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

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.

Introduction

Antimicrobial resistance is becoming one of the greatest threats to public health. According to a widely cited authoritative report,[1] drug-resistant infections by bacteria, viruses, and fungi will cause 10 million annual deaths worldwide by 2050, underscoring an urgent need to develop new classes of therapeutics to avert this global crisis. Bacteria develop drug resistance by controlling the uptake or efflux of antibiotics via altered membrane permeability, enzymatically inactivating them, or modifying their intended intervention targets, among other mechanisms.[2,3] Conventional antibiotics aim to kill, thus subjecting bacteria to evolutionary selection pressure that invariably induces drug-resistant mutations to escape the killing. In some way, ideal antibiotics are refractory to existing resistance mechanisms and can block the ability of bacteria to infect hosts without directly killing them, thus avoiding inducing drug resistance. Toward this end, targeting bacterial virulence factors that are nonessential for survival but critical for pathogenicity has emerged as one of the most attractive strategies to combat antibiotic resistance.[4−6] This antivirulence strategy, in principle, ensues a low likelihood for bacteria to develop resistance as it induces less selective pressure on them. One such virulence factor of many pathogenic Gram-negative bacteria is the type 3 secretion system (T3SS).[7,8]

The T3SS, found as cell-surface appendages, comprises ∼30 bacterial proteins,[7−9] which are classified into structural, effector, and chaperon proteins whose respective functions are largely conserved across different bacterial species. The structural proteins of the T3SS polymerize into a membrane-anchored, needlelike assembly known as “the needle complex”, through which the effector proteins are injected from the bacterial cytoplasm into host cells to promote infection. Since the structural proteins are often hydrophobic and prone to aggregation on their own, they are bound and protected, prior to their high-order assembly on the bacterial membrane, by the chaperone proteins in the cytosol to prevent premature aggregation and degradation.

Pseudomonas aeruginosa is a resistance-prone, Gram-negative pathogen often found in the intensive care unit of hospitals. It causes life-threatening nosocomial infections such as pneumonia in immune-compromised patients and poses a major risk of pulmonary deterioration to patients with chronic cystic fibrosis.[10] Various studies have demonstrated that virulence factors secreted via the T3SS promote pathogenicity of Pseudomonas aeruginosa in vitro and in vivo, which correlates to poor clinical outcomes in Pseudomonas-infected patients.[11−13] Ample evidence validates the T3SS of Pseudomonas aeruginosa as an attractive therapeutic target for antibiotic discovery and development.[14,15]

In the Pseudomonas aeruginosa T3SS[16] (Figure A), the needle is formed by multiple copies of a single protein termed PscF of 85 amino acid residues. Prior to its secretion for needle assembly, PscF is protected in a heterotrimeric complex by two chaperone proteins, PscE (67 AA) and PscG (115 AA).[17] PscG, stabilized by PscE, presents a large concave hydrophobic surface for interactions with the nonpolar residues of PscF18 (Figure B); Pseudomonas aeruginosa mutants deficient in either PscE or PscG or both fail to secrete PscF for assembly of the T3SS needle and, consequently, are noncytotoxic.[17,18] Thus, inhibitors that block PscF interactions with the PscE–PscG dimer are expected to induce premature aggregation and degradation of PscF in the bacterial cytosol, debilitating the biogenesis of the Pseudomonas aeruginosa T3SS needle. Since the survival of Pseudomonas aeruginosa is independent of the T3SS, such inhibitors are less likely to induce antibiotic resistance and therefore ideally suited for development as a novel class of therapeutics to treat multi-drug-resistant Pseudomonas aeruginosa infections. To facilitate their discovery, we designed and validated a fluorescence polarization (FP)-based high-throughput screening (HTS) system and made a surprising discovery in a proof-of-concept study.

Figure 1

Biogenesis of the Pseudomonas aeruginosa T3SS needle. (A) Schematic representation of the T3SS of Pseudomonas aeruginosa, adapted from Abrusci et al. 2014.[16] (B) Crystal structure of the heterotrimeric complex of PscE–PscF–PscG determined by Quinard et al. 2007.[18] 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.

Results and Discussion

Chemical Synthesis and Characterization of PscE, PscF, and PscG

Quinaud et al. reported the first crystal structure of the trimeric complex of PscE–PscF–PscG formed by recombinant proteins.[18] To replicate their findings, we chemically synthesized PscE, PscG, and PscF54–85 using solid-phase peptide synthesis coupled with native chemical ligation[19,20] (Figures S1–S5). [Note that the N-terminally truncated PscF54–85 was prepared because PscF1–53 is structurally disordered and does not contribute to interactions with PscE–PscG18 (Figure B).] All synthetic polypeptides were purified to homogeneity by reversed-phase (RP)-HPLC and verified by electrospray ionization mass spectrometry (ESI-MS) (Figure A). To obtain the heterotrimeric complex previously described,[18] PscE, PscG, and PscF54–85 at a 1:1:1 molar ratio were dissolved in 6 M GuHCl, followed by a 6-fold dilution with and an overnight dialysis against PBS. The resultant protein complex was analyzed by Superdex 75 size-exclusion chromatography, RP-HPLC, and ESI-MS and confirmed as a heterotrimer (Figure S6A–C). Consistent with the known structural and biophysical properties of the PscE–PscG–PscF54–85 complex,[17,18] the synthetic heterotrimer adopted a predominantly α-helical conformation in solution as determined by CD spectroscopy (Figure B) and was highly stable as made evident by a melting temperature of 61.7 °C measured in a protein thermal denaturation assay (Figure S6D). As was previously reported,[17,18] we also found that synthetic PscE and PscG could form a stable heterodimer of a helical nature with a slightly lower melting temperature (Figure B, Figure S6D).

Figure 2

Characterization of synthetic peptides/proteins by HPLC, ESI-MS, and CD spectroscopy. (A) Chemically synthesized PscF54–85, 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.

Design and Validation of an FP-Based Readout for HTS

FP assays have been widely used in HTS for low-molecular-weight inhibitors that target proteins such as enzymes and receptors in the presence of a small, fluorescently labeled natural substrate or ligand of the target protein.[21−23] For dyes attached to small, rapidly rotating molecules, FP is low as the molecules tumble fast in solution (relative to the fluorescence lifetime) and efficiently “scrambles” the polarization of emitted light. However, upon binding by a large molecule, tumbling of the dye complex is slowed, resulting in an increased polarization of fluorescence emission. The strategy for our FP assay is illustrated in Figure A, where the addition of a library compound to the high-polarization heterotrimeric complex leads to the displacement of fluorescently labeled PscF from the PscE–PscG heterodimer, resulting in a decrease in FP. Since PscF54–85 readily precipitated in aqueous solution, we truncated the peptide further by deleting 15 amino acid residues at its N-terminus (Figure S1), yielding a soluble PscF69–85 peptide, which was subsequently labeled with fluorescein (FAM). Structural studies showed that the interaction of PscF54–85 with PscE–PscG is dominated by the C-terminal amphipathic α-helix PscF69–85 rather than the N-terminal extended coil comprising residues 54–66 (Figure B).[18] For functional verification, though, we characterized the interactions of PscE, PscG, and unlabeled PscF69–85 using isothermal titration calorimetry (ITC) (Figure S7A,B). An equilibrium dissociation constant (KD) of 1.17 μM was determined for PscE and PscG. While titration of PscF69–85 to PscG alone yielded a KD value of 10.5 μM, a significantly stronger binding was observed for PscF69–85 interacting with the preformed PscE–PscG dimer (KD = 51.5 nM) (Figure B). Nearly identical KD values (10.8 μM and 52.0 nM, respectively) were obtained using FAMPscF69–85 in an FP assay (Figure C, Figure S7C). Of note, unlabeled PscF69–85 competed off FAMPscF69–85 from PscE–PscG in a dose-dependent fashion, giving rise to an IC50 value of 4.32 μM (Figure D). Collectively, these functional data fully validated the synthetic heterotrimeric complex PscE–PscG–FAMPscF69–85 as a suitable system for the development of an FP assay for HTS, and supported the structural finding as well that although PscE does not directly participate in PscF interactions, it enhances them by stabilizing the scaffold of PscG.[18]

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 PscF69–85 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 FAMPscF69–85 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 PscF69–85 (black), tanshinone 1 (TSN1) (red), dihydrotanshinone 1 (dHTSN1) (green), and dihydrotanshinone (dHTSN) (blue) with FAMPscF69–85 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.

Identification of Tanshinones as Competitive Inhibitors of PscF Binding to PscE–PscG

Armed with this validated FP assay, we screened in an ultralow throughput 10 natural herbal compounds in traditional Chinese medicine: ammonium glycyrrhizinate, astragaloside A, baicalein, curculigoside, ginsenosides Rb1 and Re, osthol, panaxadiol, quercetin, and tanshinone 1 (TSN1) (Figure S8). These compounds with various anti-infective, anti-inflammatory, or antitumor properties but no known activity against bacterial secretion systems were obtained from a collaborator’s laboratory and intended initially as “negative controls” for a proof-of-concept study. To our complete surprise, both primary and secondary screenings identified TSN1 as a positive hit, which competed with FAMPscF69–85 for PscE–PscG binding in an FP assay, yielding an IC50 value of 2.15 μM (Figures S8 and Figure D,E). Consistent with this purely serendipitous finding, TSN1, when added to the preformed heterotrimer PscE–PscG–PscF69–85, reduced not only α-helicity of the complex (Figure S9A) but also its thermal stability (Figure S9B,C). For further verification, we examined four additional commercially available tanshinone analogues (Figure S10) and identified dihydrotanshinone 1 (dHTSN1) (IC50 = 0.68 μΜ) and dihydrotanshinone (dHTSN) (IC50 = 1.50 μM) similar in structure to and more active than TSN1 (Figure D,E). By contrast, cryptotanshinone (crpTSN) and tanshinone 2A were inactive (Figure S10). Of note, fluorophores that excite and emit at longer wavelengths should in general be used for FP-based HTS assays to minimize potential spectral interference by library compounds. While FAM was not an ideal choice of fluorophore for this purpose, tanshinones, which are not fluorescent themselves, showed no spectroscopic interference with the FAM fluorescence when excited at 470 nm (Figure S11).

Structural Characterization of Tanshinone Interactions with PscE–PscG

For structural validation, we characterized by NMR spectroscopy a heterodimeric complex comprising synthetic PscE and a 15N-labeled recombinant PscG (Figure S12) in the presence and absence of the tanshinone dHTSN1. As shown in Figure A (black), the 15N–1H HSQC spectrum of 15N–PscG in complex with PscE exhibited the typical feature of an α-helical protein where its resonance peaks distributed between 7.3 and 9.0 ppm in the proton dimension. The spectrum had a good dispersion except for the broadening of some resonance peaks in the center, indicating that parts of the PscG conformation were still flexible. Nevertheless, upon addition of dHTSN1 to the 15N–PscG–PscE complex at a molar ratio of 1:1:1, those flexible resonances became much sharper (red), indicative of relatively strong interactions between dHTSN1 and the heterodimeric complex. Of note, these interactions also resulted in the broadening of a few other resonance peaks (marked in circles) beyond the center of the spectrum (Figure A).

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[15] 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.

We observed significant changes to the side-chain amide resonance peaks of Trp residues (the inset, Figure A). There are four Trp residues in PscG, three of which, Trp 67, Trp73, and Trp79, are located on the same helix involved in PscF interactions (Figure B). The four individual resonance peaks of Trp in the 15N–1H HSQC spectrum were arbitrarily labeled as W1, W2, W3, and W4 (Figure A, black). As shown in the inset of Figure A (red), upon binding of dHTSN1 to the PscG/PscE heterodimer, three amide resonance peaks of Trp became broadened, and one remained unchanged. These results indicate that dHTSN1 binding is localized to the PscF-interacting helix of PscG, inducing direct and/or indirect changes in side-chain amide resonance to the three proximal Trp residues. In fact, molecular docking studies pinpointed Trp79, among other residues of PscG (Figure S13), to be directly involved in tanshinone interactions (Figure C).

Tanshinones Block the Secretion of the T3SS Effector ExoS in Vitro

The cytotoxicity of Pseudomonas aeruginosa against host immune cells and epithelial cells is dependent on its T3SS, through which four exotoxins (effector proteins) are unleashed into the host cytoplasm: ExoS, ExoT, ExoU, and ExoY.[7−9] ExoS and ExoT are homologous exotoxins with GTPase activating and ADP ribosyltransferase activities, capable of disrupting the actin cytoskeleton and inducing apoptotic cell death, while ExoU has phospholipase A2 activity that induces rapid necrotic cell death via membrane lysis. It is anticipated that inhibitors of the biogenesis of the Pseudomonas aeruginosa T3SS needle will shut down the exotoxin transport machinery, preventing or reducing the cytotoxic and pathogenic effects of Pseudomonas aeruginosa on host cells and tissues.

We used infection of mouse macrophage cell line J774A.1 by the Pseudomonas aeruginosa reference strain PAO1[24] (ExoS, T, Y only) as an in vitro model to study the effects of tanshinones on Pseudomonas aeruginosa cytotoxicity and pathogenicity. None of the tanshinone compounds at 100 μM were directly bactericidal or bacteriostatic against PAO1, nor were they growth-inhibitory against macrophages (Figure A). However, Western blot analysis showed that treatment with the three active tanshinone compounds at 100 μM each of PAO1, grown under low-calcium conditions to transcriptionally activate the T3SS,[25] significantly reduced the secretion of ExoS (Figure B). Consistent with the biochemical findings, dHTSN and dHTSN1 were more active than TSN1 in inhibiting ExoS secretion, while crpTSN showed no inhibitory activity. These data confirmed that an impaired biogenesis of the Pseudomonas aeruginosa T3SS needle could lead to a decrease in secretion of bacterial exotoxins and other virulence factors.

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.

Tanshinones Reduce Cytotoxicity of Pseudomonas aeruginosa PAO1 to Macrophages and Inhibit Intracellular Bacterial Survival

Macrophages infected by phagocytosed Pseudomonas aeruginosa undergo rapid cell death (pyroptosis) mediated by proinflammatory caspase-1, which is activated via Nod-like receptor signaling by bacterial flagellin injected into the cytosol by the T3SS.[26] We infected J774A.1 cells with PAO1 in the presence of various tanshinone compounds, and cytotoxicity was quantified by measuring the release into the medium of the cytoplasmic enzyme lactate dehydrogenase (LDH) by dying macrophages. As shown in Figure A, TSN1, dHTSN, and dHTSN1 inhibited Pseudomonas-aeruginosa-induced cell lysis in a dose-dependent manner, whereas crpTSN was inactive. Western blot analysis implied a reduction in activated caspase-1 as the plausible cause for the survival of infected macrophages (Figure B), consistent with functional inhibition of the T3SS by tanshinones. Notably, less than 70% of inhibition of the cytotoxicity of PAO1 to macrophages was achieved by the two most active tanshinones dHTSN and dHTSN1 at 100 μM (EC50 = 12.5 and 25 μM, respectively) (Figure A), suggesting a significant residual cytotoxic effect (at the level of ∼30% LDH release) that could not be neutralized by tanshinone treatment. Since PAO1 and tanshinones were added simultaneously to macrophages before incubation, this persistent basal cytotoxicity was obviously independent of the future status of the biogenesis of the T3SS needle and likely arose from pyroptosis of macrophages induced by phagocytosed bacteria harboring T3SS-indepednent cytotoxic factors. Results from an identical LDH assay using the mutant strain PAO1 ΔpscC we constructed,[27] which is defective in the T3SS due to the lack of the outer membrane ring protein PscC, confirmed that PAO1 ΔpscC also induced LDH release from macrophages, albeit at a reduced level compared with PAO1 (Figure S14). In fact, tanshinone treatment had no effect on PAO1 ΔpscC-induced LDH release (Figure S14), consistent with the fact that tanshinones act on the T3SS. Interestingly, when LDH released from dying macrophages treated with PAO1 was normalized against that with PAO1 ΔpscC, the EC50 values of dHTSN and dHTSN1 were in the neighborhood of 3 μM (Figure S15). Thus, the lack of an appropriate “negative” control strain in the LDH assay could artificially underestimate tanshinone activity.

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.

Despite being a bona fide extracellular pathogen, phagocytosed Pseudomonas aeruginosa can transiently survive and even replicate in macrophages,[28] a cellular event enabled by bacterial virulence factors that subvert antibacterial effector functions of macrophage. Recent imaging studies revealed that Pseudomonas aeruginosa PAO1, engulfed by macrophages into phagosomal vacuoles, can subsequently escape into the cytoplasm, where it ultimately induces cell lysis (Preeti Garai et al., bioRxiv 389718; DOI: https://doi.org/10.1101/389718). Strikingly, both phagosomal escape and intracellular bacteria-mediated cell lysis are strongly dependent on ExoS—the very T3SS effector tanshinones block. We treated PAO1-infected J774A.1 cells at 2 h postinfection with gentamicin to kill off extracellular bacteria and quantified intracellular bacteria from lysed cells. As shown in Figure C, the three active tanshinones dose-dependently inhibited intracellular bacterial survival, confirming that tanshinones can indeed protect macrophage function by inhibiting ExoS secretion to prevent phagosomal escape of and cell lysis mediated by phagocytosed PAO1.

It is worth noting that Pseudomonas aeruginosa can invade and actively multiply in epithelial cells in vitro and in vivo,[29,30] where ExoS promotes its intracellular survival after invasion by (1) helping Pseudomonas aeruginosa avoid lysosomal degradation and (2) creating membrane blebs as a replicative niche for the bacterium.[31−33] Paradoxically, ExoS had long been thought to be capable of preventing Pseudomonas aeruginosa from being internalized or endocytosed by epithelial cells through destabilization of the actin cytoskeleton.[34] More recent studies, however, have reconciled these contradictory findings by linking the purported anti-internalization activity of ExoS to the use of “artificial” reporter systems, including ectopic expression of ExoS without bacteria or in trans expression of ExoS in the background of PA103, an effector-null cytotoxic strain of Pseudomonas aeruginosa.[34] When ExoS is natively encoded in Pseudomonas aeruginosa strains such as PAO1, it does not prevent bacterial internalization into epithelial cells.[34]

In Vivo Efficacy of Tanshinones in a Murine Model of Acute Pneumonia

Phagocytic macrophages and neutrophils play critical roles in bacterial clearance during acute Pseudomonas aeruginosa infection in vivo.[35,36] To subvert their antibacterial defense, Pseudomonas aeruginosa has evolved T3SS-dependent mechanisms to lyse macrophages and impair neutrophil function. Production of reactive oxygen species (ROS) by neutrophils is critical for intracellular killing of phagocytosed bacteria. A recent study demonstrated that the T3SS effectors ExoS and ExoT secreted by PAO1 independently inhibit ROS production in human neutrophils.[37] Thus, inhibition of the biogenesis of the Pseudomonas aeruginosa T3SS needle should improve phagocytic functions of macrophages and neutrophils, leading to efficient bacterial clearance from infected host.

We investigated whether or not tanshinones could protect against Pseudomonas aeruginosa infection in vivo using a murine model of acute pneumonia. As shown in Figure A (left), without treatment, 70% of C57BL/6J mice intranasally inoculated with 1 × 107 CFU of PAO1 died of acute lung infection within 48 h. While TSN1 at 100 μM given at the time of infection and every 12 h thereafter was significantly protective, dHTSN and dHTSN1 dramatically prolonged animal survival with over 90% of infected mice surviving beyond 96 h. To administer tanshinones in a clinically relevant setting, we performed another in vivo efficacy study using the same murine model of acute pneumonia where the first intranasal injection of tanshinones was made 8 h after the animals had been challenged with PAO1. As shown in Figure A (right), while 85% of mice in the mock-treated group died of infection within 48 h, only 40%, 20%, and 5% died in the groups treated with TSN, dHTSN, and dHTSN1, respectively. Further, with dHTSN1 treatment, 80% of infected mice survived beyond 96 h.

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).

In a separate in vivo experiment where infected mice received a single-dose treatment at the time of infection, we quantified PAO1 in the bronchoalveolar lavage sampled at 18 h postinfection; active tanshinones significantly reduced bacterial burden in the lung (Figure B)—an outcome likely arising, at least in part, from bacterial clearance by functional phagocytes afforded by an impaired Pseudomonas aeruginosa T3SS. Consistent with these findings, H&E staining of the lungs from mock-treated mice at 18 h postinfection revealed extensive cellular infiltration and tissue damage that occluded the airways (Figure C, Figure S16). By contrast, treatment by tanshinones and dHTSN1 in particular significantly reduced inflammation as made evident by minimal infiltration of neutrophils into the alveolar spaces of the lungs (Figure C, Figure S16). Taken together, our in vivo data support the premise that tanshinones prevent lung pathology associated with Pseudomonas infection by inhibiting the secretion via the T3SS of bacterial virulence factors.

Other Known Inhibitors of the T3SS

Of note, many studies have already validated the T3SS as an attractive drug target for antibiotic discovery and development. Two recent articles provide a comprehensive review of novel strategies for the treatment of Pseudomonas aeruginosa infections, including the targeting of the T3SS.[14,15] Passive and active immunization with T3SS structural and effector proteins can prevent or reduce T3SS-induced bacterial virulence in vitro and in vivo.[7,9,10,38,39] Although various cellular reporter assays coupled with library screening led to the identification of some small-molecule inhibitors of the T3SS of relatively low potency,[40] the lack of understanding of precise molecular targets and mechanisms of action has hampered their further development. Phenoxyacetamides are the only known class of compounds that block both the T3SS-mediated secretion and translocation of Pseudomonas aeruginosa effectors through binding to PscF to interfere with its multimerization.[41−43] We tested a prototypic phenoxyacetamide compound MBX-1641 in our in vitro and in vivo assays and found it functionally comparable to dHTSN and dHTSN1. As shown in Figure S18, MBX-1641 inhibited the secretion of ExoS in PAO1, the cytotoxicity of PAO1 to murine macrophages, and the intracellular proliferation of PAO1 as well. Further, MBX-1641 reduced bacterial burden in the bronchoalveolar lavage of PAO1-infected mice (Figure S18F). In contrast to tanshinones, however, MBX-1641 had no effect on the binding of PscF to PscE–PscG as analyzed by fluorescence polarization (Figure S18G). These findings indicate that phenoxyacetamides and tanshinones mechanistically differ as inhibitors of the Pseudomonas aeruginosa T3SS.

Conclusions

We made a serendipitous discovery of selective tanshinones as mechanistically defined inhibitors of the biogenesis of the Pseudomonas aeruginosa T3SS needle. Biochemical and biophysical as well as in vitro and in vivo functional studies of tanshinones validated these natural herbal compounds as promising drug candidates for the development of a novel class of antibiotics for the treatment of multi-drug-resistant Pseudomonas aeruginosa infections. As an active ingredient in traditional Chinese medicine widely used to treat cardiovascular and cerebrovascular diseases with a demonstrable safety profile in humans,[44] tanshinones may be used directly, upon conclusion of a comprehensive toxicology study to ascertain the safety of individual compounds, to alleviate Pseudomonas-aeruginosa-associated pulmonary infections without inducing antibiotic resistance. Our work demonstrated the feasibility of targeting the biogenesis of the T3SS needle for antibiotic discovery by developing a sensitive fluorescence polarization assay for automated HTS of library compounds. Since the T3SS is highly conserved in many other pathogenic Gram-negative bacteria such as Escherichia coli, Salmonella, Shigella, Yersinia, Vibrio, Burkholderia, and Chlamydia, our strategy for antibiotic discovery may have broad implications in combating antibiotic resistance.

Experimental Procedures

Materials

Boc-amino acids were purchased from Peptides Institute. Boc-Leu–OCH2–PAM resin and p-methyl-BHA (MBHA) resin were purchased from Applied Biosystems (Foster City, CA). N,N-Dimethylformamide (DMF), dichloromethane (DCM), N,N-diisopropylethylamine (DIEA), dimethyl sulfoxide (DMSO), methanol, 4-mercaptophenylacetic acid (MPAA), tris(2-carboxyethyl) phosphine (TCEP), p-cresol, and HPLC grade acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO). Hydrogen fluoride (HF) was purchased from APK (Shanghai, China). Trifluoroacetic acid (TFA) was purchased from HaloCarbon (River Edge, NJ). Tanshinone IIA, dihydrotanshinone 1, tanshinone 1, and cryptotanshinone were purchased from Nature Standard (Shanghai, China). Ammonium glycyrrhizinate, astragaloside A, baicalein, curculigoside, ginsenoside Rb, ginsenoside Re, osthol, panaxadiol, quercetin, and dihydrotanshinone were provided as generous gifts by Dr. Sha Liao of Northwestern University of China. All natural herbal compounds were prepared in DMSO (5–10 mM) and stored in the dark at room temperature for no more than 1 month.

Peptide Synthesis

All peptides were synthesized using the optimized HBTU activation/DIEA in situ neutralization protocol developed by Kent and colleagues for Boc-chemistry solid-phase peptide synthesis (SPPS).[45] Peptide cleavage from resin and side-chain deprotection reactions were performed at 0–4 °C for 1 h in HF. After precipitation with cold ether, crude products were purified to homogeneity on a preparative HPLC using a C18 reversed-phase column. The molecular masses were ascertained by electrospray ionization mass spectrometry (ESI-MS).

Native Chemical Ligation

Native chemical ligation[19,20] reactions were carried out in 0.1 M phosphate buffer containing 6 M guanidine hydrochloride (GuHCl), 100 mM MPAA, and 40 mM TCEP, pH 7.4. Thz was used instead of Cys to protect the δ-mercaptolysine at position 26 of PscG to avoid an intramolecular head-to-tail ligation reaction. The Thz ring opened upon treatment of the peptide by MeONH2·HCl at pH ∼ 4 for 12 h. The Trp(CHO) was deprotected using 20% piperidine and 20% tBUSH for 30 min.

Chemical Synthesis of MBX1641

Compound MBX 1641 was prepared directly from 2-(2,4-dichlorophenoxy) propanoic acid and 3,4-methylenedioxybenzylamine (Alfa Aesar) as reported.[41] Crude products were purified to homogeneity on a preparative HPLC using a C4 reversed-phase column. The molecular mass was ascertained by ESI-MS.

Heterotrimeric Complex Cofolding and Characterization

Protein folding was achieved by dissolving the polypeptides (at the same molar ratio) in 6 M GuHCl at 1 mg/mL, followed by a 6-fold dilution with phosphate buffered saline (PBS) containing 0.5 mM TCEP, pH 7.4, and an overnight dialysis against the same buffer. After dialysis, the protein complex was analyzed by size exclusion chromatography on an ÄKTA protein purification system using a Superdex 75 column at a flow rate of 0.5 mL/min at room temperature. The apparent molecular weights were calculated according to the standard calibration curve. The protein complex was also analyzed by reverse-phase HPLC on a Waters XBridge C18 column (4.6 × 150 mm, 3.5 μm) and its molecular mass ascertained by ESI-MS.

Fluorescence Polarization (FP) and FP-Based Competitive Binding Assays

All fluorescence polarization assays were done using black, low-protein-binding 96-well plates (Thermo Fisher Scientific) in a total volume of 100 μL per well of 10 mM Tris buffer containing 150 mM NaCl and 1 mM EDTA, pH 7.0, unless indicated otherwise. After a gentle mixing and incubation for 3 h, FP readings were taken at 470 nm (excitation) and 530 nm (emission) wavelengths on a Tecan Infinite M2000 fluorescence plate reader. Nonlinear regression analyses were performed to give rise to Kd and IC50 values as previously described.[46,47]

For direct binding of PscF to PscG or PscE–PscG, equal volumes of FAM–PscF69–85 (400 nM) and serially diluted PscG or PscE–PscG (0–64 μM) were mixed. For initial screening, 95 μL of FAM–PscF69–85–PscE–PscG (100 nM) in the assay buffer was mixed with 5 μL of small-molecule inhibitor in DMSO to a final molar concentration of 1, 10, 100, or 1000 μM. DMSO (5%) and 6 M GuHCl in the final assay buffer were used as the negative and positive controls, respectively. For secondary screening or competitive binding assays, 95 μL of FAM–PscF69–85–PscE–PscG (100 nM) in the assay buffer was mixed with 5 μL of serially diluted PscF69–85 or various tanshinone analogues (0–200 μM).

Isothermal Titration Calorimetry (ITC)

ITC was used to determine the binding affinity KD, enthalpy change ΔH, and binding stoichiometry n of the interaction between molecules. All ITC experiments were performed on a MicroCal ITC 200 at 25 °C in 10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 7.0. The concentrations of PscF54–85 and PscE were 300 μM each and the concentrations of PscG and PscE–PscG 30 μM each. From these initial measurements, Gibbs energy changes ΔG and entropy changes ΔS can be determined using the formula ΔG = −RT In Ka = ΔH – TΔS.

Circular Dichroism (CD) Spectroscopy and Thermal Denaturation

CD spectra of proteins at a concentration of 200 nM in 10 mM phosphate buffer (pH 7.4) were obtained at room temperature on a circular dichroism spectrometer (Jasco, Easton, MD) using a 1 mm quartz cuvette. Protein thermal denaturation was carried out in PBS using a JASCO circular dichroism spectrometer equipped with temperature controller. A 2.5 mL portion of protein solution (10 μM) prepared in PBS, pH 7.4, was aliquoted into a 3 mL cuvette. Under constant stirring, the measurement was taken at a 1° interval between 25 and 90 °C, at a heating rate of 1 °C per minute. After each 1 min heating, the solution in the cuvette was left for 20 s before signals were detected over a 16 s period. Heating and data acquisition were fully automated with the control software provided by JASCO. Data processing was performed as previously described.[48−50]

Expression of 15N-Labeled PscG and NMR Characterization of 15N-Labeled PscG in Complex with PscE and dHTSN1

PscG was uniformly labeled with 15N MOPS medium containing 1 g/L 15NH4Cl as the sole source and BME vitamins (Sigma). Cells were harvested by centrifugation at 6000g for 30 min, resuspended in a lysis buffer containing 6 M urea, and subjected to a 1 min sonication, followed by two-cycle homogenization at 4 °C. The lysates were centrifuged at 20 000 rpm for 30 min, and the supernatant was loaded onto a 10 mL Ni-NTA agarose column (Qiagen). The elute was concentrated to 10–12 mL under denaturation conditions, followed by purification on a Sep-Pak C18 column. Peak fractions containing 15N–PscG were lyophilized. The 15N–PscG protein samples were dissolved in a buffer containing 20 mM sodium phosphate (pH 7.4) and 10 mM NaCl and mixed with PscE or PscE and dHTSN1 at an equal molar ratio. The mixtures were dialyzed against the same buffer overnight and then concentrated to ∼300 μL.

All NMR samples were prepared in an NMR buffer containing 20 mM sodium phosphate (pH 7.4), 100 mM NaCl, 0.1% NaN3, 10% D2O, and 2 mM DTT. The final protein concentrations were approximately 26 and 47.8 μM for 15N–PscG–PscE and 15N–PscG–PscE–dHTSN1 complexes, respectively. All NMR spectra were collected at 25 °C on a Bruker Avance 700 MHz spectrometer equipped with a triple-resonance pulse-field gradient probe. 15N–1H HSQC NMR spectra were recorded in the echo–antiecho mode for quadrature detection. All data sets were acquired with 2048 complex points in t2 and 256 complex points in t1. Data were processed using Topspin software and displayed using NMRViewJ software.

Molecular Docking

The 3-dimensional structure of the heterotrimeric complex of PscE–PscF–PscG was used for molecular docking.[18] PscF was removed from the heterotrimeric complex to prepare the molecular target for docking and so were the four N-terminal residues (GSHM) in PscE that showed high values of b-factor in the heterotrimeric complex. The three active tanshinone compounds and the PscE–PscG complex were prepared for docking using the AutoDockTools software suite.[51] The AutoGrid module was used to create a grid box with center at 44.174, 28.238, 18.306 and size of 106, 102, 82 points along the XYZ directions with a spacing of 0.375 Å. Docking calculations were performed with AutoDock4.2.6[51] using the Lamarkian Genetic algorithm with a population size of 150, and the number of evaluations and generations set to 10 000 000. Then, 100 docking runs were performed for each compound, and docked conformations were clustered using AutoDockTools with a cutoff of 2.0 Å, yielding the largest cluster exceeding 95% of the total runs.

Bacterial Strain and Cell Line and Growth Conditions

Pseudomonas aeruginosa isolate PAO1 and its mutant strain PAO1 ΔpscC were cultured in Luria broth (LB) at 37 °C. The mouse macrophage cell line J774A.1 (ATCC TIB-67) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and incubated at 37 °C in 5% CO2.

Bactericidal Activity Assay

PAO1 overnight cultures were diluted 1:100 in LB, in the presence of tanshinone inhibitors (100 μΜ compound, 2% DMSO). After a 3 h incubation with mild agitation, bacteria were diluted and plated. Bactericidal activity was determined by colony counting and normalized against the activity under mock treatment (2% DMSO only). Results are represented as the mean ± SD percentage of input bacteria of three independent experiments.

Cytotoxicity of Tanshinones to Macrophages

A total of 1 × 104 J774A.1 cells were seeded into each well of a 96-well plate and grown for 24 h. Different tanshinone inhibitors (100 μΜ compound, 2% DMSO) were added and incubated at 37 °C in 5% CO2 for 8 h followed by CCK-8 cell viability assay (Beyotime, C0038) according to the manufacturer’s instructions. The control group was treated with 2% DMSO. Optical density (OD) was measured at 450 nm. Percentage of cell viability was calculated as follows:

Average results of three independent experiments are shown as mean ± SD.

Inhibition of T3SS-Mediated Effector Secretion

For T3SS induction, PAO1 overnight cultures were diluted to an OD at 600 nm of 0.3 in LB containing 5 mM EGTA and 20 mM MgCl2, in the presence of tanshinone inhibitors (100 μΜ compound, 2% DMSO), followed by an incubation with mild agitation of additional 3–4 h until the cultures reached an OD value of 1.5. The NC group was cultured in LB and 2% DMSO and the PC group in LB containing 5 mM EGTA, 20 mM MgCl2, and 2% DMSO. Culture supernatant was collected by centrifugation at 4000g for 15 min at 4 °C, and secreted proteins were concentrated by adding ice-cold trichloroacetic acid to a final concentration of 10% (v/v). Following a 2 h incubation on ice, the pellets were washed twice with cold acetone and suspended in an SDS-PAGE sample buffer (with 2-mercaptoethanol) according to the BCA protein assay protocol. Secreted and total proteins (supernatant and pelleted bacteria) were analyzed by immunoblotting with an anti-ExoS antibody (Agrisera, AS05056, at 1:4000 dilution) and corresponding HRP-conjugated secondary antibody. The blots were semiquantified using ImageJ 1.51k (from http://imagej.nih.gov/ij). The results were expressed as the mean ± SD percentage of secreted ExoS out of total ExoS of three independent experiments.

Cytoplasmic Lactate Dehydrogenase (LDH) Release Assay

J774a cells (1 × 104) were seeded into each well of a 96-well plate and grown for 24 h before infection. One hour before the infection, cell culture medium was changed into serum-free medium, and PAO-1 from the midexponential phase was added to the cells at a multiplicity of infection (MOI) of 8. In the presence of different concentrations of tanshinone inhibitors (0–100 μΜ compound, 2% DMSO), bacteria/cells mixtures were incubated for 5 h. LDH released into the supernatant was detected by an LDH detection kit (Beyotime, C0017) as instructed by the manufacturer. Results were normalized against the LDH released by PAO-1-infected cells with mock treatment (2% DMSO). Average results of three independent experiments are shown as mean ± SD.

Caspase-1-Mediated Pyroptosis of PAO1-Infected Macrophages

J774a cells (2 × 105) were seeded into each well of a 6-well plate and grown for 24 h before infection. One hour before the infection, cell culture medium was changed into serum-free medium, and PAO-1 from the midexponential phase was added to the cells at a multiplicity of infection (MOI) of 8. In the presence (100 μΜ compound, 2% DMSO) or absence (2% DMSO) of tanshinone inhibitors, bacteria/cells mixtures were incubated for 3 h at 37 °C in 5% CO2. Cells were collected with lysis buffer (with 2-mercaptoethanol) according to the BCA protein quantification protocol (Beyotime, P0017) and subjected to 15% SDS-PAGE gel and immunoblotting with antipro caspase-1 + p10 + p12 antibody (Abcam, ab179515, at 1:1000 dilution) and corresponding HRP-conjugated secondary antibody. β-actin was used as the internal control.

Quantification of Bacterial Internalization

J774a cells (5 × 104) were seeded into each well of a 24-well plate and grown for 24 h before infection. One hour before the infection, cell culture medium was changed into serum-free medium, and PAO-1 from the midexponential phase was added to the cells at a multiplicity of infection (MOI) of 8. In the presence of different concentrations of tanshinone inhibitors (0–100 μΜ compound, 2% DMSO), bacteria/cells mixtures were incubated for 2 h, washed, and then treated with gentamicin-containing (50 μg/mL) medium for another 2 h before being lysed for plating. Internalized bacteria were defined as the total number of intracellular bacteria in cells (extracellular bacteria were killed by gentamicin, a cell-impermeable antibiotic). Results were normalized against the intracellular bacteria number by PAO-1-infected cells with mock treatment (2% DMSO). Average results of three independent experiments are shown as mean ± SD.

Animal Studies

Female C57BL/6J mice (6 week-old) used in this study were acquired from the Experimental Animal Center of Xian Jiaotong University. All the animals were maintained in animal care facilities in the School of Life Science and Technology and provided with food and water ad libitum. The animal studies were approved by the Committee on Animal Research and Ethics, Xi’an Jiaotong University.

C57BL/6J mice were lightly anesthetized with inhaled sevoflurane and infected by intranasal instillation of PAO1 (1 × 107 CFU in 20 μL of PBS) after being lightly anesthetized with inhaled sevoflurane. Tanshinone inhibitors were administrated to the animals along with bacterial inoculation (100 μM, 1% DMSO). PBS containing 1% DMSO was used as the mock treatment. After infection (18 h), animals were sacrificed, and bronchoalveolar lavages were collected and plated to obtain the bacterial counts in the lavages. The lungs of sacrificed mice were then isolated and fixed in 10% buffered formalin, paraffin-embedded, and hematoxylin-eosin-stained for histopathological examination. Pathological scores of the tissues were assigned according to the degree of inflammation.[52]

For survival study, tanshinone inhibitors (100 μM, 1% DMSO, in 10 μL of PBS) were administrated to the infected animals intranasally at the time of infection, or 8 h after infection, and every 12 h until the death of the animal or the end of the experiment. PBS containing 1% DMSO was used as a mock treatment.

Statistical Analysis

The data were collected from at least three independent experiments in triplicate or quadruplicate, unless otherwise indicated. Data were combined and represented as mean ± SEM or mean ± SD as indicated. Results were analyzed by various statistical tests using GraphPad Prism version 7. p < 0.05 was considered statistically significant. Microscopy images are representative of at least two independent experiments.

Safety Statement

No unexpected or unusually high safety hazards were encountered.