Frequency, Spectrum, and Nonzero Fitness Costs of Resistance to Myxopyronin in Staphylococcus aureus

Abstract

The antibiotic myxopyronin (Myx) functions by inhibiting bacterial RNA polymerase (RNAP). The binding site on RNAP for Myx-the RNAP "switch region SW1/SW2 subregion"-is different from the binding site on RNAP for the RNAP inhibitor currently used in broad-spectrum antibacterial therapy, rifampin (Rif). Here, we report the frequency, spectrum, and fitness costs of Myx resistance in Staphylococcus aureus. The resistance rate for Myx is 4 ؋ 10 ؊8 to 7 ؋ 10 ؊8 per generation, which is equal within error to the resistance rate for Rif (3 ؋ 10 ؊8 to 10 ؋ 10 ؊8 per generation). Substitutions conferring Myx resistance were obtained in the RNAP ␤ subunit [six substitutions: V1080(1275)I, V1080(1275)L, E1084(1279)K, D1101(1296)E, S1127(1322)L, and S1127 (1322) Mf50 (18,20,23,49). Myx exhibits broad-spectrum antibacterial activity, with potent antibacterial activity against most Gram-positive species and some Gram-negative species. Myx is under investigation as a potential lead compound for broad-spectrum antibacterial therapy. M yxopyronin (Myx) is an ␣-pyrone antibiotic produced by Myxococcus fulvusMyx functions by inhibiting bacterial RNA polymerase (RNAP) (3,18,20,32,49). The binding site on RNAP for Myx is located in the RNAP "switch region" and comprises the RNAP switch region structural elements termed "switch 1" and "switch 2," (switch region SW1/SW2 subregion) (3,18,32,49). The binding site on RNAP for Myx is different from the binding site on RNAP for the RNAP inhibitor in current use in broad-spectrum antibacterial therapy, rifampin (Rif) (3,18,32,49). Accordingly, Myx exhibits no cross-resistance with Rif (18,19,32,33,49).Previous studies have provided information about spontaneous resistance frequencies and resistance spectra for Myx (31, 32). However, the fitness costs of resistance have not previously been assessed.In this work, we comprehensively evaluate the resistance properties of Myx in Staphylococcus aureus. We report (i) the spontaneous resistance rate for Myx in S. aureus, (ii) the spontaneous resistance spectrum of Myx in S. aureus, and (iii) the fitness costs of substitutions that confer Myx resistance. Hu et al. (19). Rif was purchased from Sigma, Inc. Bacterial strains were obtained from the American Type Culture Collection. MATERIALS AND METHODS Materials. (Ϯ)-Myx B was synthesized as described inMICs. Minimal inhibitory concentrations (MICs) in column 2 of Table 1 were quantified using broth microdilution assays (8). MICs in column 3 of Table 1, and in Tables 3 and 4, were quantified using spiral gradient endpoint assays, essentially as described previously (38,45,54). Spiral gradient endpoint assays employed 150-mm by 4-mm exponentialgradient plates containing Mueller-Hinton II cation-adjusted agar and 0.5 to 100 g/ml of Myx, 0.0008 to 0.2 g/ml of Rif, or 0.2 to 40 g/ml of Rif. Plates were prepared using an Autoplate 4000 spiral plater (Spiral Biotech, Inc.). Cells were grown to early log phase, adjusted to 1 ϫ 10 8 CFU/ml, and swabbed radially onto plates. Plates were incubated for 16 h at 3...

Rifamycin congeners kanglemycins are active against rifampicin-resistant bacteria via a distinct mechanism

“…Substitutions at just three RNAP amino acid positions, Mtb RNAP β subunit D441, H451, and S456 (corresponding to Msm / E. coli [ Eco ] RNAP β subunit D432/D516, H442/H526, and S447/S531) account for the vast majority of mutations observed in Rif R Mtb clinical isolates 3 , 29 . The antibacterial activity of the Kangs against Rif R RNAP mutants was assessed in vivo using a collection of Sau strains carrying RNAP point mutations and in vitro using purified wild-type and Rif R (S447L) Msm RNAPs 30 , 31 . The use of these models allowed us to explore the activity of the Kangs against mutations that correspond to the most commonly mutated sites in Rif R Mtb , without necessitating the use of restrictive BSL3 assay conditions.…”

Antibacterial Nucleoside-Analog Inhibitor of Bacterial RNA Polymerase

“…2A; procedures as in Srivastava et al, 2012). S. pyogenes strain ATCC 12344 (~1 × 10 9 cfu/plate) was plated on Todd Hewitt agar [Todd Hewitt broth (BD Biosciences) supplemented with 1.5% Bacto agar (BD Biosciences)] containing 64 μg/ml or 128 μg/ml PUM (8x or 16x MIC under these conditions) or 1 μg/ml or 2 μg/ml Rif (8x or 16x MIC under these conditions), and numbers of colonies were counted after 24 h at 37°C (at least six independent determinations each).…”

“…Bacterial RNAP also is the target of a class of antibacterial agents currently in preclinical development: myxopyronins, which function by binding to a site distant from the RNAP active-center and allosterically inhibiting opening of, and loading of DNA into, the RNAP active-ceneter cleft (Mukhopadhyay et al, 2008; Srivastava et al, 2011). Rifamycins, lipiarmycins, and myxopyronins are subject to spontaneous resistance emergence (Mariani and Maffioli, 2009; Ho et al, 2009; Aristoff et al, 2010; Srivastava et al 2011, 2012). Resistance to rifamycins, lipiarmycins, and myxopyronins arises from mutations that result in substitution of the respective binding sites on RNAP for the compounds, preventing binding of the compounds.…”

Structural Basis of Mycobacterium tuberculosis Transcription and Transcription Inhibition

“…Resistance rates were determined using fluctuation assays essentially as in Srivastava et al, 2012. Defined numbers of cells of M. smegmatis ATCC 19420 (~10 9 cfu/plate) were plated on Seven H11 agar containing Middlebrook ADC enrichment, 0.5% glycerol, and either 1.56 μg/ml D-AAP1, 50 μg/ml Rif, or both 1.56 μg/ml D-AAP1 and 50 μg/ml Rif; and numbers of colonies were counted after 72 h at 37°C (at least 4 determinations for each concentration of each test compound).…”