Activation of heme biosynthesis by a small molecule that is toxic to fermenting Staphylococcus aureus

Abstract

Staphylococcus aureus is a significant infectious threat to global public health. Acquisition or synthesis of heme is required for S. aureus to capture energy through respiration, but an excess of this critical cofactor is toxic to bacteria. S. aureus employs the heme sensor system (HssRS) to overcome heme toxicity; however, the mechanism of heme sensing is not defined. Here, we describe the identification of a small molecule activator of HssRS that induces endogenous heme biosynthesis by perturbing central metabolism. This molecule is toxic to fermenting S. aureus, including clinically relevant small colony variants. The utility of targeting fermenting bacteria is exemplified by the fact that this compound prevents the emergence of antibiotic resistance, enhances phagocyte killing, and reduces S. aureus pathogenesis. Not only is this small molecule a powerful tool for studying bacterial heme biosynthesis and central metabolism; it also establishes targeting of fermentation as a viable antibacterial strategy.

Keywords: glycolysis; heme oxygenase; high-throughput screen (HTS); two-component system (TCS).

Fig. 1.

A high-throughput screen identifies small molecule activators of HssRS. (A) A secondary screen consisting of an XylE reporter assay verified the activity of the top 110 hits from the primary screen, including ‘882. Triplicate cultures of S. aureus WT and ∆hssR transformed with the hrtAB promoter-xylE fusion-containing plasmid (phrt.xylE) were grown in the presence of the indicated additive, and XylE activity was measured. (B) The structure of lead compound VU0038882 (‘882). (C) ‘882 was confirmed as the top hit in a tertiary screen that measured the ability of the compound to preadapt S. aureus for growth in 20 μM heme. Triplicate cultures of WT S. aureus were grown overnight in medium containing the indicated additive and subcultured into medium containing 20 μM heme. Growth was monitored by measuring the optical density at 600 nm (OD₆₀₀) over time. (A and C) Error bars represent one SD from the mean.

Fig. 2.

‘882 acts through endogenous heme biosynthesis to activate HssRS and stimulate heme production. (A) S. aureus wild type (WT, black lines) and the heme auxotroph hemB::ermC (∆hemB, gray lines) were transformed with plasmids constitutively expressing XylE (plgt.xylE, dashed lines) or with xylE under the control of the hrtAB promoter (phrt.xylE, solid lines). Triplicate cultures of these strains were grown in the presence of ‘882, and XylE activity was measured. (B) Heme levels in triplicate cultures of S. aureus treated with the indicated additive were quantified using the pyridine hemochromogen assay and normalized to the concentration of protein in the whole cell lysates. (Inset) Pellets from a culture grown with 40 µM ‘882 are darker compared with vehicle-treated S. aureus. (A and B) Error bars represent 1 SD from the mean. (C) Exact-mass mass spectrometric analysis was used in conjunction with ultra-high performance liquid chromatography (UPLC) to detect and quantify heme in protoplasts from cells treated with 40 µM ‘882 or vehicle grown in triplicate. Measured heme molecules were referenced to estimated cfus per pellet, factoring in dilutions. Dead or lysed cells would also contribute to the measurement. Therefore the measured numbers are considered upper estimates. Samples measured in duplicate or triplicate injections had typical errors of <5% between analytical replicates. Error bars represent the SD, and significance was calculated using a two-tailed Student’s t test. (D) Adaptation by heme and ‘882 in C. diphtheriae was tested by growth analyses. Triplicate cultures were grown overnight in medium containing vehicle, 5 µM heme or 50 µM ‘882 and subcultured into medium containing 15 μM heme. The cfus were enumerated 2.5 h after inoculation and normalized to cultures unexposed to heme. Shown is the average of five replicates; error bars represent SEM and significance was determined by a two-tailed Student’s t test. (E) Adaptation by heme and ‘882 in S. haemolyticus was tested by growth analyses. Triplicate cultures were grown overnight in medium containing the indicated additive and subcultured into medium containing 30 μM heme. Growth was monitored by measuring the optical density at 600 nm (OD₆₀₀) over time. Error bars represent 1 SD from the mean.

Fig. 3.

‘882 diminishes fermentative activity. S. aureus was grown in triplicate under aerobic conditions in the presence of vehicle (black lines) or 40 µM ‘882 (gray lines). At the indicated time intervals, culture supernatants were sampled and the (A) pH, (B) d-glucose, and (C) d- and l-lactate were quantified. Error bars represent 1 SD from the mean.

Fig. 4.

Glycolytic activity regulates heme biosynthesis. S. aureus was grown in triplicate in the presence of vehicle or 10 µM ‘882. Heme levels were quantified using the pyridine hemochromogen assay and normalized to the concentration of protein in the whole cell lysates. (A) Cultures were treated with the indicated dose of 2-deoxyglucose (2dG). *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001. (B) Wild-type (WT) and ΔpfkA Newman were cultured in TSB + 1% pyruvate. #, the signal was below the limit of detection. (A and B) Error bars represent the SEM from three independent experiments. Statistical significance was determined using an unpaired Student’s t test.

Fig. 5.

‘882 inhibits fermenting S. aureus. (A) S. aureus wild type (WT, dashed lines), the menaquinone auxotroph (∆menB, dotted lines), and the heme auxotroph hemB::ermC (∆hemB, solid lines) were grown in triplicate under aerobic (aer, black lines) and anaerobic (ana, gray lines) conditions in the presence of the indicated log of the concentration of ‘882 (µM). After 18 h, the absorbance at 600 nm (OD₆₀₀) was measured and normalized to vehicle (DMSO)-treated bacteria. Curves were fit by nonlinear regression analysis and absolute logIC50 values were calculated in Prism with the top set at 1.0 and the bottom set at 0.15. LogIC50 values are indicated in parentheses in the figure key ±SEM. All logIC50 values were statistically different from WT aerobic logIC50 when analyzed by one-way ANOVA with a Dunnett post test (P < 0.001). Error bars represent the SEM. (B) Triplicate cultures of S. aureus were grown in the presence of the indicated additive. After 24 h, cfus were enumerated on tryptic soy agar (TSA) containing 5 μg/mL gentamicin (Gent) and plain TSA with a limit of detection of 100 cfus/mL (minimum y value); #, colonies were not identified above the limit of detection. Shown is the average of three independent experiments. Error bars represent 1 SD from the mean. (C) S. aureus was grown in the presence of vehicle (DMSO) or 40 µM ‘882 and coated in serum. Murine polymorphonuclear leukocytes (PMNs) were elicited with casein and harvested from the peritoneum. The ability of neutrophils to kill S. aureus in the presence of ‘882 was assessed by comparing cfus recovered from neutrophil-exposed S. aureus with those recovered from identical conditions lacking neutrophils. The mean of at least six independent experiments performed in triplicate are represented by the data; error bars represent SEM and significance was determined by a two-tailed Student’s t test.

Fig. 6.

‘373 reduces S. aureus pathogenesis in vivo. Mice infected retroorbitally with S. aureus were treated intraperitoneally with vehicle [10% (vol/vol) Tween 80] or ‘373. After 96 h, mice were euthanized and (A) cfus and (B) surface abscesses were enumerated from the livers. Each marker represents an individual mouse. Data were collected from three independent experiments resulting in n = 13 for vehicle and n = 12 for ‘373-treated mice once the highest and lowest values were removed from each group. The horizontal line indicates the mean and the error bars represent the SEM. Statistical significance was determined by a two-tailed Student’s t test. Livers from mice treated with the indicated dose of ‘373 were harvested 24 h postinfection (1 h after the second treatment), sectioned, and mounted on MALDI target plates (C). (D) Tissue sections were imaged by MALDI-MS/MS for accumulation of a fragment of ‘373 by MALDI-MS/MS (m/z 255.2→134). Spectra were acquired at 10 microscans per step. Five laser shots were acquired per pixel, and pixels were obtained every 100 µm.