Universal fluorescent sensors of high-affinity iron transport, applied to ESKAPE pathogens

Abstract

Sensitive assays of biochemical specificity, affinity, and capacity are valuable both for basic research and drug discovery. We created fluorescent sensors that monitor high-affinity binding reactions and used them to study iron acquisition by ESKAPE bacteria, which are frequently responsible for antibiotic-resistant infections. By introducing site-directed Cys residues in bacterial iron transporters and modifying them with maleimide fluorophores, we generated living cells or purified proteins that bind but do not transport target compounds. These constructs sensitively detected ligand concentrations in solution, enabling accurate, real-time spectroscopic analysis of membrane transport by other cells. We assessed the efficacy of these "fluorescent decoy" (FD) sensors by characterizing active iron transport in the ESKAPE bacteria. The FD sensors monitored uptake of both ferric siderophores and hemin by the pathogens. An FD sensor for a particular ligand was universally effective in observing the uptake of that compound by all organisms we tested. We adapted the FD sensors to microtiter format, where they allow high-throughput screens for chemicals that block iron uptake, without genetic manipulations of the virulent target organisms. Hence, screening assays with FD sensors facilitate studies of mechanistic biochemistry, as well as discovery of chemicals that inhibit prokaryotic membrane transport. With appropriate design, FD sensors are potentially applicable to any pro- or eukaryotic high-affinity ligand transport process.

Keywords: ESKAPE pathogen; fluorescence; heme; iron; ligand-binding protein; membrane transport; pathogenesis; siderophore; spectroscopy.

Figure 1.

Fluorescence observations of membrane transport. A, species-specific assay design. The target organism (e.g. K. pneumoniae) contains a Cys substitution for a surface-exposed residue in a TonB-dependent transporter (KpnFepA_T210C) labeled with FM. Fluorescence emissions depict FeEnt binding and TonB-dependent FeEnt transport in K. pneumoniae. B, universal FD sensor assay design. The fluorescence of a TonB-deficient E. coli “sensor” strain OKN13 (ΔtonB, ΔfepA)/pEcoFepA_A698C-FM reflects TonB-dependent FeEnt transport by a second test strain (e.g. K. pneumoniae strain Kp52.145) in the same solution.

Figure 2.

FD sensor analysis of FeEnt acquisition by ESKAPE pathogens. A, species-specific fluorescence assay of FeEnt uptake. KpnFepA-FM. E. coli OKN3 (ΔfepA) expressing KpnFepA-FM was exposed to FeEnt in PBS + 0.2% glucose. The tracings show emissions of living cells in the absence (dark blue) or presence (dark red or black) of FeEnt. In tonB⁺ cells (dark red), FeEnt binding to KpnFepA-FM (at 100 s) quenched emissions, but subsequent uptake depleted FeEnt from solution, restoring fluorescence. In TonB-deficient E. coli OKN13 (ΔfepA, ΔtonB)/pKpnFepA-FM; black), which cannot transport FeEnt, quenching occurred but not recovery. B, Universal fluorescence assay of FeEnt uptake using sensor strain OKN13/pEcoFepA-FM. FeEnt binding at 100 s quenched the fluorescence of the FD sensor strain E. coli OKN13/pEcoFepA-FM. It cannot transport FeEnt, so subsequent recovery did not occur. However, the fluorescence of the E. coli strain reflected FeEnt uptake by other bacteria in the same solution: as they decreased [FeEnt] by cellular transport, the intensity of OKN13/pEcoFepA-FM returned to initial levels (as described in Fig. 1B). The assay monitored FeEnt uptake by E. coli expressing chromosomal (Eco; cyan) or plasmid-mediated (Eco/pfepA⁺; blue) FepA, and ESKAPE species K. pneumoniae (Kpn; red), A. baumannii (Aba; gold), P. aeruginosa (Pae; green), and E. cloacae (Ecl; magenta). FepA-deficient K. pneumoniae (KKN4; black) does not transport FeEnt and did not recover. Hence, OKN13/pEcoFepA-FM was a universal sensor of [FeEnt] that monitored FeEnt acquisition by all the bacteria. The panels show the results of single representative experiments that were performed in triplicate, from which we calculated and plotted the mean values. Standard deviations of the mean measurements (data not shown) were 1.7–2.7%.

Figure 3.

Binding affinities from spectroscopic assays. A–E, we added FeEnt (red symbols) or FeGEnt (purple symbols) to E. coli OKN13/pEcoFepA-FM (A and D, circles) or OKN13/pKpnFepA-FM (B and D, triangles) or K. pneumoniae KKN4/pKpnFepA-FM (C and E, inverted triangles). A–C, quenching data from this species-specific test quantified binding of the iron complexes. Nonlinear fits of 1 − F/F₀ (D and E) using the one-site (with background) equation of Grafit 6.02 revealed the affinities (K_d) of the binding reactions (Table 1). F, [⁵⁹Fe]Ent binding measurements. We added varying concentrations of [⁵⁹Fe]Ent to OKN3/pEcoFepA (circles) or OKN3/pKpnFepA (triangles), measured adsorption of the ferric siderophore by filter-binding assays (26), and obtained nonlinear fits to the one-site (with background) equation of Grafit 6.02, which gave the affinities (K_d) of the binding reactions (Table 1). In each panel the error bars represent S.D. of triplicate measurements at each concentration tested.

Figure 4.

Uptake rates from FD assays. At t = 60 s we added varying concentrations of FeEnt ((0.5 (green), 1 (yellow), 5 (orange), and 10 nm (red)) to sensor strain OKN13/pEcoFepA-FM in the presence of test strain K. pneumoniae isolate Kp52.145. 10 nm FeEnt gave maximal quenching. For 1 and 5 nm FeEnt, at half-saturation (blue dashed line) we determined the slope of the time course (black dashed line) and the elapsed time (dashed red line). Either parameter estimated the transport rate of the test strain (see text).

Figure 5.

FD sensor analysis of Fc acquisition by ESKAPE pathogens. A, species-specific fluorescence assay of Fc uptake. OKN7 (ΔfhuA)/pEcoFhuA-FM reflects Fc binding and transport (orange). In OKN1 (ΔtonB; black) the same construct became an inert sensor of [Fc]: binding of Fc quenched fluorescence, and the absence of transport prevented recovery to initial levels. B, universal assay of Fc uptake. We incubated the sensor strain with 1.5 × 10⁷ cells of Gram-negative ESKAPE pathogens. Colored tracings denote the same strains as in Fig. 2. In each case, the pathogens acquired Fc, causing fluorescence recovery of the sensor strain. Uptake of Fc occurred at a slower rate than FeEnt (30), resulting in slower fluorescence recovery. The panels show the results of single representative experiments that we performed in triplicate, from which we calculated and plotted the mean values. Standard deviations of the mean measurements (data not shown) were 2.5–4%.

Figure 6.

FD sensor assay of hemin uptake. We substituted Cys for residue Ser-154 in NEAT1 of LmoHbp2, expressed and purified the binding protein, and covalently modified it with CPM. A, concentration dependence of hemin binding to LmoHbp2-CPM. Using purified LmoHbp2-CPM, plots of 1 − F/F₀ versus [hemin], analyzed by nonlinear fit to the one-site (with background) equation of Grafit 6.02, produced a saturation curve with K_d = 12 ± 3.1 nm. The error bars represent S.D. of triplicate measurements at each concentration tested. B, Hbp2-CPM detects and quantifies bacterial hemin transport. When we mixed Hbp2-CPM with heterologous bacteria in solution, the assay reflected their hemin uptake. Addition of hemin at 300 s quenched LmoHbp2-CPM emissions, but fluorescence rebounded as bacteria (L. monocytogenes (Lmo; blue), Bacillus subtilis (Bsu; gold), S. aureus (Sau; red), and V. cholerae (Vch; green)) transported the porphyrin and depleted it from solution. Conversely, hemin transport–deficient cells (EGDe Δhbp2, Δhup; black) did not elicit fluorescence recovery. The panel shows the results of a single representative experiment that was performed in triplicate, from which we calculated and plotted the mean values. Standard deviations of the mean measurements (data not shown) were 2–2.9%.

Figure 7.

A and B, FD HTS assay for Gram-negative bacterial TonB-dependent FeEnt (A) and Fc (B) transport. We used a ΔtonB host harboring either pEcoFepA-FM (A) or pEcoFhuA-FM (B), suspended in microtiter plates, to create universal FD sensors of FeEnt or Fc uptake, respectively. The inert sensors effectively monitored active iron transport in 96-well microtiter plates by both E. coli (blue) and K. pneumoniae (red). The proton ionophore CCCP (23) abrogated iron uptake, so fluorescence did not recover in the presence of these compounds. C and D, statistical comparisons of these positive and negative controls yielded Z′ factors approaching 1.0 within 10 min in the case of FeEnt (C) and 35 min in the case of Fc (D). The panels show the results of single representative experiments that were performed in triplicate, from which we calculated and plotted the mean values. Standard deviations of the mean measurements (data not shown) were 1.9–4.1%.