Inhibition of WTA synthesis blocks the cooperative action of PBPs and sensitizes MRSA to β-lactams

Abstract

Rising drug resistance is limiting treatment options for infections by methicillin-resistant Staphylococcus aureus (MRSA). Herein we provide new evidence that wall teichoic acid (WTA) biogenesis is a remarkable antibacterial target with the capacity to destabilize the cooperative action of penicillin-binding proteins (PBPs) that underlie β-lactam resistance in MRSA. Deletion of gene tarO, encoding the first step of WTA synthesis, resulted in the restoration of sensitivity of MRSA to a unique profile of β-lactam antibiotics with a known selectivity for penicillin binding protein 2 (PBP2). Of these, cefuroxime was used as a probe to screen for previously approved drugs with a cryptic capacity to potentiate its activity against MRSA. Ticlopidine, the antiplatelet drug Ticlid, strongly potentiated cefuroxime, and this synergy was abolished in strains lacking tarO. The combination was also effective in a Galleria mellonella model of infection. Using both genetic and biochemical strategies, we determined the molecular target of ticlopidine as the N-acetylglucosamine-1-phosphate transferase encoded in gene tarO and provide evidence that WTA biogenesis represents an Achilles heel supporting the cooperative function of PBP2 and PBP4 in creating highly cross-linked muropeptides in the peptidoglycan of S. aureus. This approach represents a new paradigm to tackle MRSA infection.

Figure 1

CA- and HA-MRSA ΔtarO deletion strains impaired for WTA synthesis are sensitized to β-lactam antibiotics. Sensitivity profiles of diverse antibiotics in CA-MRSA USA300 (black bars) and HA-MRSA EMRSA15 (white bars) relative to their ΔtarO deletion strains. Fold change refers to the MIC of the antibiotic in the parent strain divided by MIC in the deletion strain. The highest sensitivity was exclusively observed with certain β-lactam antibiotics.

Figure 2

Ticlopidine potentiates the activity of the β-lactam antibiotic cefuroxime against CA-MRSA USA300, but not CA-MRSA USA300 ΔtarO. (a) Chemical structure of ticlopidine. (b) Microdilution checkerboard analysis showing the combined effect of cefuroxime and ticlopidine against CA-MRSA USA300 where the extent of inhibition is shown as a heat plot. Synergistic effects are evident as both molecules alone have MICs that exceed 256 μg/mL and result in an FIC index of ≤0.063. (c) Suppression of the synergy in CAMRSA USA300 ΔtarO, leading to an additive interaction with FIC index of ≤2. (d) Galleria mellonella virulence assay. Survival curve of G. mellonella infected with CA-MRSA USA300 receiving no drug treatment (control, CTRL) or a treatment with 0.3 mg/kg cefuroxime (CEF) or 0.3 mg/kg ticlopidine (TIC) or a combination of both at 0.3 mg/kg each (CEF+TIC). After 14 days, treatment with the combination lead to significantly increased survival, compared to no drug or antibiotic treatment alone (P < 0.001).

Figure 3

Ticlopidine inhibits the initiation of wall teichoic acid biosynthesis in S. aureus. (a) At concentrations of 8 μg/mL, ticlopidine begins to antagonize the activity of targosil, a late-stage inhibitor, against CA-MRSA USA300. (b) Ticlopidine can suppress the lethality associated with late WTA stage deletion. Shown is the percent growth of a tarH conditional deletion strain normalized to the growth upon induction with 2% xylose in the presence of increasing concentrations of ticlopidine. Ticlopidine can recover approximately 40% of the growth at the highest concentration of 256 μg/mL. (c) Ticlopidine shows a dose-dependent decrease in the phosphate content of cell wall isolated from CA-MRSA USA300, with approximately 50% less phosphate when ticlopidine is present at 200 μg/mL. CA-MRSA USA300 tarO is completely devoid of WTA compared to the parental strain. (d) Membrane-based in vitro assay following the generation of a radiolabeled reaction product of TarO, undecapreny-P-P-[14C]GlcNAc, as a result of the incorporation of [¹⁴C]GlcNAc onto undecaprenyl-P-P. Assay was performed on membranes derived from E. coli cells (wecA) expressing recombinant TarO from S. aureus. The reaction product was monitored by thin layer chromatography (TLC) and shown to be dependent on the presence of recombinant TarO (Supplementary Figure 8). Ticlopidine inhibited the activity of TarO, yielding an IC₅₀ value of 238 ± 14 μM.

Figure 4

A synthetic lethal interaction when targeting TarO and native PBP2. Indirect Inhibition of PBP4, via inhibition of TarO, and inhibition of PBP2 function account for the synergistic interaction among ticlopidine and cefuroxime. (i) When not challenged with β-lactams, WTA synthesis will guide the proper localization of PBP4 and, together with PBP2, will provide highly cross-linked muropeptide species (thick arrow) that contribute to high-level β-lactam resistance. (ii) Treatment with a β-lactam with low affinity for PBP2 and ticlopidine leads to an additive interaction as sufficient highly cross-linked peptidoglycan are present to maintain β-lactam resistance (one thick arrow), even when PBP4 function is affected by the lack of WTA (one thin arrow). (iii) Due to their cooperative function, PBP4 (when challenged with ticlopidine) and PBP2 (when challenged with β-lactam with high affinity for PBP2), will be impaired in their capacities to produce a highly cross-linked cell wall (two thin arrows), contributing to enhanced β-lactam susceptibility and thus a synergistic interaction.