Pharmacological and genetic activation of cAMP synthesis disrupts cholesterol utilization in Mycobacterium tuberculosis

Abstract

There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3', 5'-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required during cholesterol metabolism. Finally, the pharmacokinetic properties of Rv1625c agonists have been optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.

Fig 1. V-59 inhibits Mtb growth in an Rv1625c-dependent mechanism.

(A and B) Impact of V-59 on Mtb growth in cholesterol media (A) and in media containing cholesterol and acetate (B). V-59 (10 μM) was added to the cultures every three days, and DMSO is the vehicle control. Data are from two experiments, with three technical replicates each (*P < 0.05, One-way ANOVA with Sidak’s multiple comparisons test). (C) Effect of V-59 on growth of Mtb in murine macrophages. Macrophages were infected at an MOI of 2 and treated with V-59 (25 μM) or DMSO. Data are from three experiments with two or more technical replicates each (****P < 0.0001, One-way ANOVA with Sidak’s multiple comparisons test on fold-change in CFU’s normalized to DMSO). All data are means ± SD.

Fig 2. Structures and activities of compounds.

MW, molecular weight; --, not determined; EC₅₀, half-maximal effective concentration; CC₅₀, 50% cytotoxic concentration; hERG, human ether-à-go-go-related gene; IC₅₀, half-maximal inhibitory concentration; ADME, absorption, distribution, metabolism, excretion; PPB, plasma protein binding; Cyp, cytochrome P450; ER, extraction ratio; PO, per oral.

Fig 3. V-59 stimulates Rv1625c to produce cAMP.

(A) Impact of V-59 on cAMP production in Mtb. Cultures were treated with V-59 or DMSO for 24 hours. Data are from two experiments with two technical replicates each (***P < 0.001, ****P < 0.0001, One-way ANOVA with Sidak’s multiple comparisons test). (B) Impact of Rv1625c agonists on cAMP production in cya^- E. coli transformed with an empty vector control or an Rv1625c expression plasmid. Supernatants were collected 18 hours after addition of V-59, mCLB073, or DMSO. Data is from one experiment, with three independent expression clones, and two technical replicates each. In (A) and (B) Data are normalized as total cAMP per 10⁸ bacteria. DMSO is the vehicle control. Data are shown as means ± SD. (C) Summary of mutations in the rv1625c gene that confer resistance to Rv1625c agonists. Mutations are grouped by their effect on the rv1625c sequence, with missense mutations (blue) and insertion or frameshift mutations (red) and mapped on the Rv1625c topology diagram to illustrate their approximate location relative to Rv1625c protein domains. Black circles represent amino acids that are essential for AC activity.

Fig 4. The transmembrane domain of Rv1625c is essential for complete degradation of cholesterol and the catalytic domain of Rv1625c is required for V-59 activity.

(A) Catabolic release of ¹⁴CO₂ from [4-¹⁴C]-cholesterol in WT, ΔRv1625c, Comp_Full, and Comp_D204 strains treated with V-59 (10 μM) or DMSO vehicle control. Data are from two experiments with three technical replicates, normalized to OD and quantified relative to WT treated with DMSO. Shown as means ± SD (**P < 0.01, ***P < 0.001, Two-way ANOVA with Tukey’s multiple comparisons test). (B) TLC comparing [4-¹⁴C]-cholesterol-derived metabolites extracted from supernatants of Mtb. Image is representative of two experiments. Equivalent counts were spotted per lane. “Chol.” = [4-¹⁴C]-cholesterol.

Fig 5. Inducing cAMP synthesis independent of V-59 and Rv1625c is sufficient to block cholesterol utilization.

(A) Total cAMP induced in TetOn-cAMP Mtb. Cultures were treated with V-59 (10 μΜ), Atc (500 ng/mL or 50 ng/mL), or EtOH and samples were collected after 24 hours. Data are normalized as total cAMP per 10⁸ Mtb and are from two experiments with two technical replicates each. (B) Impact of inducing TetOn-cAMP on the growth of Mtb in cholesterol media. Cultures were treated with V-59 (10 μM) or Atc for the duration of the experiment. Data are from two experiments with three technical replicates. (C) Catabolic release of ¹⁴CO₂ from [4-¹⁴C]-cholesterol in the TetOn-cAMP strain treated with V-59, Atc, or EtOH. Data are from two experiments with three technical replicates, normalized to OD and quantified relative to EtOH EtOH is the vehicle control throughout. All data are means ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, One-way ANOVA with Dunnet’s multiple comparisons test).

Fig 6. V-59 treatment and induction of TetOn-cAMP are associated with shared transcriptional changes to cholesterol utilization genes.

RNA-seq analysis quantifying differentially expressed genes from Mtb grown in cholesterol media, following V-59 treatment, or induction of TetOn-cAMP with Atc. Genes depicted are in the KstR regulons and involved in cholesterol utilization. MCC = methylcitrate cycle. Data are displayed as log₂ fold change in gene expression in response to cAMP-inducing vs. control treatment (“cAMP” = Tet-On cAMP Atc vs. EtOH, “WT” = WT V-59 vs. DMSO, “∆” = ΔRv1625 V-59 vs. DMSO, “C” = Comp_Full V-59 vs. DMSO). Also shown are differentially expressed genes intrinsic to ∆Rv1625 (“Δ/WT” = ∆Rv1625 DMSO vs. WT DMSO). Data are from two technical replicate samples from one experiment (*adjusted P-value ≤ 0.05).

Fig 7. Activating cAMP synthesis decreases liberation of propionyl-CoA from cholesterol.

(A) Relative GFP signal from the prpD’::GFP reporter in response to V-59 (10 μM) or DMSO treatment in murine macrophages or cholesterol media. Data are normalized to WT treated with DMSO (**P < 0.01, ***P < 0.001, Two-way ANOVA with Tukey’s multiple comparisons test). (B) Relative GFP signal from the prpD’::GFP reporter in response to inducing TetOn-cAMP with Atc treatment in murine macrophages or cholesterol media. Data are normalized to EtOH vehicle control (**P < 0.01, ***P < 0.001, One-way ANOVA with Dunnett’s multiple comparisons test). GFP MFI was quantified from 10,000 mCherry⁺ Mtb. Data are from two experiments with two technical replicates, shown as means ± SD.

Fig 8. V-59 treatment and induction of TetOn-cAMP are associated with transcriptional changes in select CRP_Mt regulon genes.

(A) Volcano plots displaying differentially expressed genes following V-59 treatment of WT Mtb relative to DMSO control (left), or following Atc treatment of TetOn-cAMP Mtb relative to EtOH control (right), based on RNA-seq. Each dot represents a single gene, genes in blue are significant (FDR < 0.05) in both data sets and genes in red are unique to their respective data set. Dashed line indicates FDR cutoff < 0.01. (B) Venn diagram showing the number of significantly differentially expressed genes shared by the V-59 and TetOn-cAMP conditions, and how many of these belong to the KstR cholesterol-related regulon. (C) RNA-seq analysis quantifying differentially expressed genes from Mtb grown in cholesterol media, following V-59 treatment, or induction of TetOn-cAMP with Atc. Genes depicted are predicted members of the CRP_MT regulon. Only genes with significant differential expression in both the V-59 and TetOn-cAMP conditions, or genes with intrinsic changes in the ∆Rv1625 strain (in italics), are shown. “cAMP” = Tet-On cAMP Atc vs. EtOH, “WT” = WT V-59 vs. DMSO, “∆” = ΔRv1625 V-59 vs. DMSO, “C” = Comp_Full V-59 vs. DMSO, “Δ/WT” = ∆Rv1625 DMSO vs. WT DMSO.

Fig 9. Chemically activating Rv1625c reduces Mtb pathogenesis in vivo.

Effect of V-59 treatment on bacterial burden and pathology in the lungs of BALB/c (A and B) or C3HeB/FeJ mice (C and D). In (A-D) mice were infected and treated with V-59, INH, or vehicle control. Data are from two independent experiments with 5 mice (A and B), or one experiment with 10 mice (C and D) per group. Outliers with CFUs below the infectious dose were excluded from the analyses (*P < 0.05, Mann-Whitney test). (E and F) Impact of mCLB073 treatment on bacterial burden (E) and pathology (F) in BALB/c mice infected and treated with the indicated doses of mCLB073, INH, or vehicle control (*P < 0.05, **P < 0.01, Kruskal-Wallis test and Dunn’s multiple comparisons test). Infections in (A-F) were by the intranasal route. Data are from one experiment with 10 mice per group. (G) Impact of mCLB073 treatment on bacterial burden in BALB/c mice infected by aerosol and treated with 5mg/kg mCLB073 or vehicle control. Data are from one experiment with 5 mice per group (*P < 0.05, Kruskal-Wallis test and Dunn’s multiple comparisons test). All data are shown as means ± SEM.