Antibiotics targeting bacterial ribosomal subunit biogenesis.

This article describes 20 years of research that investigated a second novel target for ribosomal antibiotics, the biogenesis of the two subunits. Over that period, we have examined the effect of 52 different antibiotics on ribosomal subunit formation in six different microorganisms. Most of the antimicrobials we have studied are specific, preventing the formation of only the subunit to which they bind. A few interesting exceptions have also been observed. Forty-one research publications and a book chapter have resulted from this investigation. This review will describe the methodology we used and the fit of our results to a hypothetical model. The model predicts that inhibition of subunit assembly and translation are equivalent targets for most of the antibiotics we have investigated.

An examination of the inhibitory effects of three antibiotics in combination on ribosome biosynthesis in Staphylococcus aureus.

Although a number of different antibiotics are used to combat staphylococcal infections, resistance has continued to develop. The use of rifampicin and ciprofloxacin in combination with azithromycin, known for its inhibitory effects on the bacterial ribosome, can create potential synergistic effects on ribosomal subunit synthesis rates. In this work, combination antibiotic treatments gave a significant decrease in cell numbers following growth in the presence of ciprofloxacin or rifampicin with azithromycin compared to those grown with azithromycin or rifampicin alone. DNA, RNA and protein synthesis rates were reduced with single antibiotic treatments and showed further decreases when drug combinations were used. 70S ribosome levels were reduced with every antibiotic treatment. DNA gyrase subunits A and B showed significant decreases for double and triple antibiotic-treated samples. Ribosomal subunit synthesis rates were diminished for each different antibiotic combination. Turnover of 16S and 23S rRNA was also observed in each case and was stimulated by antibiotic combinations. The frequency of spontaneous resistance was reduced in all double selections, and no triply resistant mutants were found.

Inhibition of ribosomal subunit synthesis in Escherichia coli by the vanadyl ribonucleoside complex.

The increase in antibiotic-resistant microorganisms has driven a search for new antibiotic targets and novel antimicrobial agents. A large number of different antibiotics target bacterial ribosomal subunit formation. Several specific ribonucleases are important in the processing of rRNA during subunit biogenesis. This work demonstrates that the ribonuclease inhibitor, vanadyl ribonucleoside complex (VRC), can inhibit RNases involved in ribosomal subunit formation. The ribosomal subunit synthesis rate was significantly decreased and ribosomal RNA from the subunit precursors was degraded. VRC had no inhibitory effect on translation. VRC also potentiated the inhibitory effects of an aminoglycoside and a macrolide antibiotic.

Solithromycin inhibition of protein synthesis and ribosome biogenesis in Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae.

The continuing increase in antibiotic-resistant microorganisms is driving the search for new antibiotic targets and improved antimicrobial agents. Ketolides are semisynthetic derivatives of macrolide antibiotics, which are effective against certain resistant organisms. Solithromycin (CEM-101) is a novel fluoroketolide with improved antimicrobial effectiveness. This compound binds to the large 50S subunit of the ribosome and inhibits protein biosynthesis. Like other ketolides, it should impair bacterial ribosomal subunit formation. This mechanism of action was examined in strains of Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae. The mean 50% inhibitory concentrations (IC50s) for solithromycin inhibition of cell viability, protein synthesis, and growth rate were 7.5, 40, and 125 ng/ml for Streptococcus pneumoniae, Staphylococcus aureus, and Haemophilus influenzae, respectively. The net formation of the 50S subunit was reduced in all three organisms, with IC50s similar to those given above. The rates of 50S subunit formation measured by a pulse-chase labeling procedure were reduced by 75% in cells growing at the IC50 of solithromycin. Turnover of 23S rRNA was stimulated by solithromycin as well. Solithromycin was found to be a particularly effective antimicrobial agent, with IC50s comparable to those of telithromycin and significantly better than those of azithromycin and clarithromycin in these three microorganisms.

The vanadyl ribonucleoside complex inhibits ribosomal subunit formation in Staphylococcus aureus.

OBJECTIVES: The discovery of new antibiotic targets is important to stem the increase in antibiotic resistance to most currently used antimicrobials. The bacterial ribosome is a major target for a large number of antibiotics that inhibit different aspects of translation. Most of these antimicrobial agents also inhibit ribosomal subunit formation as a second cellular target. Precise subunit assembly requires the activity of several distinct RNases for proper rRNA processing. The present work shows that the vanadyl ribonucleoside complex (VRC) inhibited RNases in Staphylococcus aureus involved in ribosomal subunit formation without an effect on translation. METHODS: Methicillin-susceptible and -resistant strains of S. aureus were examined for the inhibitory effects of VRC on cell viability by colony counting. Protein synthesis rates were measured by isotopic methionine incorporation. Ribosome synthesis was measured by radiolabelled uridine incorporation into ribosomal subunits as displayed on sucrose gradients. Pulse and chase radiolabelling was used to measure subunit synthesis rates. RNA turnover was determined by a gel on a chip assay. RESULTS: The rates of subunit synthesis and the amounts of both subunits were significantly reduced in the presence of the compound. Ribosomal RNA was degraded and cell viability was reduced as a consequence. VRC also stimulated the inhibitory effects of a macrolide and an aminoglycoside antibiotic on ribosome formation. CONCLUSIONS: Bacterial ribosomal subunit synthesis was specifically impaired in VRC-treated cells, with the rates and amounts of both subunits reduced. Cell viability was significantly reduced and rRNA turnover was stimulated.

Three methods to assay inhibitors of ribosomal subunit assembly.

The inhibition of bacterial ribosomal subunit formation is a novel target for translational inhibitors. Inhibition of subunit biogenesis has been shown to be equivalent to the inhibition of protein biosynthesis for many antibiotics. This chapter describes three methods for examining the inhibition of subunit formation in growing bacterial cells. The first method permits the determination of the IC50 value for inhibition of assembly and protein synthesis. The second is a pulse and chase labeling procedure to measure the kinetics of subunit formation. The third procedure allows an examination of ribosome reformation after antibiotic removal as a part of the post-antibiotic effect. Together these procedures give a description of the relative inhibitory effects of an antibiotic on translation and subunit formation.

Characterization of a 30S ribosomal subunit assembly intermediate found in Escherichia coli cells growing with neomycin or paromomycin.

Neomycin and paromomycin are aminoglycoside antibiotics that specifically stimulate the misreading of mRNA by binding to the decoding site of 16S rRNA in the 30S ribosomal subunit. Recent work has shown that both antibiotics also inhibit 30S subunit assembly in Escherichia coli and Staphylococcus aureus cells. This work describes the characteristics of an assembly intermediate produced in E. coli cells grown with neomycin or paromomycin. Antibiotic treatment stimulated the accumulation of a 30S assembly precursor with a sedimentation coefficient of 21S. The particle was able to bind radio-labeled antibiotics in vivo and in vitro. Hybridization experiments showed that the 21S precursor particle contained unprocessed 16S rRNA with both 5' and 3' extensions. Ten 30S ribosomal proteins were found in the precursor after inhibition by each drug. In addition, cell free reconstitution assays generated a 21S particle after incubation with either aminoglycoside. This work helps to define the features of the ribosome structure as a target for antimicrobial agents and may provide information needed for the design of more effective antibiotics.

Characteristics of a 50S ribosomal subunit precursor particle as a substrate for ermE methyltransferase activity and erythromycin binding in Staphylococcus aureus.

Erythromycin is a macrolide antibiotic that inhibits not only mRNA translation but also 50S ribosomal subunit assembly in bacterial cells. An important mechanism of erythromycin resistance is the methylation of 23S rRNA by erm methyl transferase enzymes. A model for 50S ribosomal subunit formation suggests that the precursor particle which accumulates in erythromycin treated cells is the target for methyl transferase activity. Hybridization experiments identified the presence of 23S rRNA in the 50S precursor particle. The protein content of the 50S precursor particle was analyzed by MALDI-TOF mass spectrophotometry. These studies have identified 23 of 36 50S ribosomal proteins in the precursor. Methyltransferase assays demonstrated that the 50S precursor particle was a substrate for ermE methyltransferase. Competition experiments indicated that the enzyme could displace erythromycin from the 50S precursor particle and that the methyltransferase had a higher association constant for the precursor particle compared to that of erythromycin. Inhibition experiments showed that macrolide, lincosamide and streptogramin B compounds bound to the precursor particle with similar affinity and inhibited the ermE methyltransferase activity. These studies shed light on the interaction of ermE methyltransferase and erythromycin in this clinically important pathogen.

Retapamulin inhibition of translation and 50S ribosomal subunit formation in Staphylococcus aureus cells.

Retapamulin inhibited protein biosynthesis and cell viability in methicillin-sensitive and methicillin-resistant Staphylococcus aureus organisms. A specific inhibitory effect on 50S ribosomal subunit formation was also found. Pulse-chase labeling experiments confirmed the specific inhibition of 50S subunit biogenesis. Turnover of 23S rRNA was found, with no effect on 16S rRNA amounts.

Hygromycin B inhibition of protein synthesis and ribosome biogenesis in Escherichia coli.

The aminoglycoside antibiotic hygromycin B was examined in Escherichia coli cells for inhibitory effects on translation and ribosomal-subunit formation. Pulse-chase labeling experiments were performed, which verified lower rates of ribosomal-subunit synthesis in drug-treated cells. Hygromycin B exhibited a concentration-dependent inhibitory effect on viable-cell numbers, growth rate, protein synthesis, and 30S and 50S subunit formation. Unlike other aminoglycosides, hygromycin B was a more effective inhibitor of translation than of ribosomal-subunit formation in E. coli. Examination of total RNA from treated cells showed an increase in RNA corresponding to a precursor to the 16S rRNA, while mature 16S rRNA decreased. Northern hybridization to rRNA in cells treated with hygromycin B showed that RNase II- and RNase III-deficient strains of E. coli accumulated 16S rRNA fragments upon treatment with the drug. The results indicate that hygromycin B targets protein synthesis and 30S ribosomal-subunit assembly.

The other target for ribosomal antibiotics: inhibition of bacterial ribosomal subunit formation.

The development of microbial resistance to practically all currently used antimicrobial agents has spurred efforts to develop new antibiotics and to identify novel targets in bacterial cells. This review summarizes the evidence for inhibition of bacterial ribosomal subunit formation as a target for many antibiotics distinct from their well-known inhibition of translation. Features of a model to explain this activity are explored. Results are presented to show the accumulation of both 30S and 50S ribosomal subunit precursors in antibiotic inhibited cells. These precursors have been characterized and are shown to bind radio-labeled drugs. Pulse and chase labeling studies have revealed the slower rates of subunit synthesis in drug treated cells compared with uninhibited controls. Resynthesis of subunits after antibiotic removal precedes recovery of control protein synthesis capacity, consistent with the model presented. Also certain mutant strains defective in different ribonuclease activities are more susceptible to antibiotic inhibition of assembly as predicted. Results indicating the equivalence of assembly inhibition and translational inhibition are described. Lastly, the identification of a 50S subunit precursor particle as a substrate for rRNA methyltransferase activity is shown. The weight of evidence presented clearly indicates that ribosomal antibiotics have a second target in cells. Inhibition of cell growth and subsequent cell death results from the activity of these antibiotics on the combined targets. The possibility of designing assembly specific inhibitors is discussed.

A comparison of a new oral streptogramin XRP 2868 with quinupristin-dalfopristin against antibiotic-resistant strains of haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae.

A new streptogramin antibiotic XRP 2868 was compared with quinupristin-dalfopristin for inhibitory activities against antibiotic-resistant Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. In each organism examined, XRP 2868 had an IC(50) that was twofold to fivefold lower than quinupristin-dalfopristin, for inhibition of cell viability, protein synthesis, and ribosomal subunit formation.

Accumulation and turnover of 23S ribosomal RNA in azithromycin-inhibited ribonuclease mutant strains of Escherichia coli.

Ribosomal RNA is normally a stable molecule in bacterial cells with negligible turnover. Antibiotics which impair ribosomal subunit assembly promote the accumulation of subunit intermediates in cells which are then degraded by ribonucleases. It is predicted that cells expressing one or more mutated ribonucleases will degrade the antibiotic-bound particle less efficiently, resulting in increased sensitivity to the antibiotic. To test this, eight ribonuclease-deficient strains of Escherichia coli were grown in the presence or absence of azithromycin. Cell viability and protein synthesis rates were decreased in these strains compared with wild type cells. Degradation of 23S rRNA and recovery from azithromycin inhibition were examined by 3H-uridine labeling and by hybridization with a 23S rRNA specific probe. Mutants defective in ribonuclease II and polynucleotide phosphorylase demonstrated hypersensitivity to the antibiotic and showed a greater extent of 23S rRNA accumulation and a slower recovery rate. The results suggest that these two ribonucleases are important in 23S rRNA turnover in antibiotic-inhibited E. coli cells.

An examination of the differential sensitivity to ketolide antibiotics in ermB strains of Streptococcus pyogenes and Streptococcus pneumoniae.

Several reports in the literature have described a differential sensitivity to ketolide antibiotics in ermB strains of Streptococcus pyogenes and Streptococcus pneumoniae resistant to erythromycin. Strains of S. pyogenes and S. pneumoniae carrying different erm gene alleles were examined for their susceptibility to the ketolide antibiotics cethromycin (ABT-773) and telithromycin. The effect of the antibiotics on cell growth and viability was assessed as were effects on protein synthesis and 50S ribosomal subunit formation. The susceptibility of wild-type strains of both organisms was compared with effects in strains containing the ermA and ermB methyltransferase genes. A wild-type antibiotic-susceptible strain of S. pyogenes was comparable to an ermA strain of the organism in its ketolide sensitivity, with IC(50) values for 50% inhibition of protein synthesis and 50S ribosomal subunit formation of 10 ng/mL for cethromycin and 16 ng/mL for telithromycin. An S. pneumoniae strain with the ermB gene and an S. pyogenes strain with the ermA gene were also similar in their sensitivity to ketolide inhibition. IC(50) values for inhibition of translation and subunit formation in S. pneumoniae ( ermB) were 30 ng/mL and 55 ng/mL and for the ermA strain of S. pyogenes they were 15 ng/mL and 35 ng/mL respectively. By contrast, an S. pyogenes ermB strain was significantly more resistant to both ketolides, with IC(50) values for inhibition of 50S synthesis of 215 and 380 ng/mL for the two ketolides. Experiments were conducted to examine ribosome synthesis and translational activity in the two ermB strains at intervals during growth in the presence of each antibiotic. Cell viability and 50S subunit formation were dramatically reduced in the S. pneumoniae strain during continued growth with either drug. By contrast, the ketolides had little effect on the S. pyogenes strain growing with the antibiotics. The results indicate that ketolides have a reduced inhibitory effect on translation and 50S subunit synthesis in S. pyogenes with the ermB gene compared with the other strains examined.

Structure-activity relationships for three macrolide antibiotics in Haemophilus influenzae.

A prior study examining differences in the activities of erythromycin and azithromycin on cellular functions in the Gram-negative pathogen, Haemophilus influenzae, revealed a marked difference in their inhibitory activities. The study revealed that protein synthesis and 50S ribosomal subunit assembly were equal targets for inhibition by azithromycin while erythromycin was a preferential inhibitor of translation. This contrast in inhibitory activities stimulated a comparative analysis of three additional antibiotics: clarithromycin, flurithromycin and roxithromycin. Each compound was tested over a concentration range for inhibitory effects on cellular processes. Clarithromycin was the most effective inhibitor of protein synthesis with an IC(50) of 5.6 microg/mL, followed by flurithromycin at 6 microg/mL, and roxithromycin at 9 microg/mL. IC(50) values for antibiotic effects on viable cell counts and growth rates were similar to those obtained for protein synthesis. Flurithromycin had the strongest effect on 50S ribosomal subunit formation with an IC(50) of 8 microg/mL, followed by clarithromycin and roxithromycin, at 9.0 microg/mL and 12.5 microg/mL respectively. 30S ribosomal subunit formation in cells treated with flurithromycin and roxithromycin was also reduced to some extent. Pulse-and-chase labeling kinetics examining subunit assembly rates verified the slower synthesis rate of the subunits in the presence of each macrolide. The results are discussed in terms of structural differences of these macrolides and their differential inhibitory effects on both cellular targets.

Neomycin and paromomycin inhibit 30S ribosomal subunit assembly in Staphylococcus aureus.

A number of different antibiotics that prevent translation by binding to the 50S ribosomal subunit of bacterial cells have recently been shown to also prevent assembly of this subunit. Antibacterial agents affecting 30S particle activities have not been examined extensively for effects on small subunit formation. The aminoglycoside antibiotics paromomycin and neomycin bind specifically to the 30S ribosomal subunit and inhibit translation. These drugs were examined in Staphylococcus aureus cells to see whether they had a second inhibitory effect on 30S particle assembly. A 3H-uridine pulse and chase assay was used to examine the kinetics of subunit synthesis in the presence and absence of each antibiotic. 30S subunit formation was inhibited by both compounds. At 3 microg/mL each antibiotic reduced the rate of 30S formation by 80% compared with control cells. Both antibiotics showed a concentration-dependent inhibition of particle formation, with a lesser effect on 50S particle formation. For neomycin, the IC50 for 30S particle formation was equal to the IC50 for inhibition of translation. Both antibiotics reduced the viable cell number with an IC50 of 2 microg/mL. They also inhibited protein synthesis in the cells with different IC50 values (2.5 and 1.25 microg/mL). This is the second demonstration of 30S ribosomal subunit-specific antibiotics that prevent assembly of the small subunit.

A 50S ribosomal subunit precursor particle is a substrate for the ErmC methyltransferase in Staphylococcus aureus cells.

Macrolide antibiotics like erythromycin can induce the synthesis of a specific 23S rRNA methyltransferase which confers resistance to cells containing the erm gene. Erythromycin inhibits both protein synthesis and the formation of 50S subunits in bacterial cells. We have tested the idea that the 50S precursor particle that accumulates in antibiotic-treated Staphylococcus aureus cells is a substrate for the methyltransferase enzyme. Pulse-chase labeling studies were conducted to examine the rates of ribosomal subunit formation in control and erythromycin-induced cells. Erythromycin binding to 50S subunits was examined under the same conditions. The rate of 50S subunit formation was reduced for up to 30 min after antibiotic addition, and erythromycin binding was substantial at this time. A nuclease protection assay was used to examine the methylation of adenine 2085 in 23S rRNA after induction. A methyl-labeled protected RNA sequence was found to appear in cells 30 min after induction. This protected sequence was found in both 50S subunits and in a subunit precursor particle sedimenting at about 30S in sucrose gradients. 23S rRNA isolated from 50S subunits of cells could be labeled by a ribosome-associated methlytransferase activity, with (3)H-S-adenosylmethionine as a substrate. 50S subunits were not a substrate for the enzyme, but the 30S gradient region from erythromycin-treated cells contained a substrate for this activity. These findings are consistent with a model that suggests that antibiotic inhibition of 50S formation leads to the accumulation of a precursor whose 23S rRNA becomes methylated by the induced enzyme. The methylated rRNA will preclude erythromycin binding; thus, assembly of the particle and translation become insensitive to the inhibitory effects of the drug.

Bacterial ribosomal subunit assembly is an antibiotic target.

A substantial number of antimicrobial agents target some activity of the bacterial ribosome for inhibition. Mechanistic studies and recent structural investigations of the ribosome have identified the binding sites and presumed mechanism of inhibitory activity for some compounds. A second target for many of these antibiotics has recently been examined. Formation of both 30S and 50S ribosomal subunits in bacterial cells is impaired by translational inhibitors. For many antimicrobial agents, inhibition of this target is equivalent to inhibition of translation in preventing cell growth. This review will describe features of this new target including the types of compounds which affect particle assembly and differences in the process in different microorganisms. The characteristics of this new target will be identified and aspects of a model to explain this new inhibitory activity will be explored.

Preferential inhibition of protein synthesis by ketolide antibiotics in Haemophilus influenzae cells.

The ketolide antibiotics are semi-synthetic derivatives of erythromycin A with enhanced inhibitory activity in a wide variety of microorganisms. They have significantly lower MICs than the macrolide antibiotics for many Gram-positive organisms. Two ketolides, telithromycin and ABT-773, were tested for growth-inhibitory effects in Haemophilus influenzae. Both antibiotics increased the growth rate and reduced the viable cell number with IC(50) values of 1.5 microgram/ml. Protein synthesis was inhibited in cells with a similar IC(50) concentration (1.25 microgram/ml). Macrolide and ketolide antibiotics have been shown to have a second equivalent target for inhibition in cells, which is blocking the assembly of the 50S ribosomal subunit. Pulse and chase labeling assays were conducted to examine the effect of the ketolides on subunit formation in H. influenzae. Surprisingly, both antibiotics inhibited 50S and 30S subunit assembly to the same extent, with no specific effect of the compounds on 50S assembly. Over a range of antibiotic concentrations, 30S particle synthesis was diminished to the same extent as 50S formation. H. influenzae cells seem to have only one significant target for these antibiotics, and this may help to explain why these drugs are not more effective than the macrolides in preventing the growth of this microorganism.