Retapamulin Inhibition of Translation and ACCEPTED. 50S Ribosomal Subunit Formation in. Staphylococcus aureus Cells

Transcription

1 AAC Accepts, published online ahead of print on 11 June 2007 Antimicrob. Agents Chemother. doi: /aac Copyright 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Retapamulin Inhibition of Translation and 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells W. Scott Champney* and Ward K. Rodgers Department of Biochemistry and Molecular Biology J.H. Quillen College of Medicine East Tennessee State University Johnson City, TN USA Phone: (423) Fax: (423) E mail: champney@etsu.edu Running title: Retapamulin Inhibition of Ribosome Synthesis Key Words: S. aureus, retapamulin, ribosomal subunits, antibiotics, protein synthesis Abstract 1

2 Retapamulin inhibited protein biosynthesis and cell viability in MSSA and MRSA organisms. A specific inhibitory effect on 50S ribosomal subunit formation was also found. Pulse and chase labeling experiments confirmed the specific inhibition of 50S subunit biogenesis. Turnover of 23S rrna was found with no effect on 16S rrna amounts. The emergence of antibiotic-resistant microorganisms has stimulated a search for new antibacterial agents to combat this serious crisis (1, 10, 12). New antibacterial agents with promise are derivatives of the pleuromutilin compounds. Semi-synthetic derivatives of the mutilins have been made which show enhanced antibacterial activity against a variety of pathogens. Retapamulin (SB ) is especially effective against Gram-positive cocci (13, 15, 19). Inhibition of peptide bond formation is the basis of inhibitory activity (22). A crystal structure of the antibiotic bound to the 50S subunit has recently been derived (11). It is currently in use as an FDA approved topical antimicrobial agent (20). Biogenesis of the 50S particle is prevented by many agents which bind to the large ribosomal subunit (reviewed in Champney (3)). The inhibition of 50S subunit functions in translation by the compound retapamulin stimulated a test of its ability to stop synthesis of this subunit (16, 22). Retapamulin inhibitory effects on translation and cell growth were examined over a range of concentrations in both wild type S. aureus strain RN1786 (14) and MRSA strain A1024 (6). MIC values for the two strains were 35 and 55 ng/ ml respectively. 2

3 Fig. 1A shows the inhibition of protein synthesis in the two organisms. Similar inhibitory effects on cell viability were seen (Fig. 1B), with comparable IC 50 values (Table 1). Retapamulin inhibition of 50S subunit synthesis was also examined in both strains. As Fig. 1C and 1D show, a specific inhibitory effect on large subunit formation was seen with IC 50 values of 27 ng/ ml and 20 ng/ ml for the two strains (Table 1). No inhibition of 30S particle formation was found. The effect of the antibiotic on ribosome synthesis was also examined by a pulse and chase labeling protocol. Fig. 2 indicates the rates of subunit formation in control and retapamulin-treated MRSA cells. Synthesis of the 30S subunit was unaffected by the antibiotic (Fig. 2B ), but the 50S formation rate was reduced (Fig. 2B). Specific inhibition of the rate of 50S subunit biogenesis was also observed in the wt cells (data not shown). Inhibition of 50S synthesis leads to the accumulation of stalled intermediates in the assembly pathway which are degraded by cellular ribonucleases in E coli cells (21). Retapamulin was examined for effects on 23S rrna turnover in both strains. A reduction in 23S rrna amountts was found by Agilent Bioanalyzer analysis (Fig. 3). About 25% of the 23S RNA was lost in both organisms during growth at 10 ng/ ml of the antibiotic (Table 2). A substantial increase in small RNA oligonucleotides was found for cells growing with retapamulin (Fig. 3 and Table 2). Like several other 50S subunit specific antibiotics, retapamulin has dual inhibitory effects in S. aureus cells. Both translation and 50S subunit formation were significantly affected by antibiotic treatment. Other 50S subunit antibiotics including erythromycin 3

4 (4), clarithromycin and azithromycin (5), telithromycin (9), linezolid (7), TAN1057A (8) and quinupristin-dalfopristin (17) have this same dual target specificity. In the wild type organism, retapamulin showed a preferential inhibitory effect on protein synthesis with about 5 times as much drug needed to give an equivalent inhibition of 50S formation (Table 1). In the MRSA organism 50S synthesis and translation were inhibited to the same extent by the antibiotic (Table 1). This difference in sensitivity to the drug may reflect the different clinical origins of these strains (6, 14). The close similarity of the inhibitory effects of retapamulin on methicillin-sensitive and methicillin-resistant S. aureus suggest its potential as an effective antimicrobial agent against this problematic organism. Others have also shown an equivalent inhibitory effect of retapamulin against both sensitive and resistant S. aureus strains (19, 20). It is thus a promising antimicrobial agent which should be useful against a variety of drug-resistant microorganisms. This research was supported by a grant from GlaxoSmithKline who also provided retapamulin. We are pleased to acknowledge helpful discussions with Karen O Dwyer at GSK. Literature Cited 1. Anderson, R. M The pandemic of antibiotic resistance. Nature Medicine 5:

5 2. Champney, W. S Bacterial ribosomal subunit assembly is an antibiotic target. Current Topics in Medicinal Chemistry 3: Champney, W. S The other target for ribosomal antibiotics. Inhibition of bacterial ribosomal subunit formation. Infectious Disorders - Drug Targets 6: Champney, W. S., and R. Burdine S ribosomal subunit synthesis and translation are equivalent targets for erythromycin inhibition in Staphylococcus aureus. Antimicrobial Agents Chemother 40: Champney, W. S., and R. Burdine Azithromycin and clarithromycin inhibition of 50S ribosomal subunit formation in Staphylococcus aureus cells. Curr Microbiol 36: Champney, W. S., and R. Burdine Macrolide antibiotic inhibition of translation and 50S ribosomal subunit assembly in methicillin-resistant Staphylococcus aureus cells. Microb Drug Resist 4: Champney, W. S., and M. Miller Linezolid is a specific inhibitor of 50S ribosomal subunit formation in Staphylococcus aureus cells. Curr Microbiol 44:

6 8. Champney, W. S., J. Pelt, and C. L. Tober TAN-1057A: a translational inhibitor with a specific inhibitory effect on 50S ribosomal subunit formation. Curr Microbiol 43: Champney, W. S., and C. L. Tober Structure-activity relationships for six ketolide antibiotics. Curr Microbiol 42: D'Agata, E. M Rapidly rising prevalence of nosocomial multidrug-resistant Gram-negative Bacilli: a 9-year surveillance study. Infect Control Hosp. Epidemiol. 25: Davidovich, C., A. Bashan, T. Auerbach-Nevo, R. D. Yaggie, R. R. Gontarek, and A. Yonath Induced-fit tightens pleuromutilins binding to ribosomes and remote interactions enable their selectivity. Proc. Natl Acad. Sci. U.S. 104: Diekema, D. J., B. J. BootsMiller, T. E. Vaughn, R. F. Woolson, J. W. Yankey, E. J. Ernst, S. D. Flach, M. M. Ward, C. L. Francisco, and M. A. Pfaller Antimicrobial resistance trends and outbreak frequency in United States hospitals. Clin. Infect. Diseases 38: Jones, R. N., T. R. Fritsche, H. S. Sader, and J. E. Ross Activity of retapamulin (SB ), a novel pleuromutilin, against selected resistant grampositive cocci. Antimicrobial Agents Chemother 50:

7 14. Koepsel, R. R., R. W. Murray, W. D. Rosenblum, and S. A. Khan Purification of pt181-encoded repc protein required for the initiation of plasmid replication. J Biol. Chem. 260: Kosowaska-Shick, K., C. Clark, K. Credito, P. McGhee, B. Dewasse, T. Bogdanovich, and P. C. Appelbaum Single- and multistep resistance selection studies on the activity of retapamulin compared to other agents against Staphylococcus aureus and Streptococcus pyogenes. Antimicrobial Agents Chemother 50: Long, K. S., L. H. Hansen, L. Jacobsen, and B. Vester Interaction of pleuromutilin derivatives with the ribosomal peptidyl transferase center. Antimicrobial Agents Chemother 50: Mabe, S., and W. S. Champney A comparison of a new oral streptogramin XRP2868 with quinupristin-dalfopristin against antibiotic-resistant strains of Haemophilus influenzae, Streptococcus pmeumoniae and Staphylococcus aureus. Curr Microbiol 51: McGaha, S. M., and W. S. Champney Hygromycin B inhibition of protein synthesis and ribosome biogenesis in Escherichia coli. Antimicrobial Agents Chemother 51:

8 19. Pankuch, G. A., G. Lin, D. B. Hoellman, C. E. Good, M. R. Jacobs, and P. C. Appelbaum Activity of retapamulin against Streptococcus pyogenes and Staphylococcus aureus evaluated by agar dilution, microdilution, E-test, and disk diffusion methodologies. Antimicrobial Agents Chemother 50: Rittenhouse, S., S. Biswas, J. Broskey, L. McCloskey, T. Moore, S. Vasey, J. West, M. Zalacain, R. Zonis, and D. Payne Selection of retapamulin, a novel pleuromutilin for topical use. Antimicrobial Agents Chemother 50: Silvers, J. A., and W. S. Champney Turnover and degradation of 23S ribosomal RNA in azithromycin-inhibited ribonuclease mutants of Escherichia coli. Arch Microbiol. 187: Yan, K., L. Madden, A. E. Choudhry, C. S. Voigt, R. A. Copeland, and R. R. Gontarek Biochemical characterization of the interactions of the novel pleuromutilin derivative retapamulin with bacterial ribosomes. Antimicrobial Agents Chemother 50: Figure legends Fig. 1. Inhibition of protein synthesis, cell viability and subunit formation in S. aureus cells with increasing concentrations of retapamulin. Antibiotic effects on cell growth, viability, protein synthesis and subunit formation were exmined by a four-part assay 8

9 procedure as described previously (2). Cells were grown for 2 doubling times in the presence of the antibiotic. (A) Inhibition of protein synthesis by retapamulin in wt ( ) and MRSA ( ) strains. (B) Inhibition of viable cell number (TVC) by retapamulin in wt ( ) and MRSA ( ) strains. (C) Concentration dependent inhibition of ribosomal subunit assembly in wild type S. aureus cells. Inhibition of 30S assembly ( ) and inhibition of 50S assembly ( ). (D) Concentration dependent inhibition of ribosomal subunit assembly in MRSA cells. Inhibition of 30S assembly ( ) and inhibition of 50S assembly ( ). The percent of the total gradient cpm was calculated for the 30S and 50S regions for the control and antibiotic-treated samples. Results are the mean of two determinations. Arrows indicate the IC 50 values. Fig H-uridine pulse and chase labeling kinetic analysis of ribosomal subunit formation was conducted as described previously (2). Cells were labeled with 3 H-uridine (1µCi/ ml) for 2 min, chased with uridine (50 µg/ ml) and samples were collected at the indicated time intervals for subunit formation by sucrose gradient analysis (2). (A) Assembly of 30S subunits ( ) and 50S subunits ( ) in MRSA cells without retapamulin. (B) Assembly of 30S subunits ( ) and 50S subunits ( ) in MRSA cells with retapamulin at 8 ng/ ml. Results are the mean of two determinations. Fig. 3. Agilent bioanalyzer analysis of total RNA from S. aureus cells treated with retapamulin. RNA analysis has been described previously (18). Virtual gel produced by the Agilent software based on the results from the fluorescent chromatograph. Bracket shows the region of small RNAs. Lane 1: control RNA from wt strain ; Lane 2: RNA from wt strain grown with retapamulin at 10 ng/ ml; Lane 3: RNA from wt strain grown with retapamulin at 20 ng/ ml; Lane 4: control RNA from MRSA strain; Lane 5: RNA 9

10 from MRSA strain grown with retapamulin at 10 ng/ ml; Lane 6: RNA from MRSA strain grown with retapamulin at 20 ng/ ml. Table 1. IC 50 (ng/ ml) values for inhibition of total viable cells (TVC), protein synthesis and 50S subunit synthesis by retapamulin in S. aureus strains IC 50 (ng/ ml) Strain TVC Protein Synthesis 50S Subunit Synthesis RN1786 (wt) A1024 (MRSA) The values were calculated from analyses like those shown in Fig. 1. Table 2. Relative amounts of rrna from Agilent RNA analysis Percentage of total area Retapamulin Strain (ng/ ml) 23S RNA 16S RNA Small RNA Wild type MRSA

11 The values were calculated from duplicate RNA analyses like that shown in Fig

12 % Control protein synthesis % Total gradient cpm A Retapamulin ng/ml C % Control viable cell number % Total gradient cpm Retapamulin ng/ml D B Retapamulin ng/ml Retapamulin ng/ml

13 % Total gradient cpm A Time (min) % Total gradient cpm B Time (min)

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