Novel riboswitch ligand analogs as selective inhibitors of guanine-related metabolic pathways

Abstract

Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5'-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound (PC1) binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as community-acquired bacterial infections have recently started to emerge.

Figure 1. The structure of the guanine riboswitch.

(A) Scheme representing the guanine riboswitch secondary structures in absence (ON state) or in presence (OFF state) of guanine. The formation of a guanine-riboswitch complex results in the adoption of an intrinsic terminator element that prematurely stops transcription. (B) Consensus sequence and secondary structure of the guanine riboswitch aptamer. Nucleotides indicated in blue, orange and green represent nucleotides that are conserved >90% and those colored in black are conserved >80% . Nucleotides and lines in blue and in green indicate interactions with the ligand via hydrogen bonding and base stacking, respectively. The cytosine 74 which confers ligand binding specificity is shown in orange and the bound guanine is shown as a red rounded rectangle.

Figure 2. Guanine riboswitch agonists can be used to modulate gene expression.

(A) Hydrogen bonds (left panel) and stacking interactions (right panel) formed between the bound guanine and the guanine riboswitch , . Oxygen, nitrogen, and phosphorus atoms are in red, blue, and yellow, respectively (left panel). Nucleotides follow the color scheme used in Figure 1B. Figures were prepared using PyMol (DeLano Scientific, San Francisco, CA, USA). (B) Molecular recognition features for guanine (G) and predicted ones for PC1 and PC2. Blue and red arrows represent hydrogen bond acceptors and donors, respectively. (C) In-line probing assays of the B. subtilis xpt riboswitch in the absence (−) or in the presence (+) of 1 µM guanine (G), and 1 µM or 10 µM for both PC1 and PC2. Sites of substantial ligand-induced protections (positions 49–54) are assigned on the right by a vertical bracket. Lanes NR, L and T1 correspond to molecules that were not reacted or that were partially digested by alkali or by RNase T1, respectively. Guanines are identified on the left as molecular weight markers. (D) The beta-galactosidase activity of a xpt-lacZ transcriptional fusion construct integrated in the genome of B. subtilis by recombination was assayed after 4 h of growth at 37°C in minimal medium in presence of the indicated ligand concentrations. Each experiment was performed three times and the average as well as standard deviations are shown.

Figure 3. S. aureus growth inhibition requires guaA to be under a riboswitch control.

(A) Antibiograms performed on the S. aureus ATCC 29213 strain using 75 µg of PC1 (1), PC2 (2), 2-amino-4-hydroxy-6-methylpyrimidine (3), 9-methyl guanine (4), 5-bromo-6-methyl pyrimidine (5) and PBS as control (6). Chemical structures of PC1 and PC2 are shown in Figure 2B. (B) PC1 antibiograms using bacterial strains with guanine riboswitches. While PC1-insensitive strains do not have guaA under a riboswitch control (Bs: Bacillus subtilis, Ef: Enterococcus feacium, Lm: Listeria monocytogenes, STRpy: Streptococcus pyogenes, STRd: Streptococcus dysgalactiae and STRu: Streptococcus uberis), PC1-sensitive strains control the guaA gene expression via a riboswitch mechanism (Bh: Bacillus halodurans, STAa: S. aureus ATCC 29213, STAh: S. haemolyticus, SA228a: S. aureus resistant to beta-lactam, erythromycin, ciprofloxacin, gentamicin and tetracycline but susceptible to vancomycin, MRSA COL: methicilin resistant S. aureus COL, STAe: S. epidermidis, Cb: Clostridium botulinum, CD630: C. difficile strain 630, CD6: C. difficile representing the hypervirulent NAP1/027 strain). (C) Influence of GMP on PC1 bacterial growth inhibition. Spots from serial dilutions of S. aureus cultures in cation-adjusted Muller-Hinton broth (CAMHB) in absence or presence of 600 µg/mL PC1 or 600 µg/mL PC1 supplemented with 100 µM GMP.

Figure 4. PC1 shows bactericidal activity through cellular GMP depletion.

(A) Minimal inhibitory concentrations (MIC) determination of PC1 and PC2 on S. aureus strain ATCC 29213 in CAMHB. MIC values of 600 µg/mL and 5000 µg/mL were obtained for PC1 and PC2, respectively. (B) Bactericidal activity of PC1 and other known antibiotics against S. aureus as a function of time. For determination of the bactericidal effect of PC1, bacteria were inoculated at 10⁵ CFU/mL in absence (cont) or presence of 0.5 µg/mL ciprofloxacin (cipro), 0.5 µg/mL erythromycin (erythro), 1 µg/mL vancomycin (vanco) and 600 µg/mL PC1. The concentration of each antibiotic corresponds to their MIC. (C) Bactericidal activity of PC1 against S. aureus as a function of time in absence (cont) or presence of 600 µg/mL PC1, and in presence of PC1 with 100 µM GMP or AMP. (D) Relative expression of S. aureus genes under the control of a guanine riboswitch when grown in presence of PC1 or PC1 with GMP. Results obtained in presence of PC1 are normalized using xpt gene expression. Bacteria were inoculated at 10⁸ CFU/mL in CAMHB in absence or presence of 600 µg/mL PC1 with or without 100 µM GMP. Each experiment was performed three times and the average as well as standard deviations are shown.

Figure 5. PC1 inhibits S. aureus clinical isolates in vitro and in vivo.

(A) Fold reduction in viable counts (log10 CFU/mL) for the reference S. aureus strain ATCC 29213 and for selected bovine isolates after a 4 h exposure to PC1 as compared to the untreated culture. Newbould 305 (ATCC 29740) and SHY97-3906 are bovine isolates from typical mastitis cases and isolates 3, 557 and 1290 were from cows with persisting intra-mammary infections and chronic mastitis. (B) Bacterial counts (CFU) obtained from mice mammary glands 10 h post-infection with S. aureus. Mice mammary glands were treated (intra-mammary administration) 4 h after infection with PBS with or without PC1 at 10, 50 or 100 µg/gland. Each dot represents the CFU of each individual gland (n = 6–12) and the median value for each group is indicated by the bar. Statistical differences (P<0.05) between CFU recovered from treated and untreated animals are shown by asterisks (non-parametric Kruskal-Wallis ANOVA with Dunn's post test).

Figure 6. Scheme representing the action mechanism of PC1 on S. aureus guanine pathway.

Genes highlighted in blue correspond to those of the operon xpt-pbuX-guaB-guaA that is controlled by the guanine riboswitch. Red lines indicate genes that are inhibited by PC1 via its binding to the guanine riboswitch. Of these four inhibited genes, guaA and guaB are known to be critical for guanine nucleotide biosynthesis , , , , , and their inhibition very likely lead to the repression of GMP synthesis and most probably of RNA and DNA production.