Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes

propargylcholine; synthase; Pmt, phospholipid -methyltransferase; PLE, polar lipid extract; Abstract Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPS) on the outer leaflet and phospholipids (PLs) on the inner leaflet. Loss of this asymmetry due to mutations in the lipopolysaccharide (LPS) biosynthesis or transport pathways causes externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we employed metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase (Pcs) expression in Escherichia coli allowed for incorporation of exogenous propargylcholine (PCho) into phosphatidyl(propargyl)choline (PPC) and for incorporation of exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC) as confirmed by LC-MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells, but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains, and revealed significant AEPC labeling in an E. coli LPS transport mutant ( lptD4213 ) and an LPS biosynthesis mutant ( E. coli lpxC101 ). Our results suggest that metabolic PL labeling with AECho is a promising tool to detect a compromised bacterial OM, reveal aberrant PL externalization, and identify or characterize novel cell-active inhibitors of LPS biosynthesis or transport. plasmolysis, samples of E. coli BW25113 pcs were grown in M9 medium with 500 μM AECho and fluorescently-labeled with Alexa488- DIBO as described above. After imaging the non-plasmolyzed cells for fluorescence, the samples were pelleted at 11,600 × g for 2 min, resuspended in 100 μL plasmolysis solution (containing 15% sucrose, 25 mM HEPES (pH 7.4), and 20 mM sodium azide) and deposited on an agar pad (containing 15% sucrose and 1.2% agarose) freshly prepared on a glass microscope slide and directly imaged (54,55). For membrane staining of E. coli K-12, FM 4-64 (( N -3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)-pyridinium dibromide, Invitrogen, 500 μg/mL in DMSO) was added to the plasmolyzed sample to a final concentration of 1% (v/v) before depositing a 3 μL aliquot on an agar pad. for only 4 h with AECho did not show significant fluorescent labeling (Fig. 8B). The parent strain MC4100 pcs showed no significant fluorescent labeling for any incubation time tested. Our results suggest a concentration- and time-dependence of AECho labeling in lptD4213 pcs . At least 0.5 mM AECho for 16 h incubation or at least 6 h incubation time with 1 mM AECho is needed to see significant fluorescent labeling in lptD4213 pcs , while the parent strain did not show significant fluorescent labeling.


Introduction
The widespread emergence of antibiotic resistance in pathogenic Gram-negative bacteria is a rapidly growing threat and represents one of today's greatest public health problems (1,2). The discovery of new antibiotics for Gram-negative bacteria is challenging, partly due to the action of various multidrug efflux pumps and the Gram-negative specific outer membrane (OM) (3)(4)(5). The OM is composed of an inner leaflet of phospholipids (PLs) and an outer leaflet of lipopolysaccharides (LPS or endotoxin) (6). This strict asymmetry is important for the OM to function as a permeability barrier, protecting the cells from the immune system, and hindering many toxic compounds of entering the cell (4,7). If compounds manage to penetrate through the OM, in order to reach a cytoplasmic target they also need to cross the phospholipid bilayer that makes up the inner membrane (IM). Consequently, these compounds need to cross two membranes with very different properties.
The critical OM asymmetry results from the externalization of LPS from the IM directly to the outer leaflet of the OM by the Lpt pathway (8) and also from the removal of any PLs that reach the outer leaflet of the OM by internalization (e.g. via the Mla pathway) or degradation (e.g. by PldA and PagP) (9)(10)(11)(12). Previous studies found that Escherichia coli strains with mutations in genes required for LPS biosynthesis or transport, such as lpxC (previously known as envA) (13) or lptD (also known as ostA or imp) (8), have permeability defects in their OM compared to their wild-type counterparts (14,15). A similar hyperpermeable phenotype can be observed in wild-type Gram-negative bacteria when treated with cationic OM permeabilizers, such as polymyxins, or compounds which inhibit key enzymes in the LPS biosynthesis or transport pathways (16). The inhibition of the LPS transport or biosynthesis pathway leads to lower levels of LPS on the outer leaflet and concomitant higher levels of externalized PLs. The formation of PL bilayer patches in the OM leads to higher permeability towards large and/or lipophilic compounds (4). Thus, an assay to specifically detect Gram-negative bacterial PLs would identify compounds that disrupt OM asymmetry causing an OM permeability defect (9,17,18). by guest, on May 6, 2020 www.jlr.org

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Currently available assays to identify a compromised OM by detecting bacterial PLs are indirect, low-throughput, and labor intensive. One methodology quantifies the level of OM palmitoylated lipid A, formed by PagP-dependent palmitoyl transfer from PL to LPS in the outer leaflet of the OM. This assay requires radiolabeling, acid hydrolysis, organic extraction, and thin-layer chromatography (TLC) of the lipid A anchor of LPS (9,19). Different radioactive compounds, like [ 32 P] phosphoric acid or [ 3 H]-glycerol, have been used to label PLs followed by organic extraction and liquid scintillation counting to determine indirectly the amount of PLs (20,21). A more direct assay utilizes treatment of intact bacteria by exogenous phospholipase C to hydrolyze externalized PLs, followed by organic extraction, separation by TLC, and analysis by iodine deposition and image analysis (20,22,23). More recently, a cinnamycin-conjugated fluorophore that is known to bind to curved patches of ethanolamine lipids was utilized to measure OM defects in an E. coli lysophospholipid transporter (lplT) mutants, though the interaction is non-covalent and the specificity for phosphatidylethanolamine (PE) and/or lysophosphatidylethanolamine on the outer leaflet of the OM was not evaluated (24). In mammalian systems, phosphatidylserine (PS) externalization is a hallmark of apoptosis, and various methods to visualize the externalization have been extensively used (25).
A method to quantify externalized PS makes use of the cell-impermeant N-hydroxy-sulfosuccinimidobiotin that reacts with surface exposed PS (and other primary amines). However, this methodology is a resource-intensive destructive endpoint assay that utilizes liquid chromatography-mass spectrometry (LC-MS) (26). Another methodology pioneered in mammalian systems is the selective labeling of cellular phosphatidylcholine (PC) by metabolic incorporation of propargylcholine (PCho) into phosphatidyl(propargyl)choline (PPC), which can then be fluorescently-labeled using biorthogonal "click" chemistry to visualize PPC from intact cells (27).

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Unfortunately, the head groups of these PLs are not suitable for metabolic labeling because their biosynthetic pathways do not present a clear opportunity for the introduction of an analog (Fig. S1).
While the naturally occurring E. coli lipids are not suitable, approximately 15% of all bacterial species produce PC (e.g. pathogens like Pseudomonas aeruginosa and Legionella pneumophila) (34). Two PC biosynthetic pathways are found in bacteria: the phospholipid N-methyltransferase (Pmt) pathway and the phosphatidylcholine synthase (Pcs) pathway ( Fig. S1) (34)(35)(36). While PC is synthesized starting from PE in the Pmt pathway (Fig. S1A), the Pcs enzyme catalyzes the reaction of choline (Cho) with cytidine diphosphate-diacylglycerol (CDP-DAG) to form PC (Fig. 1A). The mammalian PC biosynthetic pathway (the Kennedy pathway) uses an unrelated third pathway where Cho is phosphorylated and then activated to CDP-Cho which further reacts with DAG to form PC (Fig. S1B) (37,38). Two analogs of Cho, PCho and 1-azidoethyl-choline (AECho) (Fig. 1B), have been used to metabolically label PC in mammalian cells (27,39,40). Both analogs react with an azide or alkyne after incorporation into PC to allow further detection.
More recently, PCho has been also used to label PC in plants (41). Although Cho analogs can be incorporated into PC in eukaryotic cells, there was no certainty that the bacterial Pcs pathway would accept Cho analogs.
Here, we present for the first time the successful metabolic labeling of PC with AECho to visualize PLs in intact E. coli with a bioorthogonal fluorescent method (Fig. 1C). Exogenously supplied AECho was incorporated by Pcs into PC to form phosphatidyl(azidoethyl)choline (AEPC) (Fig. 1C, II). When the OM asymmetry and permeability were compromised by mutation, PLs (including AEPC) were exposed (Fig.   1C, III). The azide moiety in the head group was then reacted with a fluorescent alkyne reagent in a clickreaction (Fig. 1C, IV) to enable fluorescent-detection of AEPC on the OM. by guest, on May 6, 2020 www.jlr.org

Preparation of total lipid extracts from E. coli strains grown with Cho, PCho or AECho
Overnight cultures of E. coli and the corresponding strain containing the pcs plasmid (

TLC of total lipid extracts
Total lipid extracts were dissolved in chloroform-methanol (4:1, v/v) and spotted onto TLC silica gel 60 F254 plates (Millipore Sigma). Chloroform-methanol-water (65:35:8, v/v/v) was used as solvent system and was allowed to equilibrate in the TLC chamber at least 1 h before usage. TLC plates were developed by spraying them with 10% ethanolic H2SO4 solution and charring with a heat gun for about 2 min.

Normal-phase LC-MS/MS analysis of total lipid extracts from E. coli
LC-MS/MS experiments were performed on a SCIEX 4000 QTRAP mass spectrometer with Turbo V ion source coupled to an Agilent 1100 LC fitted with glass and steel capillaries and normal-phase solvent-safe pump seals. The instrument vendors were consulted prior to running these normal-phase LC-MS conditions to ensure the instruments were compatible. To avoid solvent exposure, the instruments were well-ventilated.
Chromatographic conditions and mass spectrometric settings were adapted from previous methods (46,47).
All PLs (PG, PE, PC, PPC and AEPC) were scanned for the 11 most abundant acyl chain combinations (Table S3) (33,48). Analyst® Software (SCIEX) was used for data acquisition. A detailed description of the LC-MS/MS analysis can be found in the Supplemental Methods.

Sucrose density gradient separation of IM and OM
E. coli BW25113 and BW25113 pcs were grown as described above. Separation of the IM and OM was performed as described previously (49). The fractions were collected in 1-mL steps and were used to identify the IM via nicotinamide adenine dinucleotide (NADH) assay as described (50), and the OM by LPS gel as described previously (51,52) ( Fig. S2 and S3). A detailed description of the sucrose gradient separation can be found in the Supplemental Methods.

Fluorescent labeling of E. coli strains (lptD4213 and lpxC101 mutants) grown with AECho
A bacterial overnight culture was inoculated into M9 medium containing an appropriate volume of a sterile aqueous stock solution of AECho or the same volume of sterile water to a starting OD 600 of 0.002 -0.005. (1.45 numerical aperture) was used for phase-contrast and fluorescent imaging. Images for green fluorescence were taken by using the FITC-5050A-NTE-ZERO filter set (Semrock). Images were captured by using Nikon Elements software and exported for figure preparation in ImageJ (53).

Flow cytometry analysis of click-labeled bacteria
E. coli samples were grown in M9 medium and click-labeled as described. The washed pellets were resuspended in M9+malt medium and analyzed on an Attune NxT flow cytometer (ThermoFisher), using the FITC channel to read AlexaFluor 488 with excitation wavelength 488 nm (blue laser) and emission wavelength 530 nm. Approximately 100,000 cells were analyzed for each sample.

Statistics of flow cytometry analysis
Error bars in all figures represent standard deviation from the mean (SD). Mean values were compared via two-way ANOVA using Tukey's multiple comparison tests to determine statistical significance, which is 0.0001.

Confirmation of Pcs functionality for PC production
Because E. coli does not naturally produce PC, the pcs gene of L. pneumophila was inserted into E. coli K-12 to synthesize PC, as previously described (42). To confirm that the plasmid was functional, BW25113 pcs was grown to stationary phase in M9 medium containing different concentrations of Cho (0 mM, 0.01 mM, 0.1 mM, and 1 mM Cho), followed by lipid isolation by Bligh-Dyer extraction (44,45). The total lipid extracts were separated by TLC and visualized by charring (Fig. 2). In all BW25113 pcs samples grown with Cho, PC was observed in addition to PE and PG (

Confirmation of PC in both IM and OM by sucrose gradient separation
PLs can be generally present in both leaflets of the IM as well as in the inner leaflet of the OM, thus, it was important to confirm that PC is distributed in both membranes. BW25113 and BW25113 pcs were grown to stationary phase in M9 medium in the presence of Cho. The cells were lysed and the membranes separated via sucrose gradient separation as described (49). For each strain, 12 fractions were collected and analyzed by NADH oxidase assay (50) to identify fractions containing IM and analyzed by SDS PAGE gel (51,52) to identify fractions containing OM. The results of the NADH oxidase assay (Fig. S2) indicated that the IM in both strains was present predominantly in fractions 1-4, so they were combined as the IM fraction. In the image of the LPS gel (Fig. S3), fractions 9-12 in both strains ( Fig. S3A and S3B, lanes [12][13][14][15] showed the highest abundance of rough LPS, so they were combined as OM fraction. The results observed are in agreement with the data described in the literature (49,50).
The total lipids of the IM and OM fractions were isolated by Bligh-Dyer extraction (44,45). The sucrose fractions were used as part of the aqueous phase. The total lipid extracts were separated via TLC and visualized by charring (Fig. 4).

Confirmation of PCho and AECho incorporation by E. coli pcs
Two requirements for using PC to detect exposed PL were fulfilled with the confirmation that PC is produced and present in both membranes. The next steps were to evaluate whether E. coli can uptake Cho analogs like PCho or AECho and incorporate them into PC using Pcs (27,39) (Fig. 1B). To test this, BW25113 pcs was grown to stationary phase in M9 medium supplemented with or without 1 mM PCho, followed by lipid isolation by Bligh-Dyer extraction. The total lipid extracts were first separated by TLC, and then the plate was developed by charring. However, PPC was not detected on the TLC plate (data not shown), indicating that either PPC was not produced in sufficient levels to char or that it co-migrated with an existing PL under the TLC conditions. Therefore, the total lipid extracts were analyzed for PPC by LC-MS/MS analysis by adapting the method used for PC. The sample grown with PCho showed a peak corresponding to PPC (Fig. S6B), while the sample grown without PCho did not (Fig. S6A). Both samples showed peaks for PE and PG (Fig. S7C), as expected. The retention time of PPC was shifted significantly from PC and overlapped with PG. Thus, the LC-MS/MS analyses of 11 species each for PE, PG, and PPC were not able to be combined into one LC run as had been done for PC.
Because AECho has the advantage that it reacts with fluorescent strained alkynes in a single step without the use of cytotoxic copper, AECho was also tested to determine whether it could be taken up by E. coli and incorporated into PC using Pcs. Following the same procedures used for PCho, the sample grown with AECho showed a LC-MS/MS peak corresponding to AEPC (Fig. 5B), while the sample grown without AECho did not (Fig. 5A). Both samples showed peaks for PE and PG ( Fig. S7A and S7B), as expected. As was seen for PPC, the AEPC retention time overlapped with PG and, thus, had to be analyzed independently from PE and PG. These results showed that E. coli was capable of taking up PCho and AECho and that Pcs was able to incorporate them to produce PPC and AEPC, respectively.

Confirmation of the reactivity of AEPC in lysate
After showing that Pcs is capable of incorporating AECho into AEPC, the reactivity of bacterial AEPC with a fluorescent alkyne reagent needed to be confirmed. To test this, BW25113 pcs was grown for 16 h with or without 500 μM Cho or 500 μM AECho followed by cell lysis. A green fluorescent spot corresponding to Alexa488-DIBO clicked to AEPC was only observed for the BW25113 pcs sample grown with AECho and treated with Alexa488-DIBO for 1 h (Fig. S8B,   sample 7). This spot ran slightly higher than the Alexa488-DIBO reagent itself (Fig. S8B, sample 2). All E. coli samples showed spots by charring for PE and PG (Fig. S8A, samples 3-7), while the samples grown with Cho also showed a spot for PC (Fig. S8A, samples 4  with the fluorescent click-reagent and, thus, that the azide moiety in the head group of AEPC can react with Alexa488-DIBO.

Fluorescent labeling of AEPC in E. coli lptD4213
After confirming that bacterial AEPC in E. coli lysate can react with a fluorescent strained alkyne, the last step was to test whether AEPC can be detected in live, intact E. coli cells from an isogenic pair: wild-type and a mutant that has increased PLs on the outer leaflet of the OM. E. coli lptD4213 is a well-characterized mutant, which is hyperpermeable to a range of compounds (15,56). This strain has a defect in LptD, the outer membrane component of the Lpt system, which is responsible for LPS transport from the outer leaflet of the IM to the outer leaflet of the OM (8). Due to impaired LptD function, the mutant has less LPS on the outer leaflet of the OM, and consequently more surface-exposed PLs compared to wild-type E. coli (21).
To evaluate AEPC labeling, lptD4213 pcs and its parent MC4100 pcs were grown for 16 h with or without 500 μM AECho and treated with or without the green fluorescent Alexa488-sDIBO. This commercially-available strained cyclooctyne, which replaced the discontinued Alexa488-DIBO, also reacts selectively with azides via copper-free click reaction, and allows fluorescent click-labeling with a single step. The cells were analyzed by bright-field and fluorescence microscopy. The AECho grown lptD4213 pcs strain, treated with Alexa488-sDIBO, was fluorescent (Fig. 6A6). The key control samples were not fluorescent: lptD4213 pcs treated only with Alexa488-sDIBO (Fig. 6A4) and the intact AECho-grown parent MC4100 pcs, treated with Alexa488-sDIBO (Fig. 6A2).
Fluorescent flow cytometry was used to quantitate the qualitative microscopy results. MC4100 pcs, lptD4213 without the pcs plasmid, and lptD4213 pcs (+/-rhamnose) were grown for 16 h with or without 500 μM AECho and treated with or without Alexa488-sDIBO. The cells were analyzed by flow cytometry for fluorescence. The lptD4213 pcs cells grown with AECho and rhamnose to induce Pcs production followed by the treatment with Alexa488-sDIBO were fluorescent, while the same strain treated only with Alexa488-sDIBO showed no significant fluorescence (Fig. 6B). No fluorescent labeling was detected in lptD4213 without the Pcs plasmid or in MC4100 pcs after rhamnose-induced Pcs expression, incubation with AECho and treatment with Alexa488-sDIBO (Fig. 6B). The AECho grown lptD4213 pcs (-rhamnose), by guest, on May 6, 2020 www.jlr.org Downloaded from treated with Alexa488-sDIBO, were fluorescent (Fig. 6B) despite the lack of Pcs induction, which suggested a leaky expression of Pcs. To confirm leaky expression of Pcs in the absence of rhamnose, MC4100 pcs and lptD4213 pcs were grown with and without rhamnose induction and Cho supplementation.
The total lipids were separated by TLC (Fig. S9). A spot corresponding to PC was detected for MC4100 pcs and lptD4213 pcs grown in the presence of Cho independent of rhamnose supplementation (Fig. S9, samples 4, 5, 8, and 9).
To ensure that the fluorescent flow cytometry signal observed was due to the fluorescent reagent reacting with AEPC, MC4100 pcs and lptD4213 pcs were grown for 16 h with or without 500 μM Cho or 500 μM AECho, followed by the treatment of all samples with the green fluorescent Alexa488-DIBO. The total lipids were isolated by Bligh-Dyer extraction and separated via TLC. Parallel TLC plates were visualized by charring (Fig. S10A) or green-fluorescence (Fig. S10B). A fluorescent spot corresponding to Alexa488-DIBO clicked to AEPC was only observed for the lptD4213 pcs sample grown with AECho ( Fig.   S10B, sample 6). All samples showed spots for PE and PG (Fig. S10A, samples 1-6), while the samples grown with Cho also showed a spot for PC (Fig. S10A, samples 2 and 4).
The results obtained by microscopy and flow cytometry suggest that the AEPC from the lptD4213 mutant can be detected after treatment with AECho and Alexa488-sDIBO. The TLC showed AEPC clicklabeled with Alexa488-DIBO, suggesting that the signal corresponds to AEPC crosslinked to the fluorescent click-reagent.

Evaluation of the Alexa-click labeled AEPC localization in plasmolyzed E. coli lptD4213
The green fluorescence observed for lptD4213 pcs should be localized on the OM if AEPC is externalized from the inner leaflet to the outer leaflet of the OM. However, to evaluate whether the click reagent also reaches the periplasm of the hyperpermeable lptD4213 pcs strain, the click-labeled lptD4213 pcs were imaged after osmotic shock treatment with high sucrose solution (plasmolysis). During plasmolysis, the cytoplasm shrank whereas the OM retained its shape, leading to an increased separation of the IM and OM ( Figure 7A, marked with arrows). The expanded periplasm facilitated the differential localization of fluorophores to the cytoplasm, IM, periplasm, and OM (57). In Alexa488-DIBO-labeled AECho grown by guest, on May 6, 2020 www.jlr.org Downloaded from lptD4213 pcs cells stained with the membrane dye FM 4-64, the green fluorescent Alexa488 ( Figure 7B) and the red fluorescent FM 4-64 ( Figure 7C and 7D) were co-localized to the OM. This is consistent with previous reports that FM 4-64 preferentially stains the OM and not the IM of intact wild-type E. coli (52,54).
While the OM showed consistent green fluorescent signal, there was green fluorescent signal from the IM in at least one cell (Fig. 7B), suggesting that the Alexa488-DIBO could permeate across the OM to reach the periplasmic face of the IM in the permeable lptD4213 pcs.

Dose-and time-dependence of AEPC labeling in E. coli lptD4213
For the development of a fluorescence-based assay for AEPC detection, it was important to determine how

Fluorescent labeling of AEPC in E. coli lpxC101
After showing that AECho can be used to detect AEPC in E. coli lptD4213 pcs, a second well-characterized isogenic strain pair was chosen to test whether AEPC could also be detected in the mutant, E. coli lpxC101.
This mutant has a defect in LPS biosynthesis instead of in LPS transport and is hyperpermeable to a range of compounds, similar to lptD4213 (13,14). The mutation is in lpxC, which encodes the enzyme responsible for the first committed step of the Raetz Pathway for lipid A biosynthesis (58). E. coli lpxC101 has reduced LPS on the outer leaflet of the OM and, therefore, increased surface-exposed PLs compared to wild-type E. coli (13,14).
To test whether lpxC101 shows an increased labeling of AEPC, lpxC101 pcs and its parent D21 pcs were grown for 16 h with or without 500 μM AECho and treated with or without the Alexa488-sDIBO.
The cells were analyzed by bright-field and fluorescence microscopy. lpxC101 pcs grown in the presence of AECho and treated with Alexa488-sDIBO were fluorescent (Fig. 9A6). The control samples, lpxC101 pcs grown without AECho but treated with Alexa488-sDIBO ( Fig 9A4) and the parent D21 pcs grown with AECho and treated with Alexa488-sDIBO (Fig. 9A2), showed no fluorescent labeling. These results are in agreement with the results obtained for lptD4213 pcs.
Fluorescent flow cytometry was used to quantitate the labeling. Two strains, lpxC101 without the Pcs plasmid and lpxC101 pcs (+/-rhamnose) were grown for 16 h with or without 500 μM AECho and treated with or without Alexa488-sDIBO. The cells were analyzed by flow cytometry for fluorescence. The lpxC101 pcs cells grown with AECho and rhamnose to induce Pcs expression followed by the treatment with Alexa488-sDIBO were fluorescent, while the same strain treated only with Alexa488-sDIBO showed no significant fluorescence (Fig. 9B). No fluorescent labeling was detected in lpxC101 without the Pcs plasmid or D21 pcs after rhamnose-induced Pcs expression, incubation with AECho and treatment with Alexa488-sDIBO (Fig. 9B). The lpxC101 pcs (Pcs production not induced) cells grown with AECho and treated with Alexa488-sDIBO were also fluorescent (Fig. 9B), as detected for lptD4213 pcs (see Fig. 6B), which again confirmed the leaky expression of Pcs (see also Fig. S9). The results obtained by microscopy and flow cytometry suggest that AEPC can be detected via metabolic labeling with AECho for the lpxC101 mutant, as seen in the lptD4213 mutant.

Discussion
Gram-negative OM asymmetry requires that LPS localizes exclusively on the outer leaflet and that PLs localize preferentially to the inner leaflet. This asymmetry is critical to maintain the OM permeability barrier. PLs are externalized in higher levels onto the outer leaflet in bacteria with genetic mutations in the LPS biosynthesis/transport pathways as well as bacteria treated with compounds inhibiting key enzymes in either of the two pathways (4,(14)(15)(16). Thus, an assay to detect PLs on the OM may hold promise to identify mutants with perturbed OM permeability barrier due to OM asymmetry defects as well as compounds that are able to interact with and/or enter the cell to compromise OM asymmetry (17,18).
To develop an assay to detect loss of bacterial OM asymmetry, we pursued metabolic labeling of PLs to enable a fluorescent PL detection assay with intact bacteria. Because the natural PLs in E. coli are not amenable to metabolic labeling, we explored metabolic labeling of PC by introducing Pcs into E. coli.
Results confirmed previous results that the Pcs enzyme from L. pneumophila produces PC in E. coli upon Cho supplementation, (42) and further demonstrated that PC is found in both membranes, the IM and OM.
We then showed for the first time that E. coli could take up the Cho analogs, PCho and AECho, and that bacterial Pcs could utilize these analogs to form PPC and AEPC, respectively. Most importantly, we showed that two E. coli mutants (lptD4213 and lpxC101) with defects in the LPS biosynthesis or transport pathway possessed AEPC that could be fluorescently-labeled, while the corresponding isogenic parent strains did not show significant fluorescent labeling.
Several important controls demonstrated that the fluorescent labeling of these mutants strains was due to covalent click labeling between Alexa488-DIBO and AEPC. First, there was no significant fluorescent labeling of the E. coli mutants (lptD4213 and lpxC101) without both Pcs expression and AECho supplementation, confirming that covalent click chemistry was required to fluorescently label the bacteria with Alexa488-DIBO. Second, the formation of the covalent click product AEPC-Alexa488-DIBO from intact cells was confirmed by TLC. Third, microscopy of plasmolyzed cells indicated that green fluorescent labeling was present on the OM, and to a lesser extent the IM, suggesting that fluorescent labeling derived primarily from click-labeling AEPC. Although Alexa488-DIBO does not readily permeate across the OM of E. coli K-12 under the conditions tested, it is possible that other bacterial species, mutants, or conditions might be more permeable to this reagent. In these cases, bulkier click-reagents such as DIBO-biotinstreptavidin (59) or DIBO-PEG4-bismannose-SS-biotin (60) could be evaluated.
Metabolic labeling and fluorescent detection of AEPC provides an efficient method to measure the loss of OM asymmetry in live, intact cells without the need for cell lysis or other purification steps. This methodology contrasts to the various existing methods used to directly or indirectly detect externalized PLs (9,(19)(20)(21)(22)(23)(24)(25)(26). Current methods are predominantly end-point assays that require cell lysis after labeling.
Furthermore, existing quantitative assays require some sort of extraction or purification step followed by TLC or liquid scintillation counting to determine indirectly the amount of externalized PLs. These methods are therefore highly labor-intensive and time-consuming.
While all of our data were obtained in E. coli pcs strains, it is important to point out that Cho is not an essential nutrient for E. coli growth. The only known E. coli metabolic pathway in which Cho is utilized is the osmoregulatory choline-glycine betaine pathway (61). Thus, supplementing E. coli with AECho should not have a major impact on choline metabolism, as long as the cells are not osmotically stressed. In order for Cho and its analogs to be incorporated, they need to enter the cytoplasm, where PL biosynthesis takes place. The absence of a transporter has been reported as problematic for the metabolic labeling of LPS with a 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) analog in some strains (31). Therefore, identification of the AECho transport mechanism(s) would allow determination of which bacteria might be able to uptake AECho and be metabolically labeled. Two Cho transport systems have been identified in E.
coli. BetT has been described as the high-affinity transport protein for Cho, while ProU is characterized as the low affinity uptake system (62). Preliminary results obtained with an E. coli betT mutant (63) suggest that at least PCho might be also transported by BetT (data not shown). However, further studies are required to evaluate the impact of BetT and ProU on the uptake of AECho to fully understand whether the absence of AECho uptake systems may limit the utility in other strains. In addition, because mM concentrations of AECho were necessary to see efficient PC labeling in lptD4213 pcs, the identification of the transport mechanism might reduce the concentration/time needed for optimal labeling, much as the identification of the Kdo uptake receptor NanT allowed enhanced LPS metabolic labeling with a Kdo analog (31). As PC is produced in 15% of all bacteria, including pathogens like P. aeruginosa or L. pneumophila (34), those strains that naturally express Pcs should be able to synthesize AEPC if AECho can be readily taken up by the cells. As such, strains with Pcs and naturally occurring PC may serve as even more efficient host strains for the metabolic labeling assay than E. coli.
While we envisioned PL metabolic labeling primarily as an assay to detect loss of OM asymmetry and phospholipid exposure, the labeling system could also have other uses. For example, natural PL transport between the IM and OM is critical to both provide PLs to the OM and to remove PLs from the outer leaflet of the OM. In E. coli and other organisms, the Mla system has been reported to actively transport PLs from the outer leaflet of the OM to the IM in order to prevent PL accumulation at the cell surface (9,17,18). Indeed, the mlaA strain has been reported to contain more surface-exposed PLs (9), which could be directly evaluated with our new assay. In addition, our assay may provide the opportunity to probe the internalization of fluorescent-AEPC from the outer leaflet of the OM to the IM.
Looking forward, the metabolic labeling of PC with AECho can be used in E. coli to assess mutant phenotypes, as seen in E. coli lptD4213 and lpxC101. Furthermore, this method could serve as a direct screen of AEPC accessibility for the identification of compounds which interfere with OM lipid asymmetry.
With further optimization, the fluorescent flow cytometry method should be amenable to being scaled-up to a medium-to high-throughput whole-cell screening assay for OM asymmetry, phospholipid externalization, and OM permeability defects.     Bacteria were untreated, treated with Alexa488-sDIBO only, or incubated with AECho followed by treatment with Alexa488-sDIBO. The cells were incubated overnight with 500 μM AECho. 0.2% Lrhamnose was used to induce Pcs production where indicated. Experiments were performed in biological triplicates. Error bars represent the standard deviation from the mean. The statistical significance was determined via two-.