TR-700 (torezolid), the active moiety of the novel oxazolidinone phosphate prodrug TR-701, is highly potent against gram-positive pathogens, including strains resistant to linezolid (LZD). Here we investigated the potential of Staphylococcus aureus strains ATCC 29213 (methicillin-susceptible S. aureus [MSSA]) and ATCC 33591 (methicillin-resistant S. aureus [MRSA]) to develop resistance to TR-700. The spontaneous frequencies of mutation of MSSA 29213 and MRSA 33591 resulting in reduced susceptibility to TR-700 at 2؋ the MIC were 1.1 ؋ 10 ؊10 and 1.9 ؋ 10 ؊10 , respectively. These values are ϳ16-fold lower than the corresponding LZD spontaneous mutation frequencies of both strains. Following 30 serial passages in the presence of TR-700, the MIC for MSSA 29213 remained constant (0.5 g/ml) while increasing eightfold (0.25 to 2.0 g/ml) for MRSA 33591. Serial passage of MSSA 29213 and MRSA 33591 in LZD resulted in 64-and 32-fold increases in LZD resistance (2 to 128 g/ml and 1 to 32 g/ml, respectively). Domain V 23S rRNA gene mutations (Escherichia coli numbering) found in TR-700-selected mutants included T2500A and a novel coupled T2571C/G2576T mutation, while LZD-selected mutants included G2447T, T2500A, and G2576T. We also identified mutations correlating with decreased susceptibility to TR-700 and LZD in the rplC and rplD genes, encoding the 50S ribosomal proteins L3 and L4, respectively. L3 mutations included Gly152Asp, Gly155Arg, Gly155Arg/ Met169Leu, and ⌬Phe127-His146. The only L4 mutation detected was Lys68Gln. TR-700 maintained a fourfold or greater potency advantage over LZD against all strains with ribosomal mutations. These data bring to light a variety of novel and less-characterized mutations associated with S. aureus resistance to oxazolidinones and demonstrate the low resistance potential of torezolid.Staphylococcus aureus infections pose a serious health threat worldwide. Increasing antibiotic resistance and the prevalence of methicillin (meticillin)-resistant S. aureus (MRSA) in clinical settings have created a demand for novel therapeutic agents. Linezolid (LZD) has a broad spectrum of activity against a variety of gram-positive pathogens, including MRSA, and was the first oxazolidinone antibiotic to gain FDA approval (1). LZD acts through inhibition of protein synthesis via binding to the peptidyl transferase center (PTC) of the 50S ribosomal subunit (37,65,68). Despite in vitro studies demonstrating a low resistance potential for LZD (31, 79), soon after its approval in 2000, LZD-resistant (LZD r ) MRSA (72) and LZD r , vancomycin (VAN)-resistant enterococci (22) emerged in the clinic. Although rare, resistance has most commonly occurred in patients undergoing long-term LZD therapy (10,17,22,45,72,74). Three classes of oxazolidinone resistance mechanisms have been previously characterized: mutations in the domain V region of 23S rRNA genes (69), acquisition of the ribosomal methyltransferase gene cfr (43), and mutations in the rplD gene encoding the 50S ribosomal protein L4 (76).A variety of 23S ...

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Human 11␤-hydroxysteroid dehydrogenase type I (11␤-HSD1) is an ER-localized membrane protein that catalyzes the interconversion of cortisone and cortisol. In adipose tissue, excessive cortisol production through 11␤-HSD1 activity has been implicated in the pathogenesis of type II diabetes and obesity. We report here biophysical, kinetic, mutagenesis, and structural data on two ternary complexes of 11␤-HSD1. The combined results reveal flexible active site interactions relevant to glucocorticoid recognition and demonstrate how four 11␤-HSD1 C termini converge to form an as yet uncharacterized tetramerization motif. A C-terminal Pro-Cys motif is localized at the center of the tetramer and forms reversible enzyme disulfides that alter enzyme activity. Conformational flexibility at the tetramerization interface is coupled to structural changes at the enzyme active site suggesting how the central Pro-Cys motif may regulate enzyme activity. Together, the crystallographic and biophysical data provide a structural framework for understanding 11␤-HSD1 activities and will ultimately facilitate the development of specific inhibitors.

Staphylococcal resistance to linezolid (LZD) is mediated through ribosomal mutations (23S

The ability of bacteria to regulate gene expression in response to changes in cell density is termed quorum sensing. This behavior involves the synthesis and recognition of extracellular, hormonelike compounds known as autoinducers. Here we report the structure of an autoinducer synthase, LuxS from Bacillus subtilis, at 1.6-Å resolution (Rfree ‫؍‬ 0.204; Rwork ‫؍‬ 0.174). LuxS is a homodimeric enzyme with a novel fold that incorporates two identical tetrahedral metal-binding sites. This metal center is composed of a Zn 2؉ atom coordinated by two histidines, a cysteine, and a solvent molecule, and is reminiscent of active sites found in several peptidases and amidases. Although the nature of the autoinducer synthesized by LuxS cannot be deduced from the crystal structure, features of the putative active site suggest that LuxS might catalyze hydrolytic, but not proteolytic, cleavage of a small substrate. Our analysis represents a test of structure-based functional assignment.

Following recent reports of ribosomal protein L3 mutations in laboratory-derived linezolid-resistant (LZD r ) Staphylococcus aureus, we investigated whether similar mutations were present in LZD r staphylococci of clinical origin. Sequence analysis of a variety of LZD r isolates revealed two L3 mutations, ⌬Ser145 (S. aureus NRS127) and Ala157Arg (Staphylococcus epidermidis 1653059), both occurring proximal to the oxazolidinone binding site in the peptidyl transferase center. The oxazolidinone torezolid maintained a >8-fold potency advantage over linezolid for both strains.

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Resistance to linezolid (LZD) occurs through mutations in 23S rRNA and ribosomal proteins L3 and L4 or through methylation of 23S rRNA by Cfr. Here we report novel L3 mutations, ⌬Ser145/His146Tyr and ⌬Met169-Gly174, co-occurring with cfr in LZD-resistant Staphylococcus aureus isolates recovered from a hospital outbreak in Madrid, Spain. LZD MIC values (16, 32, or 64 g/ml) correlated with the presence and severity of the L3 mutation. All isolates had TR-700 (torezolid) MIC values of <2 g/ml.