Enzymatic synthesis of aminoglycoside antibiotics: novel adenosylmethionine:2-deoxystreptamine N-methyltransferase activities in hygromycin B- and spectinomycin-producing Streptomyces spp. and uses of the methylated products

Aminocyclitols structurally related to streptamine, a 1,3-diaminocyclitol, are common components of the RNA-binding aminoglycoside antibiotics. The respective aminocyclitol cores of hygromycin B and spectinomycin are N(3)-methyl-2-deoxy-D-streptamine and N(1),N(3)-dimethyl-2-epi-streptamine. Adenosyl[methyl-(14)C]methionine:2-deoxystreptamine N-methyltransferase activities were detected in extracts of early-stationary-phase mycelia of the hygromycin B producer Streptomyces hygroscopicus subsp. hygroscopicus ATCC 27438 and the spectinomycin producer Streptomyces flavopersicus ATCC 19756. Extracts of both strains methylated the N(1)- and N(3)-amino groups of 2-deoxystreptamine, streptamine, and 2-epi-streptamine; the N(1)-amino group of N(3)-methyl-2-deoxy-D-streptamine, and the N(3)-amino group of N(1)-ethyl-2-deoxy-D-streptamine, the semisynthetic aminocyclitol of netilmicin. The mono[(14)C]methyl derivatives of 2-deoxystreptamine, streptamine, and 2-epi-streptamine were excellent substrates for L-glutamine:aminocyclitol aminotransferase and thereby provided a sensitive assay for derepression of this key enzyme, a generic biosynthetic marker that we have shown to be the only enzyme common to the biosyntheses of all major aminoglycoside antibiotics. Other prospective uses for these methyl-labeled 2-deoxystreptamine analogs are also described.     

Effects of aminoglycoside antibiotics on the coupling of protein and RNA syntheses in Escherichia coli

The interdependency of protein and RNA syntheses was studied comparatively in bacteria confronted with amino acid starvation or treated separately with various aminoglycoside antibiotics. By contrast with the concomitant inhibition of macromolecular syntheses in cells deprived of an essential amino acid, RNA production was found to continue in drug-treated cells while protein synthesis was arrested. Such uncoupling process was also observed in bacteria subjected simultaneously to amino acid starvation and treatment with certain antibiotics (neomycin, gentamicin, spectinomycin and kasugamycin) but not with others (streptomycin and kanamycin). These results were related to the intracellular concentration of guanosine polyphosphates, ppGpp and pppGpp. They were discussed in terms of interaction of aminoglycosides with ribosomes.     

Aminoglycosides versus bacteria – a description of the action, resistance mechanism, and nosocomial battleground

Since 1944, we have come a long way using aminoglycosides as antibiotics. Bacteria also have got them selected with hardier resistance mechanisms. Aminoglycosides are aminocyclitols that kill bacteria by inhibiting protein synthesis as they bind to the 16S rRNA and by disrupting the integrity of bacterial cell membrane. Aminoglycoside resistance mechanisms include: (a) the deactivation of aminoglycosides by N-acetylation, adenylylation or O-phosphorylation, (b) the reduction of the intracellular concentration of aminoglycosides by changes in outer membrane permeability, decreased inner membrane transport, active efflux, and drug trapping, (c) the alteration of the 30S ribosomal subunit target by mutation, and (d) methylation of the aminoglycoside binding site. There is an alarming increase in resistance outbreaks in hospital setting. Our review explores the molecular understanding of aminoglycoside action and resistance with an aim to minimize the spread of resistance.     

Solid-phase clean-up and thin-layer chromatographic detection of veterinary aminoglycosides

Chemical methods are needed to confirm the presence of antibiotics detected by microbial inhibition assays in fluids and tissues of farm animals. We have optimized the conditions for the isolation of hygromycin B with a copolymeric bonded solid-phase silica column followed by thin-layer chromatography (TLC) separation and detection of its fluorescence derivative after reaction with fluorescamine. The detection limit of the drug was 50 ng. Serum and plasma samples fortified with hygromycin B were acidified and passed through the copolymerized solid-phase columns previously conditioned with phosphate buffer. Hygromycin B was trapped in the columns and eluted with diethylamine-methanol and analyzed by TLC using acetone-ethanol-ammonium hydroxide as the developing solvent. Hygromycin B bands were derivatized at acidic pH with fluorescamine and visualized under ultraviolet light. Hygromycin B added to bovine plasma was detectable at 25, 50, 100, 250 and 500 ng/ml (ppb). Hygromycin B added to swine serum was detected at 50 ng/ml. However, the serum had to be deproteinized with trichloroacetic acid or acetonitrile prior to solid-phase extraction to gain accurate values. Neomycin and gentamicin (100 ng/ml aqueous solutions) could also be isolated with copolymeric solid-phase columns at a level of 50 ng. Gentamicin, neomycin, gentamicin, spectinomycin, hygromycin B and streptomycin could be separated by TLC, allowing multiresidue detection of these aminoglycosides. The respective R_F values of 0.64, 0.56, 0.52, 0.33 and 0.20 indicate the separation of these five compounds. This procedure provides a rapid and sensitive method for the semi-quantitative estimation of aminoglycosides.     

Alkamides from Echinacea disrupt the fungal cell wall-membrane complex

We tested the hypothesis that alkamides from Echinacea exert antifungal activity by disrupting the fungal cell wall/membrane complex. Saccharomyces cerevisiae cells were treated separately with each of seven synthetic alkamides found in Echinacea extracts. The resulting cell wall damage and cell viability were assessed by fluorescence microscopy after mild sonication. Membrane disrupting properties of test compounds were studied using liposomes encapsulating carboxyfluorescein. Negative controls included hygromycin and nourseothricin (aminoglycosides that inhibit protein synthesis), and the positive control used was caspofungin (an echinocandin that disrupts fungal cell walls). The results show that yeast cells exposed to sub-inhibitory concentrations of each of the seven alkamides and Echinacea extract exhibit increased frequencies of cell wall damage and death that were comparable to caspofungin and significantly greater than negative controls. Consistent with effects of cell wall damaging agents, the growth inhibition by three representative alkamides tested and caspofungin, but not hygromycin B, were partially reversed in sorbitol protection assays. Membrane disruption assays showed that the Echinacea extract and alkamides have pronounced membrane disruption activity, in contrast to caspofungin and other controls that all had little effect on membrane stability. A Quantitative Structure-Activity Relationship (QSAR) analysis was performed to study the effect of structural substituents on the antifungal activity of the alkamides. Among the set studied, diynoic alkamides showed the greatest antifungal and cell wall disruption activities while an opposite trend was observed in the membrane disruption assay where the dienoic group was more effective. We propose that alkamides found in Echinacea act synergistically to disrupt the fungal cell wall/membrane complex, an excellent target for specific inhibition of fungal pathogens. Structure-function relationships provide opportunities for synthesis of alkamide analogs with improved antifungal activities.     

Conformational changes in the ribosome induced by translational miscoding agents

Ribosomes are dynamic complexes responsible for translating the genetic information encoded in mRNAs to proteins. The accuracy of this process is vital to the survival of an organism, and is often compromised by translational miscoding agents. Aminoglycosides are a group of miscoding agents that bind to the ribosome and reduce the fidelity of translation. Previous studies have shown that aminoglycosides alter the higher order structure of the ribosome. Here, we used a toeprinting assay to how that streptomycin, neomycin, kanamycin, gentamycin, and hygromycin B trigger conformational changes within Escherichia coli ribosome. Miscoding agents viomycin and 30% ethanol also cause similar structural changes within the ribosome. In contrast, antibiotics that do not cause miscoding, such as tetracycline, chloramphenicol, erythromycin, fusidic acid and spectinomycin, do not induce the conformational changes triggered by miscoding agents. Furthermore, ribosomes isolated from strains that are either streptomycin resistant or dependent for growth do not show these conformational changes in the presence of streptomycin. These results correlate structural changes in the ribosome induced by miscoding agents in vitro with their in vivo phenotype.     

Structural basis for hygromycin B inhibition of protein biosynthesis

Aminoglycosides are one of the most widely used and clinically important classes of antibiotics that target the ribosome. Hygromycin B is an atypical aminoglycoside antibiotic with unique structural and functional properties. Here we describe the structure of the intact Escherichia coli 70S ribosome in complex with hygromycin B. The antibiotic binds to the mRNA decoding center in the small (30S) ribosomal subunit of the 70S ribosome and induces a localized conformational change, in contrast to its effects observed in the structure of the isolated 30S ribosomal subunit in complex with the drug. The conformational change in the ribosome caused by hygromycin B binding differs from that induced by other aminoglycosides. Also, in contrast to other aminoglycosides, hygromycin B potently inhibits spontaneous reverse translocation of tRNAs and mRNA on the ribosome in vitro. These structural and biochemical results help to explain the unique mode of translation inhibition by hygromycin B.     

Structural origins of gentamicin antibiotic action.

Aminoglycoside antibiotics that bind to the ribosomal A site cause misreading of the genetic code and inhibit translocation. The clinically important aminoglycoside, gentamicin C, is a mixture of three components. Binding of each gentamicin component to the ribosome and to a model RNA oligonucleotide was studied biochemically and the structure of the RNA complexed to gentamicin C1a was solved using magnetic resonance nuclear spectroscopy. Gentamicin C1a binds in the major groove of the RNA. Rings I and II of gentamicin direct specific RNA-drug interactions. Ring III of gentamicin, which distinguishes this subclass of aminoglycosides, also directs specific RNA interactions with conserved base pairs. The structure leads to a general model for specific ribosome recognition by aminoglycoside antibiotics and a possible mechanism for translational inhibition and miscoding. This study provides a structural rationale for chemical synthesis of novel aminoglycosides.     

Cell permeabilization by poliovirus 2B viroporin triggers bystander permeabilization in neighbouring cells through a mechanism involving gap junctions

Poliovirus 2B protein is a well‐known viroporin implicated in plasma membrane permeabilization to ions and low‐molecular‐weight compounds during infection. Translation in mammalian cells expressing 2B protein is inhibited by hygromycin B (HB) but remains unaffected in mock cells, which are not permeable to the inhibitor. Here we describe a previously unreported bystander effect in which healthy baby hamster kidney (BHK) cells become sensitive to HB when co‐cultured with a low proportion of cells expressing poliovirus 2B. Viroporins E from mouse hepatitis virus, 6K from Sindbis virus and NS4A protein from hepatitis C virus were also able to permeabilize neighbouring cells to different extents. Expression of 2B induced permeabilization of neighbouring cell lines other than BHK. We found that gap junctions are responsible mediating the observed bystander permeabilization. Gap junctional communication was confirmed in 2B‐expressing co‐cultures by fluorescent dye transfer. Moreover, the presence of connexin 43 was confirmed in both mock and 2B‐transfected cells. Finally, inhibition of HB entry to neighbouring cells was observed with 18α‐glycyrrhethinic acid, an inhibitor of gap junctions. Taken together, these findings support a mechanism involving gap junctional intercellular communication in the bystander permeabilization effect observed in healthy cells co‐cultured with poliovirus 2B‐expressing cells.     

Paromomycin and dihydrostreptomycin binding to Escherichia coli ribosomes.

Paromomycin binds specifically to a single type of binding site on the 70-S streptomycin-sensitive Escherichia coli ribosome. This site is different from that of dihydrostreptomycin since paromomycin binds to streptomycin-resistant ribosomes and sine dihydrostreptomycin does not compete for paromomycin binding. Paromomycin binding, unlike dihydrostreptomycin binding, is independent of changes in ribosome concentration but influenced by magnesium ion concentration. Moreover, paromomycin does not bind to the 30-S subunit of the streptomycin-sensitive ribosome, except in the presence of dihydrostreptomycin, which probably induces the conformational changes necessary for a paromomycin binding site. This induction does not occur with streptomycin-resistant ribosomes. Neither antibiotic binds to the 50-S subunit. In general, binding of the one antibiotic increases the number of sites available for binding of the other. Both antibiotics exhibit marked non-specific binding at high antibiotic/ribosome ratios. Competition studies have enabled the classification of other aminoglycosides according to their ability to compete for the paromomycin and dihydrostreptomycin binding sites. Derivatives structurally related to paromomycin compete for its binding, the degree of competition being related to antibacterial activity, but do not compete for dihydrostreptomycin binding; they, on the contrary, increase the number of dihydrostreptomycin binding sites. Neither gentamicin nor kanamycin derivatives, which induce a high level of misreading, nor kasugamycin and spectinomycin, which do not induce misreading, compete for paromomycin or dihydrostreptomycin binding sites. Other sites may be involved in the binding of these aminoglycosides and in inducing misreading.     

Interaction of cytoplasmic membrane and ribosomes in Escherichia coli: spectinomycin-induced disappearance of membrane protein I-19.

Incubation of Escherichia coli with spectinomycin caused the disappearance of a major protein from the cytoplasmic membrane. This protein, called "I-19", was not a ribosomal protein. Its disappearance was not a result of the direct action of spectinomycin on the cytoplasmic membrane, but a result of its action on ribosomes. The disappearance was specifically induced by spectinomycin, and other antibiotics such as neomycin, erythromycin, and chloramphenicol had no effect. Although growth was not required for spectinomycin-induced disappearance of protein I-19 from the cytoplasmic membrane, the disappearance was not observed under conditions where protein synthesis was inhibited completely either by the addition of chloramphenicol or by cooling in ice. It is suggested that at least some ribosomes interact with the cytoplasmic membrane and that a modification of the mode of interaction through the action of spectinomycin on ribosomes caused the deletion of membrane protein I-19.     

Effects of some antibiotics on the stringent control of RNA synthesis in Escherichia coli.

Effects of neomycin, spectinomycin, tetracycline and chloramphenicol on the stringent control RNA synthesis and on ppGpp synthesis in the rel+-cells of Escherichia coli having a temperature-sensitive valyl-tRNA synthetase were examined. Without antibiotics, ppGpp began to accumulate and both RNA and protein syntheses were inhibited by transferring the exponentially growing cells from 30 degrees C (permissive temp.) to 40 degrees C (non-permissive temp.). Tetracycline or chloramphenicol, when added after the temperature shift, caused a resumption of RNA synthesis and decay of the accumulated ppGpp, while neomycin or spectinomycin had little effect both on RNA synthesis and the level of ppGpp. When the cells were treated with these antibiotics at permissive temperature, the shift of the temperature to 40 degrees C caused neither inhibition of RNA synthesis nor an accumulation of ppGpp. When neomycin or spectinomycin was added at the beginning of the temperature shift, RNA synthesis continued with an accumulation of ppGpp. Tetracycline or chloramphenicol had no such effect under the same conditions; RNA synthesis continued without an accumulation of ppGpp.     

Plasmid-mediated resistance to aminocyclitol antibiotics in group D streptococci.

Streptococcus faecalis BM4100 was resistant to high levels of gentamicin, kanamycin, and structurally related antibiotics. The genes conferring resistance to aminocyclitols in this strain were carried by a plasmid, pIP800, self-transferable to other S. faecalis strains. The aminocyclitol resistance was mediated by constitutively synthesized phosphotransferase and acetyltransferase activities. It was inferred that phosphorylation occurred at the 2"-hydroxyl group and that acetylation occurred at the 6'-hydroxyl group of the aminocyclitols. The enzyme activities were not separable by gel filtration or by isoelectric focusing. Their apparent molecular weight was 31,000, and their isoelectric point was 5.3. With respect to substrate profile, size, and charge, the transferases from strain BM4100 resembled closely those with identical site specificity described in staphylococci. These results suggest that plasmid gene transfer may occur between the two pathogenic bacterial genera.     

Aminoglycoside sensitivity of ribosomes from the archaebacterium Methanococcus vannielii: Structure-activity relationship

Abstract The effect of about 20 aminoglycoside antibiotics comprising compounds with specific 70S or with 70S plus 80S activity on polypeptide synthesis and translational misreading by ribosomes from the archaebacterium Methanococcus vannielii was investigated. A clear structure-activity relationship was found: sensitivity was observed only to the class of 4,5-disubstituted deoxystreptamine compounds, with neomycin and paromomycin as the most active ones. The streptomycin class aminoglycosides were completely inactive whereas the gentamicin group compounds solely affected misreading and only at high concentrations. Viomycin, a specific inhibitor of the translocation reaction at the eubacterial ribosome which competes with binding of 2-deoxystreptamine aminoglycosides was inactive as well.     

Inhibition of protein synthesis by polypeptide antibiotics.. II. In vitro protein synthesis

Ennis, Herbert L. (St. Jude Children's Research Hospital, Memphis, Tenn.). Inhibition of protein synthesis by polypeptide antibiotics. II. In vitro protein synthesis. J. Bacteriol. 90:1109-1119. 1965.-This investigation has shown that the polypeptide antibiotics of the PA 114, vernamycin, and streptogramin complexes are potent inhibitors of the synthetic polynucleotide-stimulated incorporation of amino acids into hot trichloroacetic acid-insoluble peptide. The antibiotics inhibited the transfer of amino acid from aminoacyl-soluble ribonucleic acid (s-RNA) to peptide. The A component of the antibiotic complex was active alone in inhibiting in vitro protein synthesis, whereas the B fraction was totally inactive. However, the A component, when in combination with the B component, gave a greater degree of inhibition than that observed with the A fraction alone. On the other hand, the endogenous incorporation of amino acid was much less susceptible to inhibition than the incorporation of the corresponding amino acid in a system stimulated by synthetic polynucleotide. In addition, synthesis of polyphenylalanine stimulated by polyuridylic acid was inhibited to a greater extent when the antibiotics were added before the addition of polyuridylic acid to the reaction mixture than when the antibiotics were added after the polynucleotide had a chance to attach to the ribosomes. However, the antibiotics apparently did not inhibit the binding of C(14)-polyuridylic acid or C(14)-phenylalanyl-s-RNA to ribosomes. The antibiotics did not affect the normal release of nascent protein from ribosomes and did not disturb protein synthesis by causing misreading of the genetic code. The antibiotics bind irreversibly to the ribosome, or destroy the functional identity of the ribosome. The antibiotic action is apparently a result of the competition between antibiotic and messenger RNA for a functional site(s) on the ribosome.     

Preferential sensitivity of syntheses of exported proteins to translation inhibitors of low polarity in Escherichia coli.

Summary We have compared the sensitivities to different translation inhibitors of the syntheses of enzymes from various cell compartments. Alkaline and acid phosphatases were chosen as representative of periplasmic enzymes. Aminopeptidase N and -galactosidase represented peripheral membrane protein and cytoplasmic enzyme respectively. Antibiotics of low polarity such as spectinomycin and tetracycline, when used at low concentrations, caused a preferential inhibition of exported proteins compared to total cell proteins, to aminopeptidase N and to -galactosidase. Synthesis itself was inhibited as demonstrated by immunochemical assay of the amount of proteins synthesized in the presence or absence of spectinomycin. Temperature-dependance of the penetration of spectinomycin as reported by its effect, indicated that this drug does not enter the cell by simple dissolution in the membrane lipids but rather enters at specific sites of the cell envelope. In contrast to low polarity inhibitors, a polar antibiotic such as kanamycin did not cause any preferential inhibition of the syntheses of exported proteins. By using radioactive tetracycline (0.025, 0.1 and 0.2 g/ml) we have investigated the distribution of this antibiotic between free and membrane-bound polysomes isolated without the use of lysozyme. Three times as much tetracycline was found in the membrane-bound compared to free polysomes fraction.Therefore we propose that preferential sensitivity of synthesis of exported proteins to translation inhibitors of low polarity reflects a preferential binding of these antibiotics to membrane-bound polysomes fractions in Escherichia coli. We thus suggest that this preferential binding occurs since the site of entry of weakly ionized antibiotics used in this study, correspond to the site of synthesis and transfer of exported proteins.     

Control of membrane permeability in animal cells by divalent cations

The permeability of several cell lines, including HeLa, L929, 3T6 and 3T3, to various compounds is affected by the concentration of divalent cations in the culture medium. In the absence of Mg2+ ions but with 4-8 mM CaCl2 in the medium, HeLa and L929 cells become permeabilized, as measured by the entry of the aminoglycoside antibiotic hygromycin B. However, 3T3 and 3T6 cells become much more permeable when calcium and magnesium are both absent from the medium. Addition of Mg2+ above 2 mM abolishes the permeabilization induced by Ca2+. Basic pH favors permeabilization, whereas acidic pH inhibits the entry of hygromycin B. Increased entry of macromolecules, such as the toxin alpha-sarcin, horseradish peroxidase (HRP) and luciferase, is also observed under permeabilization conditions, suggesting that this method could be of general use, since it is not harmful to cells and is fully reversible. Exit of 86Rb+ ions and [3H]uridine-labelled nucleotides was also assayed. We did not observe increased release of these compounds from preloaded cells under various calcium concentrations. Finally, the effects of several inhibitors of endocytosis and other membrane functions on the permeabilization inhibitors of endocytosis and other membrane functions on the permeabilization process were also analysed. The entry of alpha-sarcin was not affected by nifedipine, dibucaine or mepacrine, but was partially inhibited by NH4Cl, amantadine and chloroquine.     

Mistranslation in a eucaryotic organism.

Previous work from our laboratory has demonstrated that a subclass of the aminoglycoside antibiotics, those containing the drug fragment paromamine, stimulates mistranslation in cell-free protein-synthesizing systems derived from eucaryotic cells. We report here experiments which show that the ciliate Tetrahymena thermophila (formerly T. pyriformis, syngen 1) is sensitive to the paromamine-containing aminoglycoside antibiotics. The drugs are active with respect to growth inhibition, inhibition of protein synthesis in the whole organism, inhibition of protein synthesis in vitro and the stimulation of mistranslation in cell-free protein-synthesizing systems. Because of their misreading properties, these drugs may be useful in isolating and propagating strains carrying mutations which can be translationally suppressed (that is, missense and nonsense mutations).     

The bactericidal action of streptomycin: membrane permeabilization caused by the insertion of mistranslated proteins into the cytoplasmic membrane of Escherichia coli and subsequent caging of the antibiotic inside the cells due to degradation of these proteins.

The mechanism by which the aminoglycoside antibiotic streptomycin permeabilizes the cytoplasmic membrane of Escherichia coli cells was reinvestigated. For this purpose, the extent of streptomycin-induced K+ loss from cells growing at low external K+ concentrations was taken as a measure of membrane permeabilization. Experiments with different K(+)-uptake mutants showed that the antibiotic specifically increased the passive permeability of the cell membrane to K+ and other ions. These permeability changes were small and the membrane potential of the treated cells remained high. The membrane permeabilization was not due to a direct interaction of the antibiotic with the cell membrane, since cells that carry an rpsL mutation and synthesize proteins in a streptomycin-insensitive way did not lose K+ after the addition of the antibiotic. Due to misreading and premature termination of translation the cells synthesized aberrant proteins under the conditions where membrane permeabilization occurred. Two conditions are described under which the cells both degraded these mistranslated proteins rapidly and reaccumulated K+, lending support to the hypothesis that membrane permeabilization is due to the presence of the mistranslated proteins in the cell membrane. Evidence is presented that the irreversibility of (dihydro)streptomycin uptake by cells washed free from the antibiotic might also be due to rapid degradation of the mistranslated proteins, leading to 'caging' of the antibiotic inside the cells.     

Effect of interferon treatment on blockade of protein synthesis induced by poliovirus infection

Treatment of HeLa cells with lymphoblastoid interferon leads to a drastic inhibition of infective poliovirus. Even relatively high concentrations of human lymphoblastoid interferon HuIFN-alpha (Ly) (400 IU/ml) do not prevent destruction of the cell monolayer after most of the cells have been infected with poliovirus. Analysis of macromolecular synthesis in a single step growth cycle of poliovirus in interferon-treated cells detected no viral protein synthesis. In spite of this inhibition of viral translation, the shut-off of host protein synthesis in interferon-treated cells is apparent when they are infected both at low and high multiplicities. Although viral RNA synthesis is inhibited considerably in cells treated with interferon, a certain amount is detected, suggesting that some viral replication takes place. Analysis of membrane permeability after poliovirus infection shows a leakage to 86Rb+ ions and modification of membrane permeability to the translation inhibitor hygromycin B at the moment when the bulk of virus protein synthesis occurs. These changes are delayed and even prevented if cells are pretreated with interferon. A situation is described in which host protein synthesis is shut-down with no major changes in membrane permeability, as studied by the two tests mentioned above. Prevention of viral gene expression by inactivation with ultraviolet light of the input virus or by treatment with cycloheximide blocks the shut-off of protein synthesis. This does not occur in the presence of 3 mM guanidine. These observations are in agreement with the idea that some poliovirus protein synthesis takes place in interferon-treated cells and this early gene expression is necessary to block cellular protein synthesis.