METABOLIC LABELING OF BACTERIAL TEICHOIC ACIDS CELL WALL

Abstract

The disclosure provides a new method for the specific metabolic labeling of bacterial teichoic acids cell wall by modified choline and click chemistry, and its use in various applications such as bio-imaging, diagnostic, vaccination or bio-materials engineering.

Claims

1. A method of labeling a bacterium that is able to metabolize choline, said method comprising a step (i) of incubating the bacterium in a culture medium containing a modified choline which is metabolized by the bacterium and covalently associated to a teichoic acid (TA) in the cytoplasm before being exported and integrated into the cell wall of the bacterium.

2. The method according to claim 1, further comprising a step (ii) of contacting the bacterium with a tag molecule to generate a binding reaction between the modified choline bound to the teichoic acid (TA) present in the cell wall of the bacterium and the tag molecule.

3. The method according to claim 2, wherein the modified choline comprises at least one reactive group X allowing the binding of the modified choline to the tag molecule, said at least one reactive group X being selected from the reactive groups consisting of an alkene group, an alkyne group, an azide group, a cyclopropenyl group and a diazirine group.

4. The method according to claim 2, wherein the modified choline is selected from the group consisting of: Propargyl-choline, 1-azidoethyl-choline, Allyl-choline, cyclopropyl-choline, azidoethyl-diazinebutyl-choline.

5. The method according to claim 2, wherein the tag molecule is selected from the group consisting of a fluorescent molecule, a luminescent molecule, a radioactive molecule, a biotin molecule or a derivative thereof and an antigen molecule.

6. The method according to claim 2, wherein the tag molecule comprises at least one reactive group Y allowing its binding to the modified choline, said at least one reactive group Y being preferably selected from the group consisting of an alkene group, an alkyne group, an azide group, a cyclopropenyl group, a tetrazine group, a dibenzocyclooctyl (DBCO) group, a dibenzocyclooctine (DIBO) group, a bicyclononine (BCN) group, a Trans-Cyclooctene (TCO) group, a strained Trans-Cyclooctene (sTCO) group.

7. The method according to claim 2, wherein the tag molecule is selected from the group consisting of: 3-Azido-7-(diethylamino)-2H-chromen-2-one, and 7-nitro-N-(prop-2-yn-1-yl)benzo[c][1,2,5]oxadiazol-4-amine.

8. The method according to claim 2, wherein said steps (i) and (ii) are performed simultaneously.

9. The method according to claim 2, further comprising a step (iii) of detection and/or quantification of the bacterium by detecting and/or quantifying the tag molecule bound to the bacterium.

10. The method according to claim 1, wherein the modified choline is chemically modified to incorporate a radioactive isotope.

11. The method according to claim 1, wherein said bacterium is a Gram-positive bacterium or a Gram-negative bacterium.

12. An in vitro method of tracking a bacterium by bio-imagery comprising labeling the bacterium with a tag molecule according to the method of claim 1, thereby allowing the tracking of the bacterium.

13. An in vitro method for the diagnosis of a bacterial infection from a biological sample of a patient comprising labeling the bacterium responsible for the infection with a tag molecule according to the method of claim 1, thereby allowing the detection of the bacterium.

14. A method for the preparation of a vaccine composition containing a bacterium or fragments thereof, comprising labeling the bacterium in vitro with an antigen according to the method of claim 2, said antigen being bound to the bacterium or to the fragments thereof.

15. An in vitro method for the preparation of a bio-material comprising: a step of labeling a first population of bacteria with a first modified choline by using the labeling method of claim 2, a step of labeling a second population of bacteria with a second modified choline by using the labeling method of claim 2, the first modified choline used to label the first population and the second modified choline used to label the second population being respectively chosen to cross-react by Click chemistry between each other, followed by a step of binding the first population with the second population by click chemistry to form a bio-material.

16. An in vitro method of identifying an agent that inhibits the bacterial cell wall synthesis, said method comprising: a step of contacting a bacterium with a test agent, a step of labeling the bacterium using the method of claim 2, wherein a test agent that inhibits the labeling of the bacterium is considered a candidate agent for inhibiting the cell wall synthesis of the bacterium.

17. A kit to label a bacterium comprising: a modified choline that (i) comprises at least one reactive group X allowing the binding of the modified choline to the tag molecule, said at least one reactive group X being selected from the reactive groups consisting of an alkene group, an alkyne group, an azide group, a cyclopropenyl group and a diazirine group, or (2) is selected from the group consisting of Propargyl-choline, 1-azidoethyl-choline, Allyl-choline, cyclopropyl-choline, and azidoethyl-diazinebutyl-choline, a tag molecule that (i) is selected from the group consisting of a fluorescent molecule, a luminescent molecule, a radioactive molecule, a biotin molecule or a derivative thereof and an antigen molecule, (ii) comprises at least one reactive group Y allowing its binding to the modified choline, said at least one reactive group Y being preferably selected from the group consisting of an alkene group, an alkyne group, an azide group, a cyclopropenyl group, a tetrazine group, a dibenzocyclooctyl (DBCO) group, a dibenzocyclooctine (DIBO) group, a bicyclononine (BCN) group, a Trans-Cyclooctene (TCO) group, a strained Trans-Cyclooctene (sTCO) group, or (iii) is selected from the group consisting of 3-Azido-7-(diethylamino)-2H-chromen-2-one, and 7-nitro-N-(prop-2-yn-1-yl)benzo[c][1,2,5]oxadiazol-4-amine, and a culture medium allowing the growth of the bacterium.

18. The method according to claim 2, wherein said binding reaction is a Click chemistry reaction.

19. The method according to claim 11, wherein the bacterium is a Gram-positive bacterium selected from the Streptococcus genus, or wherein the bacterium is a Gram-negative bacterium selected from Haemophilus or Neisseria genera.

20. The method of according to claim 19, wherein the bacterium is a Gram-negative bacterium selected from S. pneumoniae, H. influenzae and Neisseria ssp.

Description

FIGURE LEGENDS

[0128] FIG. 1. Illustration of the structure of TA from S. pneumoniae (adapted from Gisch et al., J. Biol. Chem., 288:15654, 2013)

[0129] FIG. 2. Illustration of the Copper-dependent and Copper-independent Click chemistry pathways.

[0130] FIG. 3. Bacteria coated by alkyne or azide groups are labeled in one step to provide a fluorophore (Pathway A) or a radio-tracer (pathway B).

[0131] FIG. 4. Illustration of the use of the labeling method of the invention for diagnostic applications.

[0132] FIG. 5. Illustration of the use of the labeling method of the invention to coat bacteria cell surface with immune-stimulating epitopes.

[0133] FIG. 6. Illustration of the covalent interlocking of bacteria to form prokaryote tissues and new bio-materials.

[0134] FIG. 7. A. Metabolization of modified cholines (S. pneumoniae growth with 10 g/mL). B. Exogenous GFP-LytA displays similar binding pattern on cells grown in presence of propargyl-choline and of normal choline (fluorescent and phase contrast images are shown).

[0135] FIG. 8. NMR analysis of the propargyl-choline incorporation. Sections of .sup.31P NMR (.sub.P 2.5-(1.0)) and .sup.1H NMR (water suppressed; .sub.H 6.0-0.0)) spectra of PGN-WTA preparations after LytA treatment, isolated from S. pneumoniae grown in the presence of (i) normal choline (and performed bioorthogonal reaction with 3-azido-7-(diethylamino)-2H-chromen-2-one (2)), (ii) propargyl-choline and (iii) propargyl-choline (and performed bioorthogonal reaction with 2). Incorporation of propargyl-choline in WTA is proven by the occurrence of the additional signals in panels (ii) and (iii).

[0136] FIG. 9. Detection of metabolically incorporated propargyl-choline in S. pneumoniae fixed cells before bioorthogonal reaction with coumarin. Numbers (i) to (iii) refer to different stages of division. Phase contrast and fluorescence images in absence (A) or presence (B) of propargyl-choline are shown. Scale bar=1 m

[0137] FIG. 10. TA metabolic labeling on pneumococcal live cells. A. Detection of metabolically incorporated propargyl-choline in S. pneumoniae living cells after bioorthogonal reaction. (i) and (ii) refer to two different stages of division. Membrane and septal TA labeling are indicated by black and white arrow heads, respectively. Phase contrast and fluorescence images in the presence (left panel) or absence of propargyl-choline (right panel) are shown. Scale bar=1 m. B. Electron micrographs of pneumococcal cells at division stages (i) and (ii) as shown in A. C. Demograph of a pneucococcal cell population grown in the presence of propargyl-choline and submitted to the bioorthogonal reaction shows the mid-cell positioning of TA.

[0138] FIG. 11. TA metabolic labeling on live cells after 30-min pulse of propargyl-choline. A. Numbers (i) to (iii) refer to the different stages of division. Phase contrast and fluorescence images are shown. Scale bar=1 m. B. Demograph of pneucococcal cell population grown in the presence of propargyl-choline for 30 min and labeled with propargyl-choline-coumarin showing the mid-cell positioning of TA.

[0139] FIG. 12. Specific detection of metabolically incorporated propargyl-choline in live S. pneumoniae cells. Cultures of E. coli, B. subtilis and P. aeruginosa were performed as for S. pneumoniae in the presence (+propargyl-Cho) or in the absence (+Cho) of propargyl-choline. Cultures were mixed as indicated on the left hand side of the figure and processed for biorthogonal reaction.

[0140] FIG. 13. Short pulse. The bacterium is incubated for 15 seconds (left) or 60 seconds (right) with the the azide-choline, and then incubated for 5 minutes with DIBO-ATTOS 488.

[0141] FIG. 14. Two-step and direct labeling of TA and PG in S. pneumoniae, respectively. PG is depicted by the joined ellipses and TA by black bars. Purple arrows outline the metabolization of propargyl-choline from its import to its exposure at the cell surface attached to TA. The arrow describes the click reaction between the azido- and alkyne groups or the exchange of the distal D-Ala of the PG for FDAA catalyzed by PBPs.

[0142] FIG. 15. Growth curve of S. pneumoniae R800. In C-medium without choline (*), with 30 M azidocholine () or choline ().

[0143] FIG. 16. Labeling of TA of S. pneumoniae. Demographs show the fluorescence intensity along individual cells ordered by size. (a) Bacteria grown with 30 M azido-choline for 4 hours, incubated with 25 M DIBO Alexa Fluor 594 for 5 min prior to imaging. (b) Attempt at pulse labeling by incubation for 5 min with 30 M azido-choline and subsequent addition of 25 M DIBO reagent for 5 min. (c) Pulse labeling of TA by one-pot simultaneous addition of 30 M azido-choline and 25 M reagent for 5 min.

[0144] FIG. 17. (a) Cells were treated for 5 min with 30 M choline and 25 M DIBO Alexa Fluor 488 at 37 C. prior to imaging. (b) Azido-choline and DIBO Alexa Fluor 488 were incubated overnight together prior to incubation for 5 min with cells at 30 M and 25 M, respectively, in C-medium without choline.

[0145] FIG. 18. Scheme of the two-step one-pot labeling of pneumococcal TA. Azido-choline 1 and DIBO Alexa Fluor 2 are added simultaneously to growing cells. The different rates of the azido-choline metabolization and SPAAC reaction ensure that both reagents can be added together for adequate labeling in short pulses.

[0146] FIG. 19. Bio-orthogonal labeling of WTA and LTA of S. pneumoniae. Bacteria were grown in choline free C-medium supplemented with 30 M choline for 2 hours. TA were then labeled by incubation with 30 M azido-choline and 25 M DIBO Alexa Fluor 594 for 5 min. (a) Sacculi obtained from pulse-labeled cells by boiling for 45 min in 4% SDS. (b) Spheroplasts obtained from pulse-labeled cells by digesting cells with lysozyme, mutanolysin and LytA for 60 min at 37 C. and overnight incubation at 4 C.

[0147] FIG. 20. Geography of pneumococcal cell surface. Equators (E) and division sites (D) are co-localized at the onset of a new division. Parental hemispheres are in gray, daughter hemispheres are in white. The arrows indicate that the division sites are moving away from the duplicated equators.

[0148] FIG. 21. Pulse-chase labeling of TA in S. pneumoniae expressing fluorescent FtsZ-mKate. (a) TA were labeled by a 5 min pulse of 30 M azido-choline and 25 M DIBO Alexa Fluor 488 then chased by diluting in medium with 30 M choline. (b) Demographs showing the distribution of pulse-labeled TA and FtsZ localization.

[0149] FIG. 22. Fluorescent time lapse microscopy of pulse labeled TAs of S. pneumoniae on agarose pad. (a) Overlay of bright field and 5 min labeled-TA of wild type S. pneumoniae grown on agarose pad for 50 min. (b) Overlay of bright field, 5 min labeled-TAs and FtsZ of S. pneumoniae cells expressing FtsZ-mKate grown on agarose pad for 70 min.

[0150] FIG. 23. Comparison of the pulse labeling of TA and PG, which were labeled for 5 min with 30 M azido-choline, 25 M DIBO Alexa Fluor 594 and 500 M HADA (a). (b) Demographs showing the localization of newly inserted TA and PG. Fluorescence intensities of the longest cells are enlarged to emphasize the different patterns. (c) TA (upper picture), and PG labeling (lower picture) of a representative cell. The longitudinal axis is highlighted by a line. Outlines of the fluorescent intensity over the cell length are shown with the peak intensities used to calculate the ratio division site (D)/equators (E). (d) The ratio of fluorescence intensities at sites D/E as a function of cell length. (e) The ratio of fluorescence intensities at sites D/E of labeled TA vs that of PG.

[0151] FIG. 24. Pulse-chase labeling of TAs and PG. (a) TA and PG were labeled by a 5 min pulse of 30 M azido-choline, 25 M DIBO Alexa Fluor 488 and 500 M HADA in C-medium at 37 C. then chased by diluting in C-medium supplemented with 30 M of choline. Fluorescence images of labeled TA, PG and merged images with the bright field are shown. (b) Demographs showing the distribution of pulse-labeled TA and PG in the population during the chase.

[0152] FIG. 25. TA and PG labeling of S. pneumoniae mutant strains. TA and PG were labeled by a 5 min pulse of 30 M azido-choline, 25 M DIBO Alexa Fluor 488 and 500 M HADA at 37 C. (a) Fluorescence images of TAs, PGs and the merged images with bright field are shown. (b) Demographs showing the localization of newly incorporated TA and synthesized PG for each mutant strain.

EXAMPLES

Materials and Methods

Bacterial Growth Conditions

[0153] Liquid cultures of the unencapsulated pneumococcal strain R6 were grown at 37 C.-5% CO.sub.2 in a chemically defined medium (C-medium) supplemented with 4 g/ml choline (Cmed-choline). Contrary to the original composition, the C-medium used here did not contain neither yeast extract nor albumin. Cells were harvested by centrifugation at 3,320 g for 10 min, washed three times with C-medium without choline, concentrated to OD.sub.600 nm of 2 and stored at 80 C. as aliquots containing 15% glycerol (v/v).

[0154] For bioorthogonal reactions, 10 ml of Cmed-choline and 10 ml of C-medium containing 4 g/ml of propargyl-choline were inoculated at OD.sub.600 nm of 0.05 with aliquots of cells conditioned in C-medium as described above. The growth was pursued at 37 C.-5% CO.sub.2 for 3 hours until OD.sub.600 nm of 0.2-0.25 was reached, which corresponds to the early exponential growth phase. The cells were pelleted by centrifugation at 3,320 g for 10 min and subsequently incubated with 500 l of 2% choline chloride (w/v) for 10 min at room temperature (RT) to remove the Choline-Binding Proteins (CBPs) that bind to choline residues. In the case of choline-alkyl residues, the presence of CBPs possibly impair the labeling of those molecules by the fluorescent azide reporter. The cells were washed twice with PBS (1 min centrifugation at 4,500 g) and resuspended in 400 l of PBS. A volume of 100 l was used for each bioorthogonal reaction. In pulse experiments, cells were grown in Cmed-choline for 3 h, washed twice in PBS, resuspended in C-medium containing 4 g/ml of propargylcholine or 1-azidoethyl-choline and incubated at 37 C.-5% CO.sub.2 for 30 min before proceeding to the labeling.

[0155] Escherichia coli, Bacillus subtilis and P. aeruginosa growth conditions in C-medium supplemented with both forms of choline were tested before conducting the click reactions with the same protocol as the one developed for Streptococcus pneumoniae.

Copper Catalyzed Click Chemistry

[0156] Labeling was performed on cells grown in presence of choline and choline-alkyl. A volume of 100 l of cell suspension prepared as described above was incubated with the following reagents, which final concentration is indicated: coumarin (1 mM), ascorbic acid (1 mM), Copper (II) sulfate (50 M), THPTA (tris(3-hydroxypropyltriazolylmethyl)amine) (300 M) for 30 min at RT, under mild agitation and protected from the light. Labeled cells were washed twice with PBS and resuspended in PBS for microscopy observation.

[0157] Cell fixation was performed after culture harvest. Cells from 10 ml culture were washed twice with PBS, resuspended in 500 l of 4% (w/v) paraformaldehyde for 30 min at RT followed by a 2 h-incubation at 4 C. After two washes with PBS, cells were resuspended in 400 l of PBS and aliquots of 100 l were used for the click reaction by adding the coumarin (1 mM), ascorbic acid (1 mM) and Copper (II) sulfate (100 M). The labeling proceeded for 16 h at RT under mild agitation and protected from the light. Labeled cells were washed twice with PBS and resuspended in PBS before microscopy observation.

Fluorescence Microscopy and Image Analysis

[0158] Pneumococcal cells were transferred to microscope slides and observed using an Olympus BX61 optical microscope equipped with a UPFLN 100 O-2PH/1.3 objective and a QImaging Retiga-SRV 1394 cooled charge-coupled device camera. Image acquisition and analysis were performed using the software packages Volocity and open-source Oufti, respectively and processed with Adobe Photoshop CS5. Cell population demographs were constructed by Oufti which integrates the signal values in each cell. The cells are then sorted by their length value and the fluorescence values are plotted as a heat map.

Extraction and Isolation of LTA

[0159] Pneumococcal cells were resuspended in citrate buffer (50 mM, pH 4.7) and disrupted three times by French press (Constant Cell Disruption System, Serial No. 1020) at 10 C. at a pressure of 20 kPSI. SDS was added to a final concentration of 4% to the combined supernatants. The solution was incubated for 30 min at 100 C. and was stirred afterwards overnight at room temperature. The solution was centrifuged at 30,000g for 15 min at 4 C. The pellet was washed four times with citrate buffer using the centrifugation conditions as above. The combined LTA-containing supernatants and the resulting sediment, containing the crude PGN-WTA complex, were lyophilized separately. The resulting solids were both washed five times with ethanol (centrifugation: 20 min, 20 C., 10,650g) to remove SDS and lyophilized (leading to pellet A containing LTA and pellet B containing the PGN-WTA complex). For LTA isolation, pellet A was resuspended in citrate buffer and extracted with an equal volume of butan-1-ol (Merck) at room temperature under vigorous stirring. The phases were separated by centrifugation at 4,000g for 15 min at 4 C. The aqueous phase (containing LTA) was collected, and the extraction procedure was repeated twice with the organic phase plus interphase. The combined aqueous phases were lyophilized and subsequently dialyzed for 5 days at 4 C. against 50 mM ammonium acetate buffer (pH 4.7; 3.5 kDa cut-off membrane); the buffer was changed every 24 h. The resulting crude LTA was purified further by hydrophobic interaction chromatography (HIC) performed on a HiPrep Octyl-Sepharose column (GE Healthcare; 16100 mm, bed volume 20 ml). The crude LTA material was dissolved in as little starting buffer (15% propan-1-ol (Roth) in 0.1 M ammonium acetate (pH 4.7)) as possible and centrifuged at 13,000g for 5 min at room temperature and the resulting supernatant was lyophilized. The LTA-containing pellet was dissolved in the HIC start buffer at a concentration of 30 mg/ml and purified by HIC using a linear gradient from 15% to 60% propan-1-ol (Roth) in 0.1 M ammonium acetate (pH 4.7). LTA-containing fractions were identified by a photometric phosphate test. The phosphate-containing fractions were combined, lyophilized and washed with water upon freeze-drying to remove residual buffer.

Extraction and Isolation of WTA

[0160] Pellet B (containing the crude PGN-WTA complex), which arose during LTA isolation, was resuspended at a concentration of 10 mg/ml in 100 mM Tris-HCl (pH 7.5) containing 20 mM MgSO.sub.4. DNase A and RNase I were added to final concentrations of 10 and 50 g/ml, respectively. The suspension was stirred for 2 h at 37 C. Subsequently, 10 mM CaCl.sub.2 and trypsin (100 g/ml) were added and the stirring was continued overnight at 37 C. SDS at a final concentration of 1% was added, and the mixture was incubated for 15 min at 80 C. to inactivate the enzymes. The cell wall was recovered by centrifugation for 45 min at 130,000g at 37 C. The resulting pellet was resuspended in 0.8 ml 8 M LiCl per 1 ml initially used Tris-HCl solution and incubated for 15 min at 37 C. After another centrifugation using the same conditions as above, the pellet was resuspended in 1 ml 10 mM ethylenediaminetetraacetic acid (EDTA, pH 7.0) per ml of the Tris-HCl solution used initially and this sample was incubated at 37 C. for 15 min. The pellet was washed twice with water. Finally, the pellet was resuspended in 2 to 4 ml of water and lyophilized, yielding the purified PGN-WTA complex. To remove all amino acids from the PGN, the PGN-WTA complex was dissolved in 50 mM Tris-HCl (pH 7.0; 10 mg/ml) and treated with the pneumococcal LytA amidase. Recombinant His-tagged LytA amidase (1 mg/10 g LytA) was added in three aliquots after 0, 24 and 48 h for a total period of incubation of 72 h at 37 C. Subsequently, the enzyme was inactivated by boiling for 5 min at 100 C. After centrifugation (25,000g, 15 min, 20 C.) the supernatant was collected and lyophilized. The crude LytA-treated PGN-WTA complex was further purified by GPC on a Bio-Gel P-30 (45-90 m, BioRad; column size: 1.5120 cm; buffer: 150 mM ammonium acetate (pH 4.7)) column.

Example 1. Metabolic Incorporation of Modified Choline

[0161] The choline dependency for pneumococcal growth was exploited to validate the metabolic incorporation of modified cholines. Propargyl-choline was evaluated as well as its corresponding fluorescent analogue (i.e. propargyl-choline-coumarin) obtained by the click reaction between propargyl-choline and coumarin. One interesting aspect of the coumarin comes from its fluorogenic property once coupled with alkyne that amplifies the fluorescence signal to reduce the background interference. Nonpathogenic (unencapsulated) pneumococcal R6 strain was cultured in C-medium containing propargyl-choline-coumarin or normal choline. Comparable growth rates were observed in the presence of normal choline and of propargyl-choline indicating a good metabolisation of the latter compound (FIG. 7).

Example 2. Analytical Characterization and Quantification of TA Decorated by Modified Choline

[0162] Pneumococcal cultures grown in presence of propargyl-choline or normal choline were processed to extract LTA. NMR analysis showed that propargyl-choline has been integrated in LTA (FIG. 8).

Example 3. Detection of Metabolically Incorporated Modified Choline

[0163] To prevent any modification of the structure of TA and/or their eventual re-localization at the cell surface during the chemical labeling, a preliminary study was performed with cell containing propargyl-choline fixed prior the bioorthogonal reaction. Fluorescence was specifically detected on cells grown in the presence of propargyl-choline when compared with cells grown in the presence of normal choline (FIG. 9). Bright fluorescent spots were observed at the mid-cell position in early (panels i and ii) and late division stages (panel iii).

Example 4. Metabolic Labeling on Pneumococcal Live Cells

[0164] To obtain information on the TA biosynthesis dynamics, labeling of live pneumococcal cells have been performed after optimizing the reactional conditions. Reduction of copper concentration to 50 M, addition of the catalyst THPTA and reducting the incubation time to 30 min allowed to specifically label TA (FIG. 10A). The fluorescence intensity displayed by pneumococcal cells grown in presence of propargyl-choline was measured and compared to the signal detected on cells grown with choline in five independent experiments (n=155 to 2445 in each experiment and for each culture condition). The fluorescent signal ratio propargyl-choline/choline was 4.280.8.

[0165] Fluorescence detected at the cell periphery and at the contact zone between daughter cells before they separate suggest a membrane localization of TA/LTA (FIG. 10A, stage (i), black arrow head). An electron micrograph of a pneumococcal cell of similar morphology is shown to appreciate in details the membrane topology (FIG. 10B, stage (i), black arrow head). Early-division stage (ii) is characterized by the on-set of cross-wall synthesis and membrane invagination (FIGS. 10A and 10B, white arrow heads). TA labeling is observed at the septal site in these cells indicating that TA are synthesized and/or flipped across the membrane at the same time and the same place as peptidoglycan synthesis. Image analysis of a cell population confirms the septal localization of the TA labeling on pneumococcal cells over the cell cycle (FIG. 10C): after integration of the fluorescence signal in each cell, cells were sorted by their length value and the fluorescence values was plotted as a heat map.

[0166] A pulse of propargyl-choline was performed. Pneumococcal cells were grown in medium containing choline, washed, incubated in presence of propargyl-choline for 30 min and submitted to the bioorthogonal reaction (FIG. 11). In these conditions, only TA synthesized during the 30 min pulse are labeled. Reduced membrane labeling (when compared to the 3 h culture period shown in FIG. 8) together with the septal site localization (FIGS. 11A and 11B) confirm that TA synthesis takes place in a relatively short time scale.

[0167] No fluorescent signal was detected when the bioorthogonal reaction was performed on Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa species, respectively Gram-negative and Gram-positive bacteria that do not metabolize choline (FIG. 12). This work is the first report of metabolic labeling of Gram-positive TA. These results demonstrate the specificity and selectivity of the labeling based on click chemistry. Long and short labeling pulses with propargyl-choline label TA in live cells at the septal site show that TA synthesis might be correlated to the peptidoglycan synthesis and the cell division. The success of this method offers possibility to explore mechanistic issues of pneumococcal TA biosynthesis in a more physiological context.

Example 5. High-Speed Labeling of S. pneumoniae

[0168] A quick incubation of 15 seconds with the 1-azidoethyl-choline, followed by the addition of DIBO-ATTOS 488 for 5 min, is enough to obtain a high-quality labeling (FIG. 13).

Example 6. One-Pot Two-Step Metabolic Labeling of S. pneumoniae

[0169] To monitor PG assembly, a recent method relies on the ability of bacteria to incorporate fluorescent D-amino acids (FDAA) into the growing PG (FIG. 14, direct labeling of PG). This labeling is thought to be catalyzed by the penicillin-binding proteins (PBPs) that normally catalyze the PG cross-linking. FDAAs are incorporated in a few minutes allowing labeling pulses that are significantly shorter than the bacterial generation time.

[0170] A comparable fast labeling method of TA was lacking. As shown in Example 4, the incorporation of choline-derivatives in TA of S. pneumoniae can be used to metabolically label TA in this organism (FIG. 14, two-step labeling of TA). However, this two-step approach is suited to uniformly label TA at the cell surface but is not optimal to pulse-chase experiments, as the Cu-catalyzed click reaction may be too slow relatively to the bacterial growth rate.

[0171] To study the relationship between the insertion of PG and TA in the cell wall, it is necessary to visualize both polymers with markers that can be incorporated at similar rates. Thus, to circumvent the lack of direct labeling method for TA, the Inventors have developed a choline-based two-step metabolic labeling of TA that allows pulse-chase experiments comparable to the direct method of PG labeling. This approach combines the rapid kinetics of a strain promoted azide alkyne cycloaddition (SPAAC) with the speed of a biological process.

[0172] The choice was oriented towards a rapid click reaction involving an azide function carried by the choline and the strained dibenzoannulated cyclooctyne DIBO carrying the fluorophore. Azido-choline efficiently replaced choline to allow growth of pneumococcus (FIG. 15). For subsequent click reaction, fluorescent DIBO offers a compromise between stability and rapidity with a choice of commercially available fluorophores. Uniform cell surface labeling was obtained when cells were grown in the presence of azido-choline for 4 hours prior to fast bio-orthogonal labeling with Alexa Fluor DIBO in 5 min (FIG. 16a).

[0173] Pulse-chase experiments have been performed using the two-steps approach. After growing cells in rich medium, washing cells to remove choline, the labeling pulse was carried out by adding successively the azido-choline alone and 5 min later the DIBO probe. The chase was initiated by dilution in rich medium. Cells could be adequately imaged at various chase times, but the method was not appropriate to document labeling at the pulse time (t=0). As cells continue to grow and build cell wall during the first step of the labeling process, adding the DIBO reagent even immediately after the azido-choline pulse, produced chase-like patterns with segregated bands of labeling (FIG. 16b).

[0174] Then, it was attempted to add both the metabolic and fluorescent labeling agents at the same time. This one-pot approach allowed proper recording of cell wall expansion and TA insertion during the 5 min pulse time (FIG. 16c). No labeling and negligible background fluorescence were obtained with unmodified choline (FIG. 17). Pre-incubation together of the clickable reagents also abolished labeling, likely because the resulting fluorescent choline cannot be imported and/or metabolized (FIG. 17). Therefore, when both azido and DIBO reagents were added together during the labeling pulse, the observed signal must result from the rapid metabolization and presentation of azido-choline at the cell surface followed by its reaction with the DIBO-fluorophores (FIG. 18). At the reagents concentrations used, the click reaction in solution was slow enough to allow import of unreacted free azido-choline by the bacteria, while fast enough to modify TA-exposed azido-choline during the pulse time.

[0175] When one-pot pulse-labeled cells were treated either to remove the membrane or the cell wall, site-defined labeling of the sacculi was maintained, whereas labeling of the spheroplasts membrane was uniform, respectively (FIG. 19). As expected WTA attached to the PG are not mobile, while LTAs can diffuse laterally in the membrane, at least in the absence of cell wall.

[0176] The one-pot two-step method was then applied in pulse-chase experiments to determine the localization and timing of TA insertion with respect to the cell cycle. A scheme of the cell cycle with a nomenclature of the main structural features is given in FIG. 20. A pulse-chase experiment was performed in a strain expressing a fluorescent FtsZ to serve as a marker of the cell division. During the 5 min-pulse, the new TA were mostly co-localized at the division site with the FtsZ-ring (FIG. 21a, t=0). However, in cells in late stages of division, FtsZ was not detected at the old division site, while new TA were still incorporated there. Indeed, the demographs show that FtsZ is re-localized to the equators of the daughter cells earlier than is the insertion of new TA (FIG. 21b, t=0). The localization of FtsZ was that at the time of the observation, whereas the localization of the labeled TA was that of TA incorporated during the 5 min-pulse. Although cells were imaged right after labeling and washing, it cannot be excluded that the difference observed between FtsZ and the new TA is due to this short delay. When imaged after a chase of 5 min, the TA labeling was observed as having spread or split and segregated on each side of the division site (FIG. 21a, t=5 min). The latter pattern is exemplified by the presence of FtsZ, at the time of imaging between parted bands of TA labeled during the pulse. Once the cell division is complete, as observed after 20 and 40 min (FIG. 21, t=20 and 40 min), the segregated bands of labeled TA remain at the same distance of each other if cells are forming chains. If cells have separated after their division, the TA labeling is found on free hemispheres, as shown in the demographs on one side of many cells. This behavior was also observed by time-lapse microscopy (FIG. 22).

[0177] The one-pot approach was the applied in conjunction with direct labeling with FDAA to directly compare TA insertion and PG synthesis. Dual pulse-chase labeling experiments were performed to compare the localization and timing of both processes (FIG. 23a; FIG. 24). Overall, surface presentation of new TA that were bio-orthogonally labeled was concomitant with the activity of PBPs responsible for the PG labeling. The one-pot metabolic method for labeling TA is therefore as fast as the direct method of PG labeling with FDAA. However, a difference between the two labeling is noticeable immediately after of the pulse time (t=0): simultaneous TA-labeling at closing division sites (D) or pole tips and at equators (E) is observed more often than simultaneous PG-labeling at those two sites (FIG. 23b). To quantify this observation, the fluorescence data for cells (di-cocci) that were longer than 2.1 m was analyzed by measuring the ratio of signal at the old division site (D) over that at the equators (E, future division sites). When this ratio was plotted against the cell length, it appeared that the fluorescence at the old division site due to TA-labeling was more important than that due to PG labeling. This difference diminished as cells became longer and approached cell separation (FIGS. 23c and 23d). When the D/E PG-labeling ratio was plotted against the D/E TA-labeling ratio, the data could be fitted to a straight line of slope 2.00.1 (standard error) that was significantly different from the value of 1 expected without difference between the distribution of both labeling (FIG. 23e), indicating that insertion of TA persists at the division site after PG synthesis has ceased.

[0178] Numerous mutations are known to affect the cell wall or the morphology. A simultaneous pulse-labeling of TA and PG in various mutant strains was performed to determine whether the tight coordination of these processes was affected (FIG. 25). The reasons underlying the choice of mutations are discussed in the supplementary material. The individual deletion of pgdA, adr, psr, cbpE, mapZ, gpsB, or the depletion of pbp2x and pbp2b, did not affect the co-localization of TA insertion and PG assembly.

[0179] Many bacteria in laboratory cultures have generation times shorter than an hour. To study the dynamics of molecular events at the cell surface with pulse-chase experiments therefore requires short labeling pulses. It has been found here that if metabolization of the azido-compound (first step) is significantly faster than the SPAAC reaction (second step), both reagents can be added simultaneously, making the method comparable to direct metabolic approaches.

[0180] The one-pot method is compatible with other labeling techniques and co-labeling confirmed that TA insertion and PG assembly are largely overlapping, but careful quantification revealed that incorporation of TA persists at the division site later than PG assembly.

[0181] Further insights in bacterial cell wall biology will arise by improving the spatial resolution of imaging, which is certainly possible since our approach can be used in principle with SPAAC-linked fluorophores compatible with direct stochastical optical reconstruction microscopy (dSTORM). The size of the functions tolerated on modified nutriment is generally small and is therefore a limiting factor of direct metabolic labeling methods, while dSTORM fluorophores are generally large. The new one-pot method provides a simple alternative to the problem of label size applied to the study of rapid metabolic pathways in bacteria and other organisms.