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 abacterial pathogen, but functionally different cells are usually indistinguishable from eacho ther.H erein, we introduce fluorescent activity-based probes as chemical tools for the single-cell phenotypic characterization of enzyme activity levels in Staphylococcus aureus.W es creened a1 ,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.M olecular imaging and activity-based protein profiling studies with these probes revealed adynamic network within this enzyme family involving compensatory regulation of specific family members and exposed single-cell phenotypic heterogeneity.W ep ropose the labeling of enzymatic activities by chemical probes as ageneralizable method for the phenotyping of bacterial cells at the population and single-cell level.

From Differential Stains to Next Generation Physiology: Chemical Probes to Visualize Bacterial Cell Structure and Physiology

“…This included obvious candidates such as esterases and efflux pumps, but also identified a mycobacterial divisome factor responsible for heterogeneity in polar growth. [103] Target-selective probes can also serve as reporters for phenotypic heterogeneity in bacterial population as our already mentioned studies on the distribution of the enzymatic activities of the S. aureus serine virulence factors FphB and FphE across growth conditions revealed [7,84]. While the physiological relevance and molecular mechanisms behind this observation remain unclear, it showcases how the use of chemical probes in cellular imaging can uncover new biology-both by design and by serendipity.…”

“…ABPs with different reactive electrophilic groups (e.g., fluorophosphonates [80], chloroisocoumarins [7] carbamates [81], β-lactams [82], β-lactones [82], triazole ureas [83][84][85], Michael acceptors [86], cyclophellitol epoxides and aziridine (see review [87]), disulfides and sulfonate esters [88]) have been developed to selectively study diverse enzyme families including serine hydrolases [7,74,80,89,90], cysteine proteases [91,92], kinases, metalloproteases [93], glutathione-S-transferases [94], cytochrome P450s [95], ATP-binding enzymes [96], or retaining glycosidases [87]. As discussed in more detail below, the most prominent ABP-targets in bacteria are penicillin-binding proteins [97,98], but the use of both broad-spectrum and target-selective ABPs for other bacterial enzymes in both imaging-related [12,84] and proteomic applications [90,99] is emerging.…”

“…Enzymes can be important virulence factors and chemical probe-based imaging can provide important insight into the subcellular localization and dynamic activity patterns of virulence-associated enzymes. Our recent work on the previously uncharacterized S. aureus fluorophosphonate-binding hydrolases (Fph) illustrates how ABPs can be instrumental both in the identification of new enzymatic activities, as well as in their functional validation [7,84]. Fluorescent conjugates of newly identified FphB-specific inhibitors revealed selective labeling of the target enzyme at specific sites of the cell envelope and growth-condition dependent labeling of the septal cross wall of dividing cells, suggesting its physiological role may be modification of substrates located in the cell wall [7,84].…”

“…Our recent work on the previously uncharacterized S. aureus fluorophosphonate-binding hydrolases (Fph) illustrates how ABPs can be instrumental both in the identification of new enzymatic activities, as well as in their functional validation [7,84]. Fluorescent conjugates of newly identified FphB-specific inhibitors revealed selective labeling of the target enzyme at specific sites of the cell envelope and growth-condition dependent labeling of the septal cross wall of dividing cells, suggesting its physiological role may be modification of substrates located in the cell wall [7,84]. We also demonstrated that FACS-analysis of ABP-labeled bacterial populations is suitable to dissect the dynamic distribution of enzymatic activity levels across cells in different growth environments [7,84].…”

Identification of highly selective covalent inhibitors by phage display

“…Second, we have recently identified a family of Fph hydrolases in S. aureus that function as lipid esterases and at least one of the family members has a role in host colonization 14 . We have performed screening studies using the triazole urea scaffold and found it difficult to identify highly selective inhibitors due to the highly conserved nature of this family 36 . Finally, there is currently no evidence that the DPP electrophile can function as a covalent inhibitor of lipid esterases, thus allowing us to assess the potential of the cyclic peptide to induce covalent bond formation for even sub-optimal electrophiles.…”