Fluorescent Triazole Urea Activity-Based Probes for the Single-Cell Phenotypic Characterization of Staphylococcus aureus

Abstract

Phenotypically distinct cellular (sub)populations are clinically relevant for the virulence and antibiotic resistance of a bacterial pathogen, but functionally different cells are usually indistinguishable from each other. Herein, we introduce fluorescent activity-based probes as chemical tools for the single-cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus. We screened a 1,2,3-triazole urea library to identify selective inhibitors of fluorophosphonate-binding serine hydrolases and lipases in S. aureus and synthesized target-selective activity-based probes. Molecular imaging and activity-based protein profiling studies with these probes revealed a dynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single-cell phenotypic heterogeneity. We propose the labeling of enzymatic activities by chemical probes as a generalizable method for the phenotyping of bacterial cells at the population and single-cell level.

Keywords: activity-based probes; enzymes; molecular imaging; protein profiling; single-cell analysis.

Figure 1.

Inhibitory profile of TU screening hits and labeling profile of clickable probes. A) Chemical structures of hit compounds and their regioisomers 1-4 a/b. B) Competitive FP-TMR labelling profiles of S. aureus Newman WT or indicated transposon mutant (:Tn) strains after preincubation with compounds 1-4 a/b. Cells were harvested from TSAMg plates, preincubated with inhibitors for 60 min, labelled with FP-TMR (1 μM), lysed and analysed by SDS-PAGE/fluorescence scan. Preferred targets of each probe (according to transposon mutant analysis) are highlighted by colored arrows: Orange: SAL1, blue: FphA, red: FphB, green: FphF, yellow: FphH, purple: FphE.

Figure 2.

Strategy for the design of fluorescent TU activity-based probes and labeling profiles of select clickable intermediates. A) Schematic overview of ABP design with chemical structures of the clickable probes 5-7a/b and the corresponding fluorescent probe 8a/b and 9a/b. B) Direct cellular labeling profiles of S. aureus Newman cultures with probe 5b and C) 7b. For direct visualization of labeling with clickable probes in B,C, labeled proteins were fluorescently tagged by attachment of N₃-TAMRA by click-chemistry in situ after bacterial lysis and analysed by SDS-PAGE. Data show whole cell extracts excluding secreted proteins in the culture supernatant.

Figure 3.

Labeling profiles of fluorescent triazole urea probes show selectivity for SAL1/2 or FphE. A) Labeling profile of S. aureus USA300 and its transposon mutant (:Tn) cultures in late stationary phase with fluorescent probes 8a, 8b or FP-TMR. Arrows indicate pro-form and matured forms of SAL1 (light orange) and SAL2 (dark orange). B, C) Labeling profile of S. aureus Newman and its fphE transposon mutant cultures harvested from TSAMg plates (B) or cultures in late stationary phase (C) labelled with fluorescent probes 9a, 9b or FP-TMR. All samples include full cultures with whole cell extracts and secreted culture supernatants. Arrows indicate FphE (purple), SAL1 (orange). D) Confocal micrographs of indicated S. aureus wt or transposon mutant cells. Cells were harvested from TSAMg and labelled with 300 nM 9a in TSB for 30 min, before washing and fixation. Bodipy-TMR fluorescence is shown in magenta. The red and yellow arrows highlight cells exemplifying the heterogeneity of cellular labeling (red: no or weak labeling, yellow: strong labeling). Scale bar: 1 μm.

Figure 4.

Single-cell analysis of S. aureus cells labeled with probe 9a by flow cytometry. A) Representative plot of cellular fluorescence levels for indicated S. aureus strains harvested from TSAMg and labelled with 9a or FP-TMR. Cells were analyzed by flow cytometry (552 nm laser excitation, 586 nm emission filter) after fixation. Insets show corresponding normalized mean fluorescence intensity (MFI) values for wt and transposon mutant strain fphE:Tn after subtracting MFI from unstained control samples. B, C) Plots of FphE-specific MFI values of PFA-fixed USA300 (B) or Newman (C) wt or fphH:Tn cells labeled with 300 nM of 9a at indicated growth conditions and analyzed by flow cytometry. Primary MFI values were normalized by subtracting the average MFI value of fphE:Tn control samples. Graphs shows means ± S.D. of three independent culture replicates per condition. Statistical significance was tested by unpaired two-tailed Student’s t-test.