Fluorescence High-Throughput Screening for Inhibitors of TonB Action

Abstract

Gram-negative bacteria acquire ferric siderophores through TonB-dependent outer membrane transporters (TBDT). By fluorescence spectroscopic hgh-throughput screening (FLHTS), we identified inhibitors of TonB-dependent ferric enterobactin (FeEnt) uptake through Escherichia coli FepA (EcoFepA). Among 165 inhibitors found in a primary screen of 17,441 compounds, we evaluated 20 in secondary tests: TonB-dependent ferric siderophore uptake and colicin killing and proton motive force-dependent lactose transport. Six of 20 primary hits inhibited TonB-dependent activity in all tests. Comparison of their effects on [⁵⁹Fe]Ent and [¹⁴C]lactose accumulation suggested several as proton ionophores, but two chemicals, ebselen and ST0082990, are likely not proton ionophores and may inhibit TonB-ExbBD. The facility of FLHTS against E. coli led us to adapt it to Acinetobacter baumannii We identified its FepA ortholog (AbaFepA), deleted and cloned its structural gene, genetically engineered 8 Cys substitutions in its surface loops, labeled them with fluorescein, and made fluorescence spectroscopic observations of FeEnt uptake in A. baumannii Several Cys substitutions in AbaFepA (S279C, T562C, and S665C) were readily fluoresceinated and then suitable as sensors of FeEnt transport. As in E. coli, the test monitored TonB-dependent FeEnt uptake by AbaFepA. In microtiter format with A. baumannii, FLHTS produced Z' factors 0.6 to 0.8. These data validated the FLHTS strategy against even distantly related Gram-negative bacterial pathogens. Overall, it discovered agents that block TonB-dependent transport and showed the potential to find compounds that act against Gram-negative CRE (carbapenem-resistant Enterobacteriaceae)/ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Our results suggest that hundreds of such chemicals may exist in larger compound libraries.IMPORTANCE Antibiotic resistance in Gram-negative bacteria has spurred efforts to find novel compounds against new targets. The CRE/ESKAPE pathogens are resistant bacteria that include Acinetobacter baumannii, a common cause of ventilator-associated pneumonia and sepsis. We performed fluorescence high-throughput screening (FLHTS) against Escherichia coli to find inhibitors of TonB-dependent iron transport, tested them against A. baumannii, and then adapted the FLHTS technology to allow direct screening against A. baumannii This methodology is expandable to other drug-resistant Gram-negative pathogens. Compounds that block TonB action may interfere with iron acquisition from eukaryotic hosts and thereby constitute bacteriostatic antibiotics that prevent microbial colonization of human and animals. The FLHTS method may identify both species-specific and broad-spectrum agents against Gram-negative bacteria.

Keywords: Acinetobacter baumannii; ESKAPE pathogen; FepA; TonB; antibiotic resistance; ferric enterobactin; fluorescence assays; high throughput; iron transport.

FIG 1

Candidate inhibitors of TonB action identified by primary screening. Among the 44 compounds that inhibited fluorescence >30% in the primary FLHTS screening, we selected 20 compounds for further testing and obtained them from suppliers as fresh powders. These chemicals caused dose-dependent inhibition of fluorescence recovery; we did not consider compounds that reduced initial fluorescence and/or the extent of initial quenching. We also tested compound 120304 from reference , but it had no effects in our primary or secondary tests of TonB action (see also Table 2).

FIG 2

Secondary screening by TonB-dependent siderophore nutrition assays and colicin killing tests. (A) We tested the 20 selected primary hits for interference with siderophore nutrition assays with FeEnt and Fc; the negative and positive controls were untreated bacteria and bacteria treated with CCCP, respectively. We observed several types of inhibition by the compounds: larger halos (that imply a lower rate of iron uptake [10, 44, 45]; illustrated by the control, CCCP, at 15 and 10 μM), distorted, aberrant halos, and the complete absence of a halo. Inhibition was identified by a loss of halo or an increase in the diameter of the halo around the ferric siderophore disc (Table 2). (B) We performed ColB and ColIa killing tests in the presence of the 20 selected compounds, with the same controls. The graph depicts the percent survival of bacteria in the presence of ColB or ColIa in the absence and presence of inhibitory compounds (see also Table 2). We performed each experiment 2 or 3 times and averaged the percent survival of colicin killing in the absence and presence of each compound. Error bars represent the standard deviations of the mean values. Compound abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; Bai, baicalein; CA, cadmium acetate; CD, carbidopa; CP, carboplatin; CHL, p-chloranil; DS, dideoxyscleroin; DC, dequalinium chloride; EB, ebselen; EA, ellagic acid; MC, methylcatechol; THM, thimerosal; ZP, zinc pyrithione; 120304, lead compound from reference . See Fig. 1 and Table 2 for more information about the enumerated compounds.

FIG 3

Inhibition of [⁵⁹Fe]Ent and [¹⁴C]lactose uptake. E. coli MG1655 was grown in MOPS minimal medium until late exponential phase and independently assayed for its ability to accumulate [⁵⁹Fe]Ent (A and C) and [¹⁴C]lactose (B and D) in the presence of either ebselen (A and B) or ST0082990 (C and D). We tested MG1655 in the absence of any inhibitors (circles), in the presence of CCCP at 25 μM (triangles), in the presence of ebselen at 25 (light gray squares), 200 (medium gray squares), and 250 (dark gray squares) μM, or in the presence of ST0082990 at 25 (light gray squares), 100 (medium gray squares), and 250 (dark gray squares) μM.

FIG 4

FeEnt transport by A. baumannii. (A) Growth of A. baumannii 17978 (circles) and its ΔfepA derivative (squares) in MOPS minimal medium without (open symbols) or with (gray symbols) 100 μM apoFcA (means from 3 experiments with standard deviations of the means as error bars) shows no growth defect from the ΔfepA mutation. (B) A. baumannii 17978 (●) and its ΔfepA derivative (■) were grown in LB, subcultured (1%) into MOPS minimal medium plus 100 μM apoFcA, and subcultured (1%) into MOPS minimal medium plus or minus 1 μM FeEnt (means and standard deviations from three experiments). The ΔfepA strain shows roughly half the growth of its parent. (C and D) Siderophore nutrition assays with A. baumannii 17978 and its ΔfepA mutant (C) or 17978/pWH1266 (++/p), ΔfepA/pWH1266 (ΔfepA/p), ΔfepA/pWH1266AbafepA⁺ (ΔfepA/pAbafepA⁺), and ΔfepA/p2682proAbafepA⁺ (ΔfepA/pproAbafepA⁺) (D). The bacteria were plated in top agar with 100 μM apoFcA, and discs containing FeEnt or Fc (10 μl of 50 μM) were placed on the agar surface. Both panels C and D are representative images from three separate experiments. (E) Accumulation of [⁵⁹Fe]Ent over 45 min, with each data point performed in triplicate, by 17978/pWH1266 (●), ΔfepA/pWH1266 (■), and ΔfepA/p WH1266AbafepA⁺ (▲). The graph depicts the means and standard deviations of the means from two experiments. (F) Transport of ⁵⁹Fe[Ent] by A. baumannii 17978 (●) or its ΔfepA mutant (■). The graph depicts the means and standard deviations of the means from triplicate measurements in two separate experiments.

FIG 5

Fluoresceination of Cys substitutions in AbaFepA facilitates measurement of FeEnt uptake by A. baumannii. (A and B) Cys mutagenesis of AbaFepA. The Modeller function of CHIMERA (48) predicted the tertiary structure of AbaFepA (dark beige) based on its 46% identity to EcoFepA (PDB file 1FEP; N domain, red; C domain, lime green). We selected eight amino acids in the surface loops of AbaFepA loops (colored and depicted in space-filling format) with Cys, from their similarity to residues in EcoFepA that when fluoresceinated gave substantial quenching during FeEnt binding (28). (C and D) Fluorescence spectroscopic measurement of FeEnt transport in A. baumannii. (C) After transforming mutant plasmids encoding AbaFepA Cys mutants into A. baumannii 17978 ΔfepA, we maximized expression of the AbaFepA Cys mutants by growth in iron-deficient MOPS medium and labeled the cells with FM. We monitored fluorescence voltage from FM-labeled ΔfepA/pAbafepA-Cys mutants in response to addition of 10 nM FeEnt at 100 s and normalized the data to the initial fluorescence of the bacteria (F/F₀). Binding of FeEnt to AbaFepAS279C-FM, T562C-FM and S665C-FM quenched their fluorescence; subsequent FeEnt uptake by the cells depleted the ferric siderophore from solution, resulting in a gradual increase to initial fluorescence levels (recovery). The plotted data are the averages from three separate experiments. (D) Normalized fluorescence of FM-labeled 17978 ΔfepA/pAbafepAS279C following exposure to various concentrations of CCCP (black, no CCCP; blue, 2.5 μM CCCP; green, 5 μM CCCP; red, 10 μM CCCP). The plotted data are the averages from two separate experiments.

FIG 6

FeEnt uptake by fluoresceinated AbaFepA-Cys mutants. (A) FM labeling. A. baumannii 17978 ΔfepA harboring plasmids expressing AbaFepA Cys mutants were labeled with 5 μM FM and subjected to SDS-PAGE, followed by fluorescent imaging. We included E. coli OKN3/pITS23-S271C as a positive control. The imaged gel was representative of three experiments. (B) FeEnt transport V_max screening. All unlabeled mutants normally utilized FeEnt in siderophore nutrition tests (data not shown), but quantitative measurements of [⁵⁹Fe]Ent uptake showed that certain Cys substitutions (T223C, A326C, T383C, and S482C) impaired iron transport, especially when fluoresceinated. The graph depicts the transport of [⁵⁹Fe]Ent by the mutant strains before and after FM labeling, relative to wild-type A. baumannii that was untreated but harboring the empty plasmid vector. S279C, T562C, and T665C mutants transported [⁵⁹Fe]Ent with 38 to 75% of the efficiency of wild-type AbaFepA. Each bar derives from the average of 2 or 3 experiments, performed in triplicate.

FIG 7

Adaptation of fluorescence assay to HTS format in A. baumannii. We tested 17978 ΔfepA expressing AbaFepAS279C-FM, T562C-FM, and S665C-FM (not shown) in the fluorescence assay in a 96-well microtiter plate format. The tracings depict normalized fluorescence of FM-labeled ΔfepA/pAbafepAS279C (A) or T562C mutant (B) following exposure to 10 μM CCCP (negative control; filled symbols) or an equivalent volume of DMSO; open symbols. Following three readings of initial fluorescence (F₀), we injected 20 nM FeEnt in the absence (gray symbols) or presence (filled symbols) of CCCP and measured changes in the ensuing fluorescence (F) over time. We also included cells to which no FeEnt was added (positive control; open symbols). The graph averages data from two separate experiments. (C) Z′ factors for each mutant's controls were calculated at each read during experiment, averaging between 0.6 and 0.8 for the controls from 8 to 15 min.