Tanshinones: First-in-Class Inhibitors of the Biogenesis of the Type 3 Secretion System Needle of Pseudomonas aeruginosa for Antibiotic Therapy

Abstract

The type 3 secretion system (T3SS) found as cell-surface appendages of many pathogenic Gram-negative bacteria, although nonessential for bacterial survival, is an important therapeutic target for drug discovery and development aimed at inhibiting bacterial virulence without inducing antibiotic resistance. We designed a fluorescence-polarization-based assay for high-throughput screening as a mechanistically well-defined general strategy for antibiotic discovery targeting the T3SS and made a serendipitous discovery of a subset of tanshinones-natural herbal compounds in traditional Chinese medicine widely used for the treatment of cardiovascular and cerebrovascular diseases-as effective inhibitors of the biogenesis of the T3SS needle of multi-drug-resistant Pseudomonas aeruginosa. By inhibiting the T3SS needle assembly and, thus, cytotoxicity and pathogenicity, selected tanshinones reduced the secretion of bacterial virulence factors toxic to macrophages in vitro, and rescued experimental animals challenged with lethal doses of Pseudomonas aeruginosa in a murine model of acute pneumonia. As first-in-class inhibitors with a demonstrable safety profile in humans, tanshinones may be used directly to alleviate Pseudomonas-aeruginosa-associated pulmonary infections without inducing antibiotic resistance. Since the T3SS is highly conserved among Gram-negative bacteria, this antivirulence strategy may be applicable to the discovery and development of novel classes of antibiotics refractory to existing resistance mechanisms for the treatment of many bacterial infections.

Figure 1

Biogenesis of the Pseudomonas aeruginosa T3SS needle. (A) Schematic representation of the T3SS of Pseudomonas aeruginosa, adapted from Abrusci et al. 2014. (B) Crystal structure of the heterotrimeric complex of PscE–PscF–PscG determined by Quinard et al. 2007. Shown in red are residues 54–85 of PscF, which makes direct interactions with PscG (but not PscE). The major α-helix at the C-terminus of PscF energetically dictates PscF binding to the stable heterodimeric complex of PscE–PscG.

Figure 2

Characterization of synthetic peptides/proteins by HPLC, ESI-MS, and CD spectroscopy. (A) Chemically synthesized PscF^54–85, PscE, and PscG characterized by RP-HPLC and ESI-MS. RP-HPLC analyses were performed at 40 °C on a Waters XBridge C18 column (4.6 × 150 mm, 3.5 μm) running a 30 min, 5–65% linear gradient of acetonitrile in water containing 0.1% TFA at a flow rate of 1 mL/min. The molecular masses were ascertained by ESI-MS, in agreement with the calculated values. (B) Circular dichroism spectra obtained at 25 °C of synthetic PscE, PscF, PscG, PscE–PscG heterodimer, and PscE–PscF–PscG heterotrimer at 20 μM each in 10 mM phosphate buffer, pH 7.4.

Figure 3

Identification of tanshinone derivatives as inhibitors of the biogenesis of the Pseudomonas aeruginosa T3SS needle. (A) Strategy for the design of a fluorescence polarization assay for high-throughput screening (HTS). The difference in fluorescence polarization between PscE–^FAMPscF–PscG (high) and ^FAMPscF (low) forms the basis of a physical readout for HTS. (B) Representative quantification by isothermal titration calorimetry of the interaction of PscF^69–85 with a preformed PscE–PscG heterodimer at 25 °C in 10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.0. (C) Representative quantification by fluorescence polarization of the interaction of ^FAMPscF^69–85 with a preformed PscE–PscG heterodimer at room temperature in 10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.0. (D) Representative competition of PscF^69–85 (black), tanshinone 1 (TSN1) (red), dihydrotanshinone 1 (dHTSN1) (green), and dihydrotanshinone (dHTSN) (blue) with ^FAMPscF^69–85 for binding to PscE–PscG heterodimer as quantified by fluorescence polarization at room temperature in 10 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% DMSO, pH 7.0. (E) Chemical structures of TSN1, dHTSN1, and dHTSN.

Figure 4

Structural characterization of tanshinone derivatives interacting with PscE–PscG by NMR spectroscopy and molecular modeling. (A) ¹⁵N–¹H HSQC spectra of ¹⁵N-labeled PscG of the PscE–PscG heterodimer in the presence (red) and absence (black) of dHTSN1. Circled are the resonance peaks broadening or shifting upon binding to dHTSN1. Inset: amide resonance peaks of tryptophan side-chains in ¹⁵N-labeled PscG. (B) Crystal structure of the PscE–PscF–PscG heterotrimer displaying four Trp residues of PscG (green), three of which, W67, W73, and W79, are located in the same α-helix involved in direct interactions with PscF (red). (C) TSN1, dHTSN1, and dHTSN docked in the PscF-binding pocket of PscG. Molecular modeling identifies W79 as the most probable Trp residue involved in direct interactions with tanshinones.

Figure 5

Functional characterization of tanshinone derivatives as inhibitors of the biogenesis of the Pseudomonas aeruginosa T3SS needle in vitro. (A) Effects of tanshinone 1 (TSN1), dihydrotanshinone 1 (dHTSN1), dihydrotanshinone (dHTSN), and cryptotanshinone (crpTSN) at 100 μM on cell viability of Pseudomonas aeruginosa strain PAO1 and murine macrophage cell line J774A.1. The data are averages of three independent experiments. (B) Effects of tanshinone compounds at 100 μM on the secretion of ExoS by PAO1 grown under low-calcium conditions where the T3SS is transcriptionally activated. The data are averages of three independent experiments. Note that tanshinones were initially dissolved in DMSO and diluted into culture medium for in vitro assays, where 2% DMSO in culture medium was used as the negative control.

Figure 6

Functional characterization of tanshinone derivatives as inhibitors of the biogenesis of the Pseudomonas aeruginosa T3SS needle in vitro. (A) Inhibition of the cytotoxicity of PAO1 to murine macrophages by different concentrations of tanshinone compounds as measured by the LDH release assay. The data are averages of three independent experiments. (B) Western blot analysis of caspase-1 activation in PAO1-infected murine macrophages treated with tanshinone compounds at 100 μM. (C) Inhibition of intracellular proliferation of PAO1 in murine macrophages by different concentrations of tanshinone compounds. The data are averages of three independent experiments. Note that tanshinones were initially dissolved in DMSO and diluted into culture medium for in vitro assays, where 2% DMSO in culture medium was used as the negative control.

Figure 7

Functional characterization of tanshinone derivatives as inhibitors of the biogenesis of the Pseudomonas aeruginosa T3SS needle in vivo. (A) Effects of TSN1, dHTSN, and dHTSN1 on the survival of C57BL/6J mice (n = 20 in each group) intranasally challenged with PAO1. Left panel: tanshinones were administered simultaneously with PAO1. Right panel: tanshinones were administered 8 h postinfection. (B) Reduction of bacterial burden in the bronchoalveolar lavage of PAO1-infected mice by tanshinone compounds. (C) H&E staining of the lungs from normal mice and PAO1-infected mice treated with tanshinone compounds and control. Note that tanshinones were initially dissolved in DMSO and diluted into PBS for in vivo assays, where 1% DMSO in PBS was used as the negative control. The aqueous solubility of tanshinones in the presence of 1% DMSO ranges from 200 to 300 μM (Figure S17).