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Antimicrobial Agents and Chemotherapy, September 2007, p. 3385-3387, Vol. 51, No. 9
0066-4804/07/$08.00+0     doi:10.1128/AAC.00475-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Retapamulin Inhibition of Translation and 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells{triangledown}

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, Tennessee 37614

Received 6 April 2007/ Returned for modification 7 May 2007/ Accepted 30 May 2007


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ABSTRACT
 
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.


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TEXT
 
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. Semisynthetic derivatives of the mutilins which show enhanced antibacterial activity against a variety of pathogens have been made. Retapamulin (SB 275833) is especially effective against gram-positive cocci (13, 15, 19). Inhibition of peptide bond formation is the basis of its inhibitory activity (22). A crystal structure of the antibiotic bound to the 50S subunit was recently derived (11). This antibiotic 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 by Champney [3]). The inhibition of 50S subunit functions in translation by the compound retapamulin stimulated a test of this compound's 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 (wt) Staphylococcus aureus strain RN1786 (14) and methicillin-resistant S. aureus (MRSA) strain A1024 (6). MICs for the two strains were 35 and 55 ng/ml, respectively.

Figure 1A shows the inhibition of protein synthesis in the two organisms. Similar inhibitory effects on cell viability were seen (Fig. 1B), with comparable 50% inhibitory concentrations (IC50 values) (Table 1). Retapamulin inhibition of 50S subunit synthesis was also examined in both strains. As Fig. 1C and D show, a specific inhibitory effect on large subunit formation was seen, with IC50 values of 27 ng/ml and 20 ng/ml for the two strains (Table 1). No inhibition of 30S particle formation was found.


Figure 1
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  		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 examined by a four-part assay 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 ({blacksquare}) strains. (B) Inhibition of viable cell number (total viable cells [TVC]) by retapamulin in wt (•) and MRSA ({blacksquare}) strains. (C) Concentration-dependent inhibition of ribosomal subunit assembly in wt S. aureus cells. Inhibition of 30S assembly ({blacksquare}) and inhibition of 50S assembly (•) are shown. (D) Concentration-dependent inhibition of ribosomal subunit assembly in MRSA cells. Inhibition of 30S assembly ({blacksquare}) and inhibition of 50S assembly (•) are shown. The percentages of the total gradient cpm were calculated for the 30S and 50S regions for the control and antibiotic-treated samples. Results are the means of two determinations. Arrows indicate the IC50 values.


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  		TABLE 1. IC50 values of retapamulin for inhibition of TVC, protein synthesis, and 50S subunit synthesis in S. aureus strainsa

The effect of the antibiotic on ribosome synthesis was also examined by a pulse-chase labeling protocol. Figure 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).


Figure 2
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  		FIG. 2. [3H]uridine pulse-chase labeling kinetic analysis of ribosomal subunit formation was conducted as described previously (2). Cells were labeled with [3H]uridine (1 µCi/ml) for 2 min and then 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 ({blacksquare}) and 50S subunits (•) in MRSA cells without retapamulin. (B) Assembly of 30S subunits ({blacksquare}) and 50S subunits (•) in MRSA cells with retapamulin at 8 ng/ml. Results are the means of two determinations.

Inhibition of 50S synthesis leads to the accumulation of stalled intermediates in the assembly pathway, which are degraded by cellular ribonucleases in Escherichia coli cells (21). Retapamulin was examined for effects on 23S rRNA turnover in both strains. A reduction in 23S rRNA amounts was found by Agilent Bioanalyzer analysis (Fig. 3). About 25% of the 23S rRNA 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).


Figure 3
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  		FIG. 3. Agilent Bioanalyzer analysis of total RNA from S. aureus cells treated with retapamulin. RNA analysis has been described previously (18). A virtual gel was produced by the Agilent software, based on the results from the fluorescence chromatograph. The bracket shows the region of small RNAs. Lane 1, control RNAs from wt strain; lane 2, RNAs from wt strain grown with retapamulin at 10 ng/ml; lane 3, RNAs from wt strain grown with retapamulin at 20 ng/ml; lane 4, control RNAs from MRSA strain; lane 5, RNAs from MRSA strain grown with retapamulin at 10 ng/ml; lane 6, RNAs from MRSA strain grown with retapamulin at 20 ng/ml.


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  		TABLE 2. Relative amounts of rRNA from Agilent RNA analysisa

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 (4), clarithromycin and azithromycin (5), telithromycin (9), linezolid (7), TAN1057A (8), and quinupristin-dalfopristin (17), have the same dual-target specificity.

In the wt organism, retapamulin showed a preferential inhibitory effect on protein synthesis, with about five 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 S. aureus and MRSA suggests its potential as an effective antimicrobial agent against these problematic organisms. Others have also shown equivalent inhibitory effects 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.


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ACKNOWLEDGMENTS
 
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 GlaxoSmithKline.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, J. H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614. Phone: (423) 439-2022. Fax: (423) 439-2030. E-mail: champney{at}etsu.edu Back

{triangledown} Published ahead of print on 11 June 2007. Back


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Antimicrobial Agents and Chemotherapy, September 2007, p. 3385-3387, Vol. 51, No. 9
0066-4804/07/$08.00+0     doi:10.1128/AAC.00475-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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