expulsion of the macrolide antibiotics from the ribosome. The consensus sequence of peptides conferring erythromycin resistance is similar to the sequence of the leader peptide involved in translational attenuation of erythromycin resistance genes, indicating that a similar type of interaction between the nascent peptide and antibiotics can occur in both cases.

Ribosomal peptidyl transferase can withstand mutations at the putative catalytic nucleotide.

Peptide bond formation is the principal reaction of protein synthesis. It takes place in the peptidyl transferase centre of the large (50S) ribosomal subunit. In the course of the reaction, the polypeptide is transferred from peptidyl transfer RNA to the alpha-amino group of amino acyl-tRNA. The crystallographic structure of the 50S subunit showed no proteins within 18 A from the active site, revealing peptidyl transferase as an RNA enzyme. Reported unique structural and biochemical features of the universally conserved adenine residue A2451 in 23S ribosomal RNA (Escherichia coli numbering) led to the proposal of a mechanism of rRNA catalysis that implicates this nucleotide as the principal catalytic residue. In vitro genetics allowed us to test the importance of A2451 for the overall rate of peptide bond formation. Here we report that large ribosomal subunits with mutated A2451 showed significant peptidyl transferase activity in several independent assays. Mutations at another nucleotide, G2447, which is essential to render catalytic properties to A2451 (refs 2, 3), also did not dramatically change the transpeptidation activity. As alterations of the putative catalytic residues do not severely affect the rate of peptidyl transfer the ribosome apparently promotes transpeptidation not through chemical catalysis, but by properly positioning the substrates of protein synthesis.

A novel site of antibiotic action in the ribosome: interaction of evernimicin with the large ribosomal subunit.

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.

Interactions between 23S rRNA and tRNA in the ribosomal E site.

Interactions between tRNA or its analogs and 23S rRNA in the large ribosomal subunit were analyzed by RNA footprinting and by modification-interference selection. In the E site, tRNA protected bases G2112, A2392, and C2394 of 23S rRNA. Truncated tRNA, lacking the anticodon stem-loop, protected A2392 and C2394, but not G2112, and tRNA derivatives with a shortened 3' end protected only G2112, but not A2392 or C2394. Modification interference revealed C2394 as the only accessible nucleotide in 23S rRNA whose modification interferes with binding of tRNA in the large ribosomal subunit E site. The results suggest a direct contact between A76 of tRNA A76 and C2394 of 23S rRNA. Protections at G2112 may reflect interaction of this 23S rRNA region with the tRNA central fold.

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.

Mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA affect the function of the decoding center of the ribosome.

A dynamic structural rearrangement in the phylogenetically conserved helix 27 of Escherichia coli 16S rRNA has been proposed to directly affect the accuracy of translational decoding by switching between "accurate" and "error-prone" conformations. To examine the function of helix 27 in eukaryotes, random and site-specific mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA have been characterized. Mutations at positions of yeast 18S rRNA corresponding to E. coli 886 (rdn8), 888 (rdn6), and 912 (rdn4) increased translational accuracy in vivo and in vitro, and caused a reduction in tRNA binding to the A-site of mutant ribosomes. The double rdn4rdn6 mutation separated the killing and stop-codon readthrough effects of the aminoglycoside antibiotic, paromomycin, implicating a direct involvement of yeast helix 27 in accurate recognition of codons by tRNA or release factor eRF1. Although our data in yeast does not support a conformational switch model analogous to that proposed for helix 27 of E. coli 16S rRNA, it strongly suggests a functional conservation of this region in tRNA selection.

Introduction of a mini-gene encoding a five-amino acid peptide confers erythromycin resistance on Bacillus subtilis and provides temporary erythromycin protection in Proteus mirabilis.

A 15-bp mini-gene was introduced into Bacillus subtilis and into stable protoplast-like L-forms of Proteus mirabilis. This mini-gene encoded the peptide MVLFV and modeled a fragment of Escherichia coli 23S rRNA responsible for E. coli erythromycin (Ery) resistance. Expression of the introduced mini-gene conferred permanent Ery resistance on B. subtilis. In L-forms of P. mirabilis, the Ery-protective effect was maintained in the course of several generations. Herewith, the mechanism of Ery resistance mediated by expression of specific short peptides was shown to exist in evolutionary distant bacteria. Three new plasmids were constructed containing the gene under study transcriptionally fused with the genes encoding glutamylendopeptidase of Bacillus licheniformis or delta-endotoxin of Bacillus thuringiensis. The Ery resistance pentapeptide (E-peptide) mini-gene served as an efficient direct transcriptional reporter and allowed to select bacillar glutamylendopeptidase with improved productivity. The mini-genes encoding E-peptides may be applied as selective markers to transform both Gram-positive and Gram-negative bacteria. The small size of the E-peptide mini-genes makes them attractive selective markers for vector construction.

Antibiotics and ribosomes: interaction and resistance.

This session was convened by Dr Joyce Sutcliffe (Pfizer Central Research, Groton, CT, USA) and Dr R Leclerq (Hôpital Côte de Nacre, Caen, France), with the assistance of the Committee Member Advisor, Dr Patrice Courvalin (Institut Pasteur, Paris, France). It included several presentations in which recent advances on understanding interactions of antibiotics with the ribosome, and mechanisms of action of such drugs, were discussed. The majority of speakers focused on antibiotic interaction with the large ribosomal subunit. Exciting new developments in the field of ribosomal research were highlighted by publication, just several weeks before the conference, of high-resolution crystallographic structures of the large and small ribosomal subunits. With this in mind, several speakers discussed biochemical and genetic data on antibiotic-ribosome interactions within the context of newly available structural information.

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.

Effect of antibiotics on large ribosomal subunit assembly reveals possible function of 5 S rRNA.

Functional large ribosomal subunits of Thermus aquaticus can be reconstituted from ribosomal proteins and either natural or in vitro transcribed 23 S and 5 S rRNA. Omission of 5 S rRNA during subunit reconstitution results in dramatic decrease of the peptidyl transferase activity of the assembled subunits. However, the presence of some ribosome-targeted antibiotics of the macrolide, ketolide or streptogramin B groups during 50 S subunit reconstitution can partly restore the activity of ribosomal subunits assembled without 5 S rRNA. Among tested antibiotics, macrolide RU69874 was the most active: activity of the subunits assembled in the absence of 5 S rRNA was increased more than 30-fold if antibiotic was present during reconstitution procedure. Activity of the subunits assembled with 5 S rRNA was also slightly stimulated by RU69874, but to a much lesser extent, approximately 1.5-fold. Activity of the native T. aquaticus 50 S subunits incubated in the reconstitution conditions in the presence of RU69874 was, in contrast, slightly decreased. The presence of antibiotics was essential during the last incubation step of the in vitro assembly, indicating that drugs affect one of the last assembly steps. The 5 S rRNA was previously shown to form contacts with segments of domains II and V of 23 S rRNA. All the antibiotics which can functionally compensate for the lack of 5 S rRNA during subunit reconstitution interact simultaneously with the central loop in domain V (which is known to be a component of peptidyl transferase center) and a loop of the helix 35 in domain II of 23 S rRNA. It is proposed that simultaneous interaction of 5 S rRNA or of antibiotics with the two domains of 23 S rRNA is essential for the successful assembly of ribosomal peptidyl transferase center. Consequently, one of the functions of 5 S rRNA in the ribosome can be that of assisting the assembly of ribosomal peptidyl transferase by correctly positioning functionally important segments of domains II and V of 23 S rRNA.

Suppression of nonsense mutations induced by expression of an RNA complementary to a conserved segment of 23S rRNA.

We identified a short RNA fragment, complementary to the Escherichia coli 23S rRNA segment comprising nucleotides 735 to 766 (in domain II), which when expressed in vivo results in the suppression of UGA nonsense mutations in two reporter genes. Neither UAA nor UAG mutations, examined at the same codon positions, were suppressed by the expression of this antisense rRNA fragment. Our results suggest that a stable phylogenetically conserved hairpin at nucleotides 736 to 760 in 23S rRNA, which is situated close to the peptidyl transferase center, may participate in one or more specific interactions during peptide chain termination.

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.

A ketolide resistance mutation in domain II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre.

Ketolides represent a new generation of macrolide antibiotics. In order to identify the ketolide-binding site on the ribosome, a library of Escherichia coli clones, transformed with a plasmid carrying randomly mutagenized rRNA operon, was screened for mutants exhibiting resistance to the ketolide HMR3647. Sequencing of the plasmid isolated from one of the resistant clones and fragment exchange demonstrated that a single U754A mutation in hairpin 35 of domain II of the E. coli 23S rRNA was sufficient to confer resistance to low concentrations of the ketolide. The same mutation also conferred erythromycin resistance. Both the ketolide and erythromycin protected A2058 and A2059 in domain V of 23S rRNA from modification with dimethyl sulphate, whereas, in domain II, the ketolide protected, while erythromycin enhanced, modification of A752 in the loop of the hairpin 35. Thus, mutational and footprinting results strongly suggest that the hairpin 35 constitutes part of the macrolide binding site on the ribosome. Strong interaction of ketolides with the hairpin 35 in 23S rRNA may account for the high activity of ketolides against erythromycin-resistant strains containing rRNA methylated at A2058. The existence of macrolide resistance mutations in the central loop of domain V and in hairpin 35 in domain II together with antibiotic footprinting data suggest that these rRNA segments may be in close proximity in the ribosome and that hairpin 35 may be a constituent part of the ribosomal peptidyl transferase centre.