The mechanism of action of auranofin analogs in B. cenocepacia revealed by chemogenomic profiling.

Drug repurposing efforts led to the discovery of bactericidal activity in auranofin, a gold-containing drug used to treat rheumatoid arthritis. Auranofin kills Gram-positive bacteria by inhibiting thioredoxin reductase, an enzyme that scavenges reactive oxygen species (ROS). Despite the presence of thioredoxin reductase in Gram-negative bacteria, auranofin is not always active against them. It is not clear whether the lack of activity in several Gram-negative bacteria is due to the cell envelope barrier or the presence of other ROS protective enzymes such as glutathione reductase (GOR). We previously demonstrated that chemical analogs of auranofin (MS-40 and MS-40S), but not auranofin, are bactericidal against the Gram-negative Burkholderia cepacia complex. Here, we explore the targets of auranofin, MS-40, and MS-40S in Burkholderia cenocepacia and elucidate the mechanism of action of the auranofin analogs by a genome-wide, randomly barcoded transposon screen (BarSeq). Auranofin and its analogs inhibited the B. cenocepacia thioredoxin reductase and induced ROS but did not inhibit the bacterial GOR. Genome-wide, BarSeq analysis of cells exposed to MS-40 and MS-40S compared to the ROS inducers arsenic trioxide, diamide, hydrogen peroxide, and paraquat revealed common and unique mediators of drug susceptibility. Furthermore, deletions of gshA and gshB that encode enzymes in the glutathione biosynthetic pathway led to increased susceptibility to MS-40 and MS-40S. Overall, our data suggest that the auranofin analogs kill B. cenocepacia by inducing ROS through inhibition of thioredoxin reductase and that the glutathione system has a role in protecting B. cenocepacia against these ROS-inducing compounds.IMPORTANCEThe Burkholderia cepacia complex is a group of multidrug-resistant bacteria that can cause infections in the lungs of people with the autosomal recessive disease, cystic fibrosis. Specifically, the bacterium Burkholderia cenocepacia can cause severe infections, reducing lung function and leading to a devastating type of sepsis, cepacia syndrome. This bacterium currently does not have an accepted antibiotic treatment plan because of the wide range of antibiotic resistance. Here, we further the research on auranofin analogs as antimicrobials by finding the mechanism of action of these potent bactericidal compounds, using a powerful technique called BarSeq, to find the global response of the cell when exposed to an antimicrobial.

Maltose-Derivatized Fluorescence Turn-On Imaging Probe for Bacteria Detection.

We report a maltose-derivatized fluorescence turn-on imaging probe, Mal-Cz, to detect E. coli and Staphylococci. The fluorescence turn-on is achieved through an intramolecular C-H insertion reaction of the perfluoroaryl azide-functionalized carbazole to give a fluorescent product. Confocal fluorescence microscopy confirmed the successful uptake of Mal-Cz by E. coli and Staphylococci upon photoactivation. The Mal-Cz probe could selectively detect E. coli and S. epidermidis in the presence of P. aeruginosa and M. smegmatis without interference from these bacteria. Both the photoactivation and bacteria detection can be accomplished using a hand-held UV lamp at 365 nm, with the limit of detection of 103 CFU/mL by the naked eye. Mal-Cz could also be used to detect E. coli and S. epidermidis spiked in milk by the naked eye under a hand-held UV lamp. The uptake of Mal-Cz requires metabolically active bacteria: the uptake was reduced in stationary phase bacteria and was diminished in bacteria that were killed by heating or treating with antibiotics or sodium azide. The uptake decreased with increasing concentration of added free maltose, indicating that Mal-Cz hijacked the maltose uptake pathways. In E. coli, the maltose transport systems, including maltoporin LamB, maltose binding protein MBP, and the maltose ATP binding cassette (ABC) transporter MalFGK2, are all critical for the transport of Mal-Cz. The uptake was diminished in the deletion mutants ΔLamB, ΔMalE, ΔMalF, and ΔMalK.

Azide-Masked Fluorescence Turn-On Probe for Imaging Mycobacteria.

A fluorescence turn-on probe, an azide-masked and trehalose-derivatized carbazole (Tre-Cz), was developed to image mycobacteria. The fluorescence turn-on is achieved by photoactivation of the azide, which generates a fluorescent product through an efficient intramolecular C-H insertion reaction. The probe is highly specific for mycobacteria and could image mycobacteria in the presence of other Gram-positive and Gram-negative bacteria. Both the photoactivation and detection can be accomplished using a handheld UV lamp, giving a limit of detection of 103 CFU/mL, which can be visualized by the naked eye. The probe was also able to image mycobacteria spiked in sputum samples, although the detection sensitivity was lower. Studies using heat-killed, stationary-phase, and isoniazid-treated mycobacteria showed that metabolically active bacteria are required for the uptake of Tre-Cz. The uptake decreased in the presence of trehalose in a concentration-dependent manner, indicating that Tre-Cz hijacked the trehalose uptake pathway. Mechanistic studies demonstrated that the trehalose transporter LpqY-SugABC was the primary pathway for the uptake of Tre-Cz. The uptake decreased in the LpqY-SugABC deletion mutants ΔlpqY, ΔsugA, ΔsugB, and ΔsugC and fully recovered in the complemented strain of ΔsugC. For the mycolyl transferase antigen 85 complex (Ag85), however, only a slight reduction of uptake was observed in the Ag85 deletion mutant ΔAg85C, and no incorporation of Tre-Cz into the outer membrane was observed. The unique intracellular incorporation mechanism of Tre-Cz through the LpqY-SugABC transporter, which differs from other trehalose-based fluorescence probes, unlocks potential opportunities to bring molecular cargoes to mycobacteria for both fundamental studies and theranostic applications.

Trehalose-Grafted Glycopolymer: Synthesis via the Staudinger Reaction and Capture of Mycobacteria.

A new trehalose-grafted poly(2-hydroxyethyl methacrylate) (HEMA) glycopolymer was synthesized via the perfluorophenyl azide (PFPA)-mediated Staudinger reaction between poly(HEMA-co-HEMA-PFPA) and a diphenylphosphine-derivatized trehalose. The reaction occurred rapidly at room temperature without the use of any catalyst, giving the trehalose glycopolymers over 68% yield after 1 h. The grafting density of trehalose can be controlled by the copolymer composition in poly(HEMA-co-HEMA-PFPA), resulting in 6.1% (TP1) or 37% (TP2) at 10:1 and 1:1 HEMA/HEMA-PFPA feed ratio, respectively. The trehalose glycopolymer was covalently attached on glass slides or silicon wafers using a thin film of poly(HEMA-co-HEMA-PFPA) as the adhesion layer, achieved through the C-H insertion reaction of the photogenerated singlet perfluorophenyl nitrene. To demonstrate the ability of the trehalose glycopolymer to capture mycobacteria, arrays of the trehalose glycopolymer were fabricated and treated with Mycobacterium smegmatis. Results from the optical, fluorescence, and scanning electron microscopy showed that mycobacteria were indeed captured on the trehalose glycopolymer. The amount of mycobacteria captured increased with the percent trehalose in the trehalose glycopolymer and also with the concentration of the trehalose glycopolymer. In addition, the captured bacteria could be visualized by the naked eye under the illumination of a hand-held UV lamp.

New Auranofin Analogs with Antibacterial Properties against Burkholderia Clinical Isolates.

Bacteria of the genus Burkholderia include pathogenic Burkholderia mallei, Burkholderia pseudomallei and the Burkholderia cepacia complex (Bcc). These Gram-negative pathogens have intrinsic drug resistance, which makes treatment of infections difficult. Bcc affects individuals with cystic fibrosis (CF) and the species B. cenocepacia is associated with one of the worst clinical outcomes. Following the repurposing of auranofin as an antibacterial against Gram-positive bacteria, we previously synthetized auranofin analogs with activity against Gram-negatives. In this work, we show that two auranofin analogs, MS-40S and MS-40, have antibiotic activity against Burkholderia clinical isolates. The compounds are bactericidal against B. cenocepacia and kill stationary-phase cells and persisters without selecting for multistep resistance. Caenorhabditis elegans and Galleria mellonella tolerated high concentrations of MS-40S and MS-40, demonstrating that these compounds have low toxicity in these model organisms. In summary, we show that MS-40 and MS-40S have antimicrobial properties that warrant further investigations to determine their therapeutic potential against Burkholderia infections.

Quantification of binding affinity of glyconanomaterials with lectins.

Carbohydrate-mediated interactions are involved in many cellular activities including immune responses and infections. These interactions are relatively weak, and as such, cells employ multivalency, i.e., the presentation of multiple monovalent carbohydrate ligands within a close proximity, for cooperative binding thus drastically enhanced binding affinity. In the past two decades, the field of glyconanomaterials has emerged where nanomaterials are used as multivalent scaffolds to present multiple copies of carbohydrate ligands on the nanomaterial surface. At the core of glyconanomaterial research is the ability to control and modulate multivalency through ligand display. For the quantitative evaluation of multivalency, the binding affinity must be determined. Quantification of the binding parameters provides insights for not only the fundamental glyconanomaterial-lectin interactions, but also the rational design of effective diagnostics and therapeutics. Several methods have been developed to determine the binding affinity of glyconanomaterials with lectins, including fluorescence competitive assays in solution or on microarrays, Förster resonance energy transfer, fluorescence quenching, isothermal titration calorimetry, surface plasmon resonance spectroscopy, quartz crystal microbalance and dynamic light scattering. This Feature Article discusses each of these techniques, as well as how each technique is applied to determine the binding affinity of glyconanomaterials with lectins, and the data analysis. Although the results differed depending on the specific method used, collectively, they showed that nanomaterials as multivalent scaffolds could amplify the binding affinity of carbohydrate-lectin interactions by several orders of magnitude, the extent of which depending on the structure of the carbohydrate ligand, the ligand density, the linker length and the particle size.