Development of a novel target-based cell assay, reporter of the activity of Mycobacterium tuberculosis protein-O-mannosyltransferase

The Protein-O-mannosyltransferase is crucial for the virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This enzyme, called MtPMT (Rv1002c), is responsible for the posttranslational O-mannosylation of mycobacterial proteins. It catalyzes the transfer of a single mannose residue from a polyprenol phospho-mannosyl lipidic donor to the hydroxyl groups of selected Ser/Thr residues in acceptor proteins during their translocation across the membrane. Previously, we provided evidence that the loss of MtPMT activity causes the absence of mannoproteins in Mycobacterium tuberculosis, severely impacting its intracellular growth, as well as a strong attenuation of its pathogenicity in immunocompromised mice. Therefore, it is of interest to develop specific inhibitors of this enzyme to better understand mycobacterial infectious diseases. Here we report the development of a "target-based" phenotypic assay for this enzyme, assessing its Omannosyltransferase activity in bacteria, in the non-pathogenic Mycobacterium smegmatis strain.

Robustness of the quantitative contribution of this assay was evaluated by intact protein mass spectrometry, using a panel of control strains, overexpressing the MtPMT gene, carrying different key point-mutations. Then, screening of a limited library of 30 compounds rationally chosen allowed us to identify 2 compounds containing pyrrole analogous rings, as significant inhibitors of MtPMT activity, affecting neither the growth of the mycobacterium nor its secretion of mannoproteins. These molecular cores could therefore serve as scaffold for the design of new pharmaceutical agents that could improve treatment of mycobacterial diseases. We report here the implementation of a miniaturized phenotypic activity assay for a glycosyltransferase of the C superfamily.

INTRODUCTION

Mycobacterium tuberculosis (Mtb) remains the world's deadliest bacterial pathogen in 2020, with nearly 10 million newly infected people and 1.5 million deaths due to tuberculosis (TB), including 214,000 HIV-positive people (WHO, 2021). Moreover, contrasting with the steady decline of the disease burden resulting from decades of global efforts and commitments, we are currently witnessing an alarming annual 10% increase in the number of cases of antimicrobial-resistant (AMR) TB. This threat unveils the risk of shortage in our effective anti-tuberculosis therapeutic arsenal and urges the research and development for new treatments to prevent future out-of-control epidemics of drugresistant TB. A reason for this setback is the strong selection pressure maintained by protracted and burdensome anti-tuberculosis treatments targeting essential physiological processes and hence promoting the emergence of AMR bugs. Therefore, several "non-traditional" antimicrobial therapeutic strategies are currently explored to circumvent this drawback associated with classical antibiotics, including the very appealing anti-virulence approach that aims at targeting pathways not essential in in vitro culture but indispensable for host colonization and/or infection [START_REF] Clatworthy | Targeting virulence: a new paradigm for antimicrobial therapy[END_REF][START_REF] Theuretzbacher | Non-traditional Antibacterial Therapeutic Options and Challenges[END_REF][START_REF] Rex | Designing development programs for non-traditional antibacterial agents[END_REF][START_REF] Gigante | Recent advances and challenges in antibacterial drug development[END_REF].

In this regard, we previously reported that secreted O-mannosylated proteins are not essential for Mtb growth in laboratory conditions, but crucial for adhesion to host cells, intracellular survival and Mtb virulence in a rodent animal model [START_REF] Ragas | The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A[END_REF][START_REF] Liu | Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis[END_REF]. To date, more than sixty Omannosylated proteins secreted by Mtb have been identified from standard laboratory cultures, many of which are important effectors contributing to the bacilli defenses against the host immune response [START_REF] Tonini | Potential Plasticity of the Mannoprotein Repertoire Associated to Mycobacterium tuberculosis Virulence Unveiled by Mass Spectrometry-Based Glycoproteomics[END_REF]. These mannoproteins (such as Apa/Rv1860, MPB/T83/Rv2873, LpqH/Rv3763, SodC/Rv0432 or PstS-1/Rv0934) can modulate the inflammatory response, in particular by interacting with lectin receptors on macrophages, allowing bacteria to enter and survive longer in these cells [START_REF] Pitarque | Deciphering the molecular bases of Mycobacterium tuberculosis binding to the lectin DC-SIGN reveals an underestimated complexity[END_REF][START_REF] Ragas | The Mycobacterium tuberculosis cell-surface glycoprotein apa as a potential adhesin to colonize target cells via the innate immune system pulmonary C-type lectin surfactant protein A[END_REF][START_REF] Schnappinger | Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment[END_REF][START_REF] Stewart | Effect of deletion or overexpression of the 19-kilodalton lipoprotein Rv3763 on the innate response to Mycobacterium tuberculosis[END_REF][START_REF] Sartain | N-Terminal clustering of the O-glycosylation sites in the Mycobacterium tuberculosis lipoprotein SodC[END_REF][START_REF] Esparza | PstS-1, the 38-kDa Mycobacterium tuberculosis glycoprotein, is an adhesin, which binds the macrophage mannose receptor and promotes phagocytosis[END_REF][START_REF] Torrelles | Diversity in Mycobacterium tuberculosis mannosylated cell wall determinants impacts adaptation to the host[END_REF].

Although the relevance of the mannoprotein mannosyl appendages has never been clearly and definitively confirmed as decisive for the biological activity of any of these glycosylated proteins [START_REF] Alonso | Protein O-mannosylation deficiency increases LprG-associated lipoarabinomannan release by Mycobacterium tuberculosis and enhances the TLR2-associated inflammatory response[END_REF], as a whole, the mannoproteome meets the definition of non-essential virulence factor by contributing to the infectiousness and pathogenicity of the bacilli.

To date, protein-O-mannosylation is the only protein glycosyl post-translational modification demonstrated formally in Mtb and is initiated by the Protein-O-Mannosyltransferase MtPMT encoded by the Rv1002c gene. This enzyme catalyzes the transfer of single mannose units from polyprenol phospho-mannosyl lipidic donors to the hydroxyl groups of selected threonines or serines (S/T) of acceptor proteins during their translocation across the membrane. These initial single mannosyl entries are then generally progressively elongated through successive addition of mannosyl units by the (α12) mannosyl transferase PimE (Rv1159) [START_REF] Liu | Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis[END_REF] to form linear mannooligosaccharides containing up to 8 residues [START_REF] Parra | Scrutiny of Mycobacterium tuberculosis 19 kDa antigen proteoforms provides new insights in the lipoglycoprotein biogenesis paradigm[END_REF] (Fig. 1A, B andC). This process generates substantial repertoires of proteoglycoforms differing by the total number of mannosyl residues but also the length and position of the different mannooligosaccharides, as previously illustrated by the mass spectrometry analysis of the P27-51 mannosylated peptide from LpqH (Fig. 1C) [START_REF] Parra | Scrutiny of Mycobacterium tuberculosis 19 kDa antigen proteoforms provides new insights in the lipoglycoprotein biogenesis paradigm[END_REF]. Since these different proteoglycoforms may differ in their biological activity, the whole blockade of the protein-O-mannosylation process by chemical inhibition of the sole protein mannosylating enzyme MtPMT would constitute a valuable non-traditional alternative to abrogate Mtb virulence in-vivo [START_REF] Van Els | Toward Understanding the Essence of Post-Translational Modifications for the Mycobacterium tuberculosis Immunoproteome[END_REF][START_REF] Mehaffy | Mycobacteria and their sweet proteins: An overview of protein glycosylation and lipoglycosylation in M. tuberculosis[END_REF]. PMTs are polytopic membrane proteins belonging to the GT-C glycosyl transferase family to which also belongs the mycobacterial arabinosyl transferase EmbA, target of the first line antituberculous drug ethambutol. However, unlike EmbA, there is no known specific inhibitor for MtPMT. Moreover, the only PMT inhibitor reported to date is selective of the eukaryotic PMT-1 isotype family [START_REF] Orchard | Rhodanine-3-acetic acid derivatives as inhibitors of fungal protein mannosyl transferase 1 (PMT1)[END_REF]. In addition, the complete lack of knowledge concerning the mode of action of this compound, as well as the limited knowledge of the GT-C family enzyme, preclude any attempt for a rational discovery of potential MtPMT inhibitor candidates from this initial lead. As an alternative, high-throughput screening (HTS) remains the method of choice to discover potential MtPMT inhibitors without a priori. However, this blind approach often remains challenging, particularly for glycosyltransferases such as PMTs that are not easily amenable to HTS approaches [START_REF] Chao | High-Throughput Approaches in Carbohydrate-Active Enzymology: Glycosidase and Glycosyl Transferase Inhibitors, Evolution, and Discovery[END_REF]. Indeed, a major obstacle is the difficulty to read out the transfer of mannose residues on target proteins. This has been assessed by different approaches, including monitoring the transfer of radiolabeled mannose onto an acceptor peptide [START_REF] Schultz | The role of mannosylphosphorylpolyisoprenol in glycoprotein biosynthesis in Mycobacterium smegmatis[END_REF][START_REF] Cooper | Characterization of mycobacterial protein glycosyltransferase activity using synthetic peptide acceptors in a cell-free assay[END_REF], detecting protein glycosylation by western blotting using mannose binding concanavalin A lectin (ConA) [START_REF] Garbe | Expression of the Mycobacterium tuberculosis 19-kilodalton antigen in Mycobacterium smegmatis: immunological analysis and evidence of glycosylation[END_REF], assessing mannosylation-induced protein apparent-mass-increase by gel electrophoretic mobility delay [START_REF] Girrbach | Structure-function analysis of the dolichyl phosphate-mannose: protein O-mannosyltransferase ScPmt1p[END_REF], or characterizing peptide glycosylation by mass spectrometry [START_REF] Dobos | Evidence for glycosylation sites on the 45-kilodalton glycoprotein of Mycobacterium tuberculosis[END_REF][START_REF] Vanderven | Export-mediated assembly of mycobacterial glycoproteins parallels eukaryotic pathways[END_REF][START_REF] Smith | O-linked glycosylation sites profiling in Mycobacterium tuberculosis culture filtrate proteins[END_REF][START_REF] Tonini | Potential Plasticity of the Mannoprotein Repertoire Associated to Mycobacterium tuberculosis Virulence Unveiled by Mass Spectrometry-Based Glycoproteomics[END_REF]. Although efficient, most of these approaches are poorly quantitative and rather cumbersome due to multiple sample handlings that are poorly compatible with HTS robotic platforms.

The second major hurdle comes from the membrane-embedded intrinsic nature of MtPMT, that makes the mannose transfer activity depend on a complex lipidic surrounding. Indeed, this environment ensures the spatial colocalization of the MtPMT polytopic enzyme with both the mannose donor embedded in the lipid bilayer, and the acceptor polypeptide chain being translocated into the periplasm by the SecYEG translocon [START_REF] Loibl | Protein Omannosyltransferases associate with the translocon to modify translocating polypeptide chains[END_REF]. The difficulty to reconstitute to functionality of such a system from its isolated components or to control the relative concentrations of its different constituents within a bacterial crude lysate or membrane extract, strongly hampers the development of a robust cell-free assay amenable to high throughput [START_REF] Cooper | Characterization of mycobacterial protein glycosyltransferase activity using synthetic peptide acceptors in a cell-free assay[END_REF]. These shortcomings are generally circumvented by cell-based inhibitor screening assays, which in addition allow assessing transport and bioavailability of the candidate effector, limiting the severe attrition typically seen with candidates issued from cell-free assays [START_REF] Clatworthy | Targeting virulence: a new paradigm for antimicrobial therapy[END_REF][START_REF] Gigante | Recent advances and challenges in antibacterial drug development[END_REF]. However, these so-called "phenotypic" approaches are typically non-target specific, and the growth-inhibitory activity (live-dead bacteria) on which they are commonly based does not apply to MtPMT, as by definition it is non-essential and its arrest does not affect growth or kill Mtb under laboratory conditions [START_REF] Theuretzbacher | Non-traditional Antibacterial Therapeutic Options and Challenges[END_REF].

Nonetheless, in view of the potential therapeutic relevance of the pleiotropic interruption of protein-O-mannosylation, we focused our efforts on the development of a down-sizable MtPMT activity assay, compatible with medium to high throughput screening of chemicals able to inhibit in cellulo the mannosylation by MtPMT of mycobacterial-secreted proteins.

With this aim, and to circumvent the stringent and burdensome cultivation conditions of Mtb, MtPMT was expressed ectopically in the nonpathogenic fast-growing Mycobacterium smegmatis (M. smegmatis) invalidated for its own MsPMT. The resulting recombinant strain was modified further to over-express the poly-histidine tagged secreted protein Fasciclin (FasC His ) as a substrate acceptor of MtPMT. The biological function of the fasciclin domains present in some mycobacterial proteins (FasC from M. smegmatis ; MPT83/Rv2873 and MPT70/Rv2871 from Mtb) remains unclear. However, on the basis of structural homology with human fasciclin, it has been proposed that these domains are involved in cell adhesion [START_REF] Carr | Solution structure of the Mycobacterium tuberculosis complex protein MPB70: from tuberculosis pathogenesis to inherited human corneal desease[END_REF][START_REF] Wiker | MPB70 and MPB83--major antigens of Mycobacterium bovis[END_REF]. Functionality and workability of the reporter strain were assessed by conventional characterization of mannosylation of the secreted FasC His and the level of ConA binding to the affinity-purified FasC His was validated as a surrogate value for quantification of the MtPMT activity. Reporter strain cultivation, FasC purification and ConA binding quantification steps were further optimized and downsized to be performed in 96-well microtiter plate format. As a proof of concept, validation of this automation compatible MtPMT activity cell-based assay was achieved by mid-throughput scanning of a preliminary library of about thirty rationally chosen compounds. This reporter strategy constitutes an example of a target-based cell assay to monitor the activity in cellulo of a non-essential polytopic bacterial membrane enzyme involved in the post-translational modifications of secreted proteins.

RESULTS

Construction of a mycobacterial MtPMT activity reporter strain

To develop a readily workable cellular assay to monitor the in situ activity of MtPMT, we generated an appropriate surrogate reporter strain to overcome the restrictive safety and time constraints raised by cultivation of the pathogenic and slow growing Mtb. With this aim, we chose to use as starting material the non-pathogenic M. smegmatis mc 2 155 for its rapid growth phenotype, its genetic proximity to Mtb and its natural capacity to produce and secrete mannoproteins. As a preliminary step, the M. smegmatis endogenous protein-O-mannosylating enzyme coded by the MSMEG_5447 was disrupted by allelic exchange, to generate the recombinant recipient strain named (Msm-Δ(MsPMT)) (Fig. 2) [START_REF] Liu | Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis[END_REF]. This mannoprotein-defective strain was transformed in turn with an episomal vector bearing the Mtb Rv1002c gene under the control of the mycobacterial pBlaF* promotor [START_REF] Spratt | Identification of strong promoter elements of Mycobacterium smegmatis and their utility for foreign gene expression in mycobacteria[END_REF] allowing constitutive ectopic expression of MtPMT in M. smegmatis [START_REF] Lee | Mycobacteriophage L5 integrase-mediated site-specific integration in vitro[END_REF].

Notably, the two amino acid sequences of MtPMT and MsPMT share 93% similarity and 75% identity.

Finally, the latter recombinant strain (Msm-ΔMsPMT::MtPMT) was further modified with a multicopy replicative plasmid harboring the MSMEG_5196 gene fused to the hexa-histidine coding sequence, to over-express constitutively an easy-to-detect secreted acceptor substrate of MtMPT: the FasC His . This naturally secreted mannoprotein of M. smegmatis was chosen because of its high abundance in the culture medium and high degree of mannosylation (up to 13 hexoses), which makes it a sensitive probe of the MtPMT mannosyl transfer activity [START_REF] Liu | Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis[END_REF] (Fig. 1 and2). Thanks to this, the ectopic expression and heterologous activity of MtPMT in M. smegmatis can be verified by analysis of the glycosylation pattern of the NiNTA-purified FasC His secreted by the recombinant reporter strain (Msm-ΔMsPMT::MtPMT).

As shown by SDS-PAGE revealed by Coomassie blue staining or by Western blot with an anti-HisTag mAb (Fig. 3A), FasC His purified from the culture medium of the recipient strain (Msm-ΔMsPMT, called no-PMT) transformed with the empty vector and defective for protein mannosylation appears as a thin band as expected for this non-glycosylated control. In contrast, FasC His purified from the reporter strain (Msm-ΔMsPMT::MtPMT, called WT) exhibits a broader band profile consistent with mass dispersion induced by multiple and heterogeneous glycosylation.

Further accurate inspection by Liquid Chromatography-Electro Spray Ionisation Mass Spectrometry (LC-ESI-MS) confirms this hypothesis by highlighting the precise differences in molecular weight (MW) profile of the proteins purified from the two strains (Fig. 3A and Sup. fig. 1). Indeed, consistent with the protein mannosylation defect of the recipient strain, the deconvoluted mass spectrum of the Fas His purified from the Msm-ΔMsPMT (called no-PMT) strain shows a single peak at 23,845 Da attributable to the non-glycosylated protein (theoretical average MW 23,845 Da). This aglycosylated form of the protein also represents the most intense peak of the mass spectrum of the Fas His purified from the reporter strain Msm-ΔMsPMT::MtPMT (WT). However, in addition to this peak at 23,845 Da, a series of peaks regularly spaced by 162 Da mass shifts indicates the presence of at least 13 FasC His glycoforms, accounting for almost 80% of the cumulated peak area. This complex mass profile resulting from the accumulative decoration of the protein by oligomannosyl appendages confirms the orthogonal mannosyl transferase activity of Mtb MtPMT in M. smegmatis.

To verify whether the MS profile of the purified FasC His could reflect quantitative alteration of the enzymatic activity, we generated several similar reporter strains expressing a single amino acid mutated sequence of MtPMT, L366A, Y371A and Y444A. These mutations, predicted to be located in soluble external loops by sequence homology with the fungal enzymes ScPMT1/2 [START_REF] Bai | Structure of the eukaryotic protein Omannosyltransferase Pmt1-Pmt2 complex[END_REF], were chosen for their regularly spaced deleterious effects on MtPMT activity. Comparison of the MS profiles of the FasC His purified from these different mutated strains reveals that the MtPMT substitutive mutations are associated with significant decreases in the number and intensities of the glycosylated form signals relative to those of the unsubstituted protein. Since these differences are solely attributable to the impact of the mutations on the initial mannose transfer by MtPMT onto the acceptor substrate, the FasC His MS profiles therefore appear as reliable semi-quantitative indicators of the relative activities of the mutated enzymes in vivo (Fig. 3B andC, and Sup. fig. 2). In conclusion, altogether these results confirm the effectiveness of the reporter cell system to assess the in situ activity of MtPMT in M. smegmatis.

However, the LC-MS workflow requires the pre-analytical purification of the reporter FasC His that is poorly compatible with the search for MtPMT inhibitors in massive chemical libraries.

Alternatively, multi-well plate format quantitative enzyme linked lectin assay (ELLA) turned out to be an alternative for quantifying FasC His relative mannosylation, because of its multiplexing capabilities allowing for the simultaneous quantification of multiple samples in a high-throughput manner, such as heterogeneous complex bacterial supernatants.

Development of a miniaturized MtPMT activity phenotypic assay

In order to develop an assay for quantifying FasC His mannosylation directly from crude microculture supernatants, we implemented the detection of Concanavalin A (ConA) binding to FasC His oligomannosides. Adjustment of the assay to the multi-well plate format implies down-scaling the entire workflow, including (1) the culture of reporter cells and production of the MtPMT acceptor substrate FasC His , (2) the isolation of FasC His from the pool of ConA binding manno-conjugates present in the culture supernatant, and (3) the quantification of FasC His mannosylation. Keeping in mind the possibility to transfer the assay to a robotic platform for middle/high-throughput screening for potential MtPMT inhibitors we chose to demonstrate the feasibility based on the 96-well plate format.

Reporter cell culture and FasC His production. Since protein expression of proteins are highly dependent on bacterial culture conditions, we first monitored the production of secreted FasC His by the reporter strain grown in the reduced volume of the microplate well (250 µL). Best results were obtained with reporter cells generated extemporaneously, to minimize potential instability of FasC His production that tends to decrease over repeated cultures of the recombinant strain. In brief, newly transformed reporter cells picked up from selective solid medium are expanded once in liquid culture for 7 days before use. Then, the 96-well plates filled with 200 µL/well of fresh medium are seeded at a final OD600 0.1 with an aggregate-free cell suspension prepared from the primary liquid culture. Cells are grown for 7 days and the level of FasC His production in the crude cell culture supernatant is monitored over the time course of the culture, either by Coomassie blue stained SDS-PAGE, or by ELISA using anti Histag antibodies. Both approaches reveal a time dependent increase in FasC His abundance as a result of the accumulation of newly secreted protein in the culture medium. Interestingly, the downscaling of the culture volume from 10 mL flask to 250 µL microplate wells only marginally affects cell growth (OD600nm ~1 at day 7). In contrast, quantification by anti His-tag ELISA after 7 days of cultivation shows a slightly higher FasC His concentration close to 200 µg/mL in microplate reduced volume, compared to cultures in greater volume flasks (Sup. fig. 3 A andB). Hence, this reproducible result confirms in fine that the production of FasC His by the recombinant reporter strain is only slightly affected by the 40-fold reduction of the culture volume from 10 mL flasks to 250 µL microplate wells.

FasC His isolation and mannosylation quantification. The relative quantification of FasC His

mannosylation by ConA binding requires the preliminary isolation of the FasC His reporter protein from the bulk pool of glycoconjugates present in the culture supernatant, mainly mannophosphoinositides, lipoglycans and polysaccharides that are likely to interact with the lectin. This is readily achieved simultaneously on multiple samples by selective capture of the His-tagged protein using multi-well plates precoated with specific anti-His-tag antibodies. The rate of mannose branching of the proteins captured this way can then be estimated with HRP-conjugated ConA that binds preferentially to terminal mannose residues directly linked to the protein or localized at the non-reducing end of small linear manno-oligosaccharides (Fig. 4A). The performances in terms of sensitivity and reliability of the approach for the quantification of FasC His mannosylation, are clearly attested by the relative dose dependent binding of ConA to the FasC His contained in the culture supernatant of reporter strains harboring mutated MtPMT (Fig. 4B).

The ConA-binding dose-response curve to FasC His from the culture supernatant of the Msm-ΔMsPMT::MtPMT reference strain shows a hyperbolic shape with a likely initial linear dose response (at the highest supernatant dilution) that rapidly reaches a plateau for FasC His concentration above 4 ng/μL. At the opposite end, the non-glycosylated form of FasC His obtained from the Msm-ΔMsPMT and Msm-ΔMsPMT::MtPMTY444A strains shows ConA binding signals undiscernible from the basal background, as expected from the absence or quasi absence of mannosyl appendages on these molecules. Interestingly, the in-between signal of ConA binding of 80% and 60% observed for the FasC His produced by the recombinant strains harboring mutated MtPMT, Msm-ΔMsPMT::MtPMTL366A and Msm-ΔMsPMT::MtPMTY371A, is fully consistent with their intermediate glycosylation level evidenced by LC-MS analyses (Fig. 4C). In addition, the ConA binding curves obtained for these lesser glycosylated forms of FasC His show similar profiles compared to the fully glycosylated FasC His , with a plateau occurring at the same saturating concentration, which corresponds to a quantity of about 200 ng of FasC His per well, consistent with the maximum binding capacity range claimed by the manufacturer (150-300 ng of poly-histidine tagged protein/well). Thus, provided one works above this saturating concentration, this intrinsic limitation in the amount of immobilized His-tagged ligand allows convenient normalization of the ConA binding response versus the concentration of Fasc His to quantify the protein mannosylation and to assess the mannosyltransferase activity of MtPMT in vivo.

Finally, a remarkable correlation is observed between the data obtained by ELLA and LC-MS analysis though they differ radically in that the latter approach is performed on purified FasC His only while the former operates directly on crude microculture supernatants. Both quantitations provide similar patterns of protein-O-mannosylation attenuation among the different genetic backgrounds (WT>L366A>Y371A>Y444A>No-PMT), and thus confirm the quantitative ELLA as a valid and convenient approach to assess the MtPMT activity in cellulo.

Implementation of the mycobacterial MtPMT phenotypic assay

To validate the approach for medium-throughput screening (MDS) of potential protein-Omannosylation inhibitory compounds, a set of 30 chemicals were assessed with this novel target-based cell assay, according to the experimental workflow schematized in Fig. 5. These 30 compounds were rationally chosen for their putative inhibitory effect on MtPMPT activity, as biomimetics of proline-enriched mycobacterial manno-peptides [START_REF] Laqueyrerie | Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47kilodalton secreted antigen complex[END_REF][START_REF] Smith | O-linked glycosylation sites profiling in Mycobacterium tuberculosis culture filtrate proteins[END_REF]. Briefly, freshly prepared M. smegmatis Msm-ΔMsPMT::MtPMT WT-reporter cells are cultured in duplicate in 96-well microplates in the presence of increasing concentrations (2.5, 25 and 250 µg /mL) of the selected compounds (Fig. 5A). After 7 days, the OD600nm are measured and the supernatants are collected to verify the putative effect of the compound on the MtPMT activity by quantification of FasC His relative mannosylation by ConA ELLA (Fig. 5A andB). In a first instance, inactive compounds or those inducing growth inhibition are disregarded. Besides, those associated with reproducible and coherent dosedependent decrease in the level of mannose binding to ConA are considered for additional controls of the FasC His production (Fig. 5C). Indeed, the aim of the latter control is to exclude any bias due to a decline in the expression or secretion of the mannosylated FasC His below the saturating concentration required to normalize the responses for comparison.

Among the 30 chosen compounds, we first wanted to test several rhodamine derivatives (Fig. 6A, Table II), including R3A-5a also referenced as OGT2599, previously reported as inhibitor of yeast and mice PMTs [START_REF] Szafranski-Schneider | Msb2 shedding protects Candida albicans against antimicrobial peptides[END_REF][START_REF] Orchard | Rhodanine-3-acetic acid derivatives as inhibitors of fungal protein mannosyl transferase 1 (PMT1)[END_REF]. We also included two rhodamine containing chemicals reported to selectively kill non-replicating MTb [START_REF] Bryk | Selective killing of nonreplicating mycobacteria[END_REF] in order to verify whether this specific bactericidal activity could be associated with protein-O-mannosylation deficiency. Beside this class of substances, we chose to test several pyrrole or pyrrolidine-containing compounds that could present analogies with the proline-rich sequences surrounding the Thr/Ser targeted by the MtPMT [START_REF] Smith | O-linked glycosylation sites profiling in Mycobacterium tuberculosis culture filtrate proteins[END_REF]. The rationale here is that this kind of biomimetic may compete with the acceptor substrate for the binding to the enzyme (Fig. 6A, Table II).

Finally, we blind-tested several molecules without prior knowledge of their structures, including glycosyltransferase transition-state substrate-analogues, or rhodamine derivatives (Table II). The effects of each of these 30 chemicals on mycobacterial growth, the FasC His relative-O-mannosylation and FasC His secretion were analyzed thoroughly. The results confirm that neither the rhodamine 3-acetic acid derivative R3A-5a (OGT2599) nor the rhodamine-containing chemicals are likely to alter the bacterial MtPMT activity, at these concentrations.

Of the 30 compounds tested, 8 show probable Fasc His relative mannosylation reduction, among which 3 molecules induce a statistically significant dose-dependent inhibitory effect (Z317129322, Z66495095, and D-Proline, see Fig. 6B and Sup. fig. 3). These 8 compounds all contain pyrrole analogs, and most significantly, with D-Proline, a maximum inhibition of 29±2% was observed with concentrations ranging from 2.5 μM to 250 μM. Interestingly, the two compounds with the highest inhibitory effects on MtPMT activity (compounds Z66495095 and D-Proline) had no effect on either bacterial growth or FasC His secretion (Fig. 6C andD), two crucial criteria for avoiding the induction of drug resistance mechanisms. In contrast, OD600nm measurements revealed a dose-dependent inhibitory effect on mycobacterial growth for four compounds (Z280700946, Z5816758, Z269692472, and Z317129322, Fig. 6C). However, despite these dose-dependent growth declines, the concentrations of FasC His in the different supernatants were above the maximum capture-capacity of the anti-His-tag antibody-coated well (i.e., more than 200 ng/well, see Fig. 6D), confirming that the observed relative ConA binding decrease results from a lower FasC His mannosylation rather than from a diminution of the FasC His concentration. This control validates our interpretation that the decrease in ConA binding (observed for the 8 pyrrolederived compounds) reflects a lower mannosylation of FasC His glycoforms, attributable to an inhibitory effect exerted on MtPMT activity. Notably, compounds Z5816758, Z269692472, and Z317129322 appeared to inhibit FasC His secretion, in a dose-dependent manner (Fig. 6D), while neither L-Proline nor pyrrolidine showed any significant inhibitory effect compared to D-Proline under the same test conditions (not shown), suggesting that the stereochemistry and the conformation of these molecules are determinant factors in the inhibition of MtPMT (Fig. 6A).

Overall, our results demonstrate the operability and reliability of this unique multiplexed target-based cell assay to evidence the eventual MtPMT activity alterations induced by chemicals in cellulo. This achievement opens the way for the development of MDS/HTS campaigns to identify specific inhibitors of bacterial PMTs of pharmacologic potential, for the development of non-conventional antituberculous adjuvant therapies.

DISCUSSION

The MtPMT enzyme, encoded by the Rv1002c Mtb gene, is non-essential for the microorganism growth, but crucial for its virulence [START_REF] Liu | Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis[END_REF]. This protein is the only Mtb enzyme that catalyzes the initial transfer of mannoside units onto mannoproteins acting as "acceptor" substrates [START_REF] Vanderven | Export-mediated assembly of mycobacterial glycoproteins parallels eukaryotic pathways[END_REF] and being subsequently either secreted or anchored at the membrane [START_REF] Tonini | Potential Plasticity of the Mannoprotein Repertoire Associated to Mycobacterium tuberculosis Virulence Unveiled by Mass Spectrometry-Based Glycoproteomics[END_REF].

After transfer of the first mannoside units by MtPMT, the oligomannosides are then extended by elongating enzymes in a linear, unbranched fashion [START_REF] Liu | Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis[END_REF]. Therefore, in mycobacteria, measurement of the rate of terminal mannosylation of oligomannosylated-proteins reflects that of initial O-mannosylation, i.e., the activity of MtPMT. In the present work, we sought to exploit this relationship to measure MtPMT activity, via the measurement of the binding rate of ConA, a lectin that specifically recognizes terminal mannoside units. Our goal was to initiate the search for MtPMT inhibitors that would be effective in a native biological context. Therefore, we first constructed a reporter strain heterologously producing MtPMT in M. smegmatis, a non-pathogenic mycobacterium.

Then we developed a high-throughput phenotypic MtPMT activity-assay, based on the detection of ConA binding to FasC, chosen as secreted, highly mannosylated mycobacterial reporter of MtPMT activity. As a proof of concept, we applied our phenotypic assay to the screening of a reduced panel of rationally selected compounds.

Remarkably, the catalytic site of PMT enzymes is highly conserved among species, consisting primarily of a triad of essential amino acids, which catalyze the transfer of mannoside units from a glycolipidic substrate, "donor" of mannoside units [START_REF] Bai | Structure of the eukaryotic protein Omannosyltransferase Pmt1-Pmt2 complex[END_REF]. Due to this high conservation, the catalytic site appeared to us unattractive as a specific molecular target for inhibitors of MtPMT activity, which should be free of host toxicity for future therapeutic applications. In contrast, the species-specific donor and acceptor substrate binding sites seemed more relevant to target. Given these pharmacological imperatives, we noticed that the literature on PMTs mentioned the F664S substitution in the PpPMT2 enzyme sequence of the yeast Pichia pastoris, as related to strong fungal resistance to rhodamine analogs [START_REF] Argyros | A phenylalanine to serine substitution within an O-protein mannosyltransferase led to strong resistance to PMT-inhibitors in Pichia pastoris[END_REF]. Together with recent information provided by the resolution of the 3D structures of Saccharomyces cerevisiae ScPMT1/2 in complex with their donor and acceptor substrates [START_REF] Bai | Structure of the eukaryotic protein Omannosyltransferase Pmt1-Pmt2 complex[END_REF], this observation suggested that rhodamine-derived compounds might bind to the enzymatic active sites. Indeed, F664 of PpPMT2 from Pichia pastoris align with S644 of ScPMT1 from S. cerevisiae, which is in direct contact with the periplasmic extremity of the dolichol carbon chain, and in close proximity with the phosphomannoside unit [START_REF] Bai | Structure of the eukaryotic protein Omannosyltransferase Pmt1-Pmt2 complex[END_REF].

Unfortunately, according to our results, no rhodamine derivatives were found to inhibit MtPMT activity, whereas the compound R3A-5A, in particular, had been reported to inhibit the activities of several yeast PMTs, such as Candida albicans [START_REF] Orchard | Rhodanine-3-acetic acid derivatives as inhibitors of fungal protein mannosyl transferase 1 (PMT1)[END_REF], Saccharomyces cerevisiae [START_REF] Arroyo | Functional and genomic analyses of blocked protein O-mannosylation in baker's yeast[END_REF], and Pichia pastoris [START_REF] Argyros | A phenylalanine to serine substitution within an O-protein mannosyltransferase led to strong resistance to PMT-inhibitors in Pichia pastoris[END_REF]. These negative results could be explained by the impermeability of the mycobacterial membrane to these highly hydrophobic compounds, or alternatively by the low or no affinity of these molecules for our mycobacterial target enzyme. The latter interpretation therefore encourages further investigations dedicated to the inhibition of glycolipid donor substrate binding, in order to discern the targeting of mycobacterial PMT enzymes vs. eukaryotic ones (including host PMTs), which use distinct lipidic substrates (i.e. polyprenol vs. dolichol) [START_REF] Richards | The transfer of mannose from guanosine diphosphate mannose to dolichol phosphate and protein by pig liver endoplasmic reticulum[END_REF][START_REF] Babczinski | Involvement of dolicholmonophosphate in the formation of specific mannosyl-linkages in yeast glycoproteins[END_REF][START_REF] Verma | Formation of alpha-1,2-mannosyl-mannose by an enzyme preparation from rabbit liver[END_REF][START_REF] Herscovics | Mannosyltransferase activity in calf pancreas microsomes. Formation of 14C-labeled lipid-linked oligosaccharides from GDP-D-[14C]mannose and pancreatic dolichyl beta-D-[14C]mannopyranosyl phosphate[END_REF]. More promisingly, our preliminary screening allowed us to identify a small panel of weak MtPMT inhibitors, all containing pyrrole analogs, chosen as biomimetics of short proline-enriched peptides.

Indeed, this residue is particularly abundant in the vicinity of mannosylation-sites of mycobacterial glycoproteins [START_REF] Smith | O-linked glycosylation sites profiling in Mycobacterium tuberculosis culture filtrate proteins[END_REF], namely the hydroxyl groups of serine or threonine side chains, as illustrated by the secreted protein rich in Alanine-Proline ("APA"), a major mannosylated antigen of Mtb [START_REF] Laqueyrerie | Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47kilodalton secreted antigen complex[END_REF]. In particular, our results have led us to consider the two molecules Z66495095 and D-Proline as relevant basic molecular structures for the selection or future design of even more effective inhibitors, with no effect on mycobacterial growth or mannoprotein secretion, in order to avoid the emergence of drug resistance. Remarkably, both of these compounds possess a 5atom ring, including one nitrogen, and devoid of double bonds. However, it seems that the stereochemistry and the conformation are also important to exert such an inhibitory effect, since remarkably neither L-Proline nor pyrrolidine showed a significant inhibitory effect under the same test conditions. Whether promising or even negative, these results strongly validate the implementation of our MtPMT phenotypic assay for the identification of inhibitors of our mycobacterial target-enzyme.

CONCLUSION AND PERSPECTIVES

Overall, these results provide evidence that our quantitative phenotypic protein-O-mannosylation assay is sufficiently robust to detect significant inhibitory effects of MtPMT activity in mycobacteria.

This assay also appears to be easily adaptable for MtPMT inhibitor screening campaigns at even higher throughput scales, being easily automated and potentially even more miniaturizable. Our preliminary data obtained from the screening of 30 selected compounds supported our hypothesis that the presence of proline near the glycosylation sites of protein substrates probably contributes to their binding to this enzyme, since structurally related compounds inhibit MtPMT activity, probably through a competition effect. This observation therefore encourages us to search in the future, in larger chemical libraries, for potent inhibitors of the molecular recognition between MtPMT and its protein substrates, whose molecular structure could therefore include in priority analogues of pyrrolic rings.

Such compounds, capable of preventing the multiplication of Mtb in-vivo, by targeting a non-essential virulence factor of mycobacteria with pleiotropic activity, could thus lead to the development of new therapeutic strategies, adjuvant to current anti-tuberculosis treatments, with a reduced risk of inducing bacterial drug-resistance.

In the perspective of identifying MtPMT inhibitors capable of crossing the Mtb cell wall to reach their membrane-inserted target-enzyme, the results obtained with this MtPMT activity assay in M. smegmatis could be confirmed directly in pathogenic mycobacteria, by simple transposition of the genetic tools (i.e. the plasmids) into Mtb for example. MtPMT inhibitors could then be tested under infection conditions, in cell or mice models. In the future, it might also be interesting to develop similar assays for other relevant biological systems including PMTs. For example, eukaryotes express multiple PMT isoforms that could thus be characterized concomitantly but independently, by co-overexpressing tagged manno-proteins, specific reporter-substrates for each isoform. In particular, this type of assay could be transferred into mammalian reporter cells and applied to the various human PMTs (POMT1/2, TMTC1-4, TMEM260) [START_REF] Manya | Demonstration of mammalian protein O-mannosyltransferase activity: coexpression of POMT1 and POMT2 required for enzymatic activity[END_REF][START_REF] Larsen | Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins[END_REF][START_REF] Larsen | The SHDRA syndromeassociated gene TMEM260 encodes a protein-specific O-mannosyltransferase[END_REF]. Differential screening would thus enable assessment of the effects of the mycobacterial hits on host O-mannosylation, in order to avoid any undesired inhibition of this vital process. Importantly, the functional data provided by our new phenotypic activity assay allows for a precise analysis of the effects of mutations of MtPMT key amino acids or peptide regions, in an in-vivo context. Thus, this assay or its derivatives could also be used to shed light on the modes of molecular interaction between PMT enzymes and their substrates, paving the way for the elucidation of their catalytic mechanism. To achieve this goal, it would also be possible to specifically use the lectin ELLA assay presented in this article to determine more broadly kinetic parameters in vitro, and so complete the preliminary work of Cooper et al. [START_REF] Cooper | Characterization of mycobacterial protein glycosyltransferase activity using synthetic peptide acceptors in a cell-free assay[END_REF]. Indeed, by using membrane extracts from M. smegmatis, this early study showed PMT activities with Km values in the mM range, favored in the presence of Ala residues close to the Thr mannosylation sites of synthetic peptide substrates. Furthermore, the spatial organization and regulation of PMTs could be studied by analyzing their activities resulting from macromolecular complexes similar to those described for oligosaccharyltransferases [START_REF] Wild | Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation[END_REF], including possible associations as homo-or hetero-oligomers, or with complementary glycosyltransferases, transporters, chaperones, etc., all embedded into their native membrane environment.

More generally, GT-C glycosytransferases are responsible for the production of extracellular glycans involved in many human congenital disorders of glycosylation [START_REF] Ng | Perspectives on Glycosylation and Its Congenital Disorders[END_REF], or essential for the virulence of bacterial, viral and protozoan pathogens [START_REF] Petrou | Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation[END_REF][START_REF] Puschnik | A Small-Molecule Oligosaccharyltransferase Inhibitor with Pan-flaviviral Activity[END_REF][START_REF] Yadav | Targeting the GPI biosynthetic pathway[END_REF][START_REF] Sjodt | Structure of the peptidoglycan polymerase RodA resolved by evolutionary coupling analysis[END_REF][START_REF] Zhang | Structures of cell wall arabinosyltransferases with the anti-tuberculosis drug ethambutol[END_REF][START_REF] Ashraf | Structural basis of lipopolysaccharide maturation by the O-antigen ligase[END_REF]. The development and implementation of miniaturized phenotypic activity assays such as those reported here therefore represent an opportunity to refine our knowledge on the fundamental molecular mechanisms governing GT-C functions, which should stimulate the development of innovative therapeutic strategies targeting these enzymes.

MATERIALS AND METHODS

Construction of M. smegmatis control strains

Construction of the four plasmids pWM19, pMV361, pWM158, and pWM218, was described previously [START_REF] Malaga | Production of unmarked mutations in mycobacteria using site-specific recombination[END_REF][START_REF] Stover | New use of BCG for recombinant vaccines[END_REF][START_REF] Liu | Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis[END_REF]. The construction of the mutant strain PMM143, was also described previously [START_REF] Liu | Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis[END_REF]. To construct the new PMM186 kanamycin-sensitive strain, named Msm-Δ(MsPMT), the res-Ωkm-res cassette, was recovered from PMM143 by transforming the latter with the plasmid pWM19 and then selecting a clone with a PCR profile consistent with the res-Ωkm-res cassette excision. Plasmids pWM158_L366A, pWM158_Y371A, and pWM158_Y444A were obtained from pWM158, applying Stratagene's QuikChange® site-directed mutagenesis method, performed using Phusion DNA polymerase (ThermoFisher) with commercial high-GC buffer, as well as inverted-complementary oligonucleotide-primer pairs containing the desired mutation (Table I). All new DNA constructs were validated by restriction profiling and subsequent global sequencing of the entire MtPMT gene, performed by Eurofins Genomics. The strains expressing no PMT, or WT, L366A, Y371A or Y444A MtPMT genes, were constructed by transferring first either the pMV361, or pWM158, pWM158_L366A, or pWM158_Y371A, or pWM158_Y444A plasmids respectively, into the Msm-Δ(MsPMT) strain, and secondly the pWM218 plasmid, as follow.

Transformation and growth conditions of M. smegmatis derived strains

M. smegmatis cells were grown aerobically overnight at 37°C in Luria-Bertani broth and 0.05% Tween 80 (called LBT) until OD600nm = 0.5, followed by 3 washes with cold washing-buffer (0.05% Tween 80, 10% glycerol). Electroporation was performed at 2,500 V, 25 μf, 200 Ω, in the presence of the plasmids pMV361, or pWM158, or pWM158_L366A, or pWM158_Y371A, or pWM158_Y444A, encoding respectively no PMT, or WT, or site-mutated-MtPMT (at ratio 200 ng plasmid/200 µL cells at OD600nm = 25, in washing-buffer). Transformed bacteria were then selected on LB-agar containing 25 µg/mL kanamycin, and incubated at 37°C for 4 days. Isolated colonies were grown in LBT-kanamycin (25 µg/mL) for 7 days (OD600nm = 0.5), before being transformed with the second plasmid (pWM218, encoding FasC His ), plated on LB-agar containing 50 µg/mL hygromycin, and then grown again for 7-10 days in LBT-kanamycin (25 µg/mL)-hygromycin (50 µg/mL) (LBT-KH).

LC-ESI-MS analysis of FasC His glycoforms

FasC His was purified from 10-day-old 0.22 µm-filtered 10 mL flask-culture media of M. smegmatis control strains (no PMT, WT, L366A, Y371A, and Y444A), by using Ni-NTA spin columns (Qiagen). The concentration of purified proteins was determined using the Micro BCA TM Bicinchoninic Acid Protein Assay Kit (ThermoScientific). SDS-PAGE analyses were performed with 10 µg protein/well on 13% Trisglycine-polyacrylamide gels, with a Mini-Protean 3 electrophoresis system (Bio-Rad). Samples were run at 160 V for 1 h. Purified proteins were visualized after staining with InstantBlue (Expedeon), whereas for western-blot analysis, semi-dry transfer onto nitrocellulose membrane was performed (at 25 V for 30 min), followed by Anti-Histidine tag detection using a monoclonal-HRP-conjugated antibody (clone 3D5, Invitrogen), following the manufacturer's instructions. NanoLC-MS analyses were performed with 5 μL of purified proteins diluted in loading buffer (2% acetonitrile (CAN), 0.1% trifluoro acetic acid (TFA)) to final concentration of 0.4 μM, by employing a nanoRS UHPLC system (Dionex), coupled to a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Samples were loaded onto a reverse-phase C4 pre-column (300 μm i.d. x 5mm; Thermo Fisher Scientific) at 20 μL/min in 2% ACN and 0.05% TFA. After 5 min of desalting, the precolumn was switched online to a home-made C4 analytical nanocolumn (75 μm i.d. x 15 cm) packed with C4 Reprosil (Cluzeau CIL), equilibrated in 95% solvent A (0.2% formic acid (FA)) and 5% solvent B (0.2% FA in ACN). Proteins were eluted using a linear gradient from 5% to 100% B in 38 min, at a flow rate of 300 nL/min. MS scans were acquired in positive mode in the 800-2,000 m/z range with a resolution set at 60,000. The spectra were deconvoluted and semi-quantified with Unidec (10.1021/acs.analchem.5b00140.) using the following parameters: m/z range: 1,000-2,000 Th; no background subtraction, no smoothing; charge range: 10-25; mass range: 20,000-30,000 Da; sample mass: every 10 Da; peak detection range: 140 Da, and peak detection threshold: 0.04.

96-well microplate cultures of M. smegmatis-derived control strains

After transformation with pWM218, one colony of each M. smegmatis control strain was transferred into 10 mL LBT-KH medium in a flask. After 4 days, 10 mL of fresh LBT-KH were added, and culture prolonged for 3 additional days until OD600nm 1.5. In order to inoculate the wells with a homogeneous aggregate-free bacterial suspension, the 20 mL pre-culture was centrifuged for 10 min at 4°C, at 500 g. 5 sterile glass beads (4 mm) were added to the pellet to gently dislodge it. 20 mL of cold Luria-Bertani Tween medium (LBT) were added, then the clear suspension was centrifuged for 10 min at 100 g, harvested and assayed at OD600nm. Duplicate microplates where then inoculated at OD600nm 0.1 in 250 µL/well of LBT-Kanamycin-Hygromycin (LBT-KH). Plastic covers were placed on the microplates to limit medium evaporation. Microplates were incubated at 37°C in a humidity-controlled oven. OD600nm was measured with the BMG LABTECH Clariostar, after homogenizing 25 µL/well of culture in 75 µL of mQ water, by gentle pipetting/filling. Further direct-and/or lectin-ELISA analyses could then be performed from culture supernatants, obtained by centrifugation of the whole microplates at 2,000 g at 4°C for 15 min.

Direct quantification of FasC His concentration and mannosylation from microculture supernatants

For both assays, supernatants were analyzed in triplicate in 96-well microplates. FasC His concentrations were determined by Direct Anti-His tag ELISA. A standard range from 50 to 2,000 ng/mL (in PBS) of purified FasC His was prepared for each microplate. 50 µL/well of culture supernatant, diluted 100x in PBS, were mixed with 50 µL/well of carbonate coating-buffer (Na2CO3 0.53%/NaHCO3 0.42%, pH 9.6) and then incubated overnight at room temperature. After washing 3 times with 200 µL/well PBS, the plates were incubated for 1 h at room temperature with 200 µL/well PBS containing 0.05% Tween 20 and 5% milk. After 3 washes with 200 µL/well of PBS containing 0.05% Tween 20, the plate was incubated for 2 h at room temperature with 50 µL/well of anti-His antibody (anti-His C-term-HRP antibody, Invitrogen), diluted 1,000x in PBS containing 0.05% Tween 20 and 0.5% milk. Four washes with 200 µL/well of PBS were performed before revelation.

Quantifications of FasC His mannosylation were determined by enzyme linked lectin assay (ELLA). For this assay, 96-well microplates coated with anti-His antibody (Genscript) were used. 100 µL/well of culture-supernatants (diluted twice in PBS) were placed in the wells. The plate was incubated for 3 h at room temperature and then washed 4 times with 260 µL/well of PBS containing 2% Tween 20. 100 µL of Concanavalin A-HRP solution (0.5 µg/mL ConA-HRP, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 0.05% Tween20, in PBS) were added and then the plate was incubated at room temperature for 2 h and washed 4 times with 260 µL/well of PBS. Values of 100% and 0% were defined as the ones measured for the WT and No PMT M. smegmatis control strains, respectively.

For both assays, revelation was performed with 50 µL/well of TMB solution (eBioscience). 25 µL/well of stop solution (1 mM sulfuric acid) was added after 20 min incubation. Absorbances were measured at 450 nm using the BMG LABTECH Clariostar microplate reader. Saturation of the pre-coated antihistidine antibody of the ELLA was ensured from at least 200 ng/well of FasC His , amount possibly inferred from concentrations determined by Direct-ELISA.

Cell treatment with putative MtPMT inhibitors

Microplates were inoculated at OD600nm 0.1 in 250 µL/well of LBT-KH, in the presence of 2.5 µL of 100xconcentrated compound (prepared in DMSO, then added into wells at final concentrations ranging from 2.5 to 250 µM) and then incubated at 37°C for 7 days in a humidity-controlled oven. The microcultures in rows A and H, as well as columns 1, 2, and 12 were not treated with any compound, nor were they analyzed, to avoid considering possible "edge effects." The wells in row D and column 7 were filled with compound-free DMSO to allow data normalization by the values obtained for these "untreated" microcultures. The microcultures in row E were inoculated with the No PMT strain to serve as blank for the ELLA assay.

Statistical analysis of the effects of MtPMT inhibitors

The statistical analysis of the effect of varying concentrations of D-Proline, Z317129322, or Z66495095 on ConA binding was carried out in R, v4.2.1 (RCoreTeam, 2022), using the default stats package to perform a full two-way ANCOVA where the concentration of the molecule was used as numeric covariable. Data handling and graphic output of the result was produced using dplyr, tidyr, and ggplot2 packages, all within the tidyverse metapackage, v2.0.0 [START_REF] Wickham | Welcome to the tidyverse[END_REF]. R codes are available upon request.

Screened compounds

-Pyrrole derivatives: the following compounds were obtained from the commercial suppliers indicated below and used without further purification.

oFigure 1 .

 1 ABBREVIATIONS

Figure 2 .

 2 Figure 2. Construction of the mycobacterial MtPMT activity reporter strain. The original M. smegmatis Δ(MsPMT) strain, derived from mc 2 155 and deficient for its endogenous PMT, was complemented with two plasmids encoding MtPMT and FasC His , for permanent and transient expression respectively. The annotated sequence of FasC His , used as mannosylated secreted mycobacterial reporter protein-substrate for the Protein-O-mannosylation activity of MtPMT, is presented. The amino acids previously detected by mass spectrometry as mannosylated in FasC His from M. smegmatis mc 2 155, are boxed in red (unpublished). The signal peptide and the C-terminal 6xHis tag are shown in italics and light gray letters. The Fascilin domain is indicated by bold font. Glycosylation sites predicted as putative mannosylation sites by the NetOglyc 4 software are shown in blue, while T and S amino acids not identified by the software are in orange.

Table I .

 I Name and main features of sense-primers, plasmids and strains used in this study

	Relative Intensitiy of	His glycoforms (%) FasC	1.0 Name 1.5 Sp-L366A Sp-Y371A Sp-Y444A 0.5 pWM19 1 H e x 2 H e x 3 H e x 4 H e x 5 CC TGG CCC ATG GCG TTG CGG CCG GTG CTC TAC GCC ATC G Relevant characteristics Trivial name Antibiotic resistance MtPMT strain 1 Source MtPMT strain 2 Sequences of sense-primers (5'-3') This study G TTG CGG CCG GTG CTC GCC GCC ATC GAC CAG This study GCC GAC ATC GAT CGG CAG ATG GCC TTC TTC TAC This study Plasmids Thermosensitive Escherichia coli /mycobacteria shuttle plasmid containing the Hygromycin and (Malaga et al., Res Gentamicin 2003) resolvase gene from transposon γδ H e x 6 H e x 7 H e x 8 H e x 9 H e x 1 0 H e x 1 1 H e x 1 2 H e x 1 3 H e x 1 4 H e x 1 5 H e x 1 6 H e x 1 7 H H e x 0.0 pMV361 Integrative Escherichia coli/mycobacteria shuttle Empty (Stover et al., Kanamycin 1991) vector Mycobacterial integrative e x 1 8 plasmid, derived from
			pWM158	pMV361, containing the Glycoform wild-type MtPMT gene from WT-MtPMT	Kanamycin	(Liu et al., 2013)
				Mtb (Rv1002c) under the
				control of the pBlaF*
				promotor
				derived from pWM158,
			pWM158_L366A	containing the L366A-mutated-MtPMT gene from	L366A-MtPMT	Kanamycin	This study
				Mtb
				derived from pWM158,
			pWM158_Y371A	containing the Y371A-mutated-MtPMT gene from	Y371A-MtPMT	Kanamycin	This study
				Mtb
				derived from pWM158,
			pWM158_Y444A	containing the Y444A-mutated-MtPMT gene from	Y444A-MtPMT	Kanamycin	This study
				Mtb
				Mycobacterial replicative
				plasmid containing the
			pWM218	FasC His gene under the	FasCHis	Hygromycin	(Liu et al., 2013)
				control of the pBlaF*
				promotor
				Strains
			PMM143	M. smegmatis mc 2 155 Msmeg_5447::res-Ωkm-res	Msm-Δ(MsPMT)_km R	Kanamycin	(Liu et al., 2013)
			PMM186	M. smegmatis mc 2 155 Msmeg_5447::res	Msm-Δ(MsPMT)	-	This study
	PMM186::pMV361:pWM218	PMM186 transformed with pMV361 and pWM218	No PMT	Kanamycin and Hygromycin	This study
	PMM186::pWM158:pWM218	PMM186 transformed with pWM158 and pWM218	WT	Kanamycin and Hygromycin	This study
	PMM186::pWM158_L366A:pWM218	PMM186 transformed with pWM158_L366A and pWM218	L366A	Kanamycin and Hygromycin	This study
	PMM186::pWM158_Y371A:pWM218	PMM186 transformed with pWM158_Y371A and pWM218	Y371A	Kanamycin and Hygromycin	This study
	PMM186::pWM158_Y444A:pWM218	PMM186 transformed with pWM158_Y444A and pWM218	Y444A	Kanamycin and Hygromycin	This study

Table II .

 II Chemical structure of the 30 compounds tested in this study with the MtPMT activity assay 750

	Class	Compound	Structure	Ref.

22 compounds with no effect on MtPMT activity

FUNDING

This work was supported by the Fondation pour la Recherche Médicale (Equipes FRM DEQ20180339208 to Nigou J.), the Fondation MSDAVENIR (grant FIGHT-TB to Nigou J.), the Fondation Rolland Garrigou pour la culture et la santé (FONROGA, supporting this work for Master internship grant), and the Agence Nationale de la Recherche (grant ANR-20-PAMR-0005 to Nigou J.). This work was also funded in part by grants from the Région Occitanie, European funds (FEDER, Fonds Européens de Développement Régional), Toulouse Métropole, and the French ministry of research with the

Supplementary Figures

Supplementary figure 1. Reproducibility validation of LC-ESI-MS analysis of FasCHis glycoforms. The reporter-substrate mannoprotein was purified from supernatants of 10-day-old flask cultures of the WT-MtPMT activity reporter strain. Relative intensities of glycoform peaks (normalized to the intensity of the aglycosylated form, 0 Hex) are shown for two biological duplicates, analyzed as technical duplicates. Supplementary figure 2. Homologous structures of MtPMT, ScPMT1 and ScPMT2. The AlphaFold model of MtPMT as well as the cryo-EM structures of ScPMT1 and 2, in complex with donor and acceptor substrates respectively [START_REF] Bai | Structure of the eukaryotic protein Omannosyltransferase Pmt1-Pmt2 complex[END_REF] The two-way ANCOVA analysis revealed that the effect of the dose was significant (p-value = 1.04*10-5, n = 2), with no significant differences between the three compounds.


Benoit Maxit

Clara Catros

Florian Aubrit

Julien Le Mener

Olivier Sandre

David Jacob

Sylvain Boj

New methodology for anisotropic nanoparticles characterization by polarization light scattering: length and diameter determination of rod-like nanoparticles

Keywords: anisotropic nanoparticles, DLS, DDLS

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Indirect optical methods like dynamic light scattering (DLS) measurement exhibit real benefits: limited sample preparation, ease of use, short experimental time, strong statistical relevance of the results, etc.

However, standard DLS analysis assumes that particles are spherical and thus can only provide information on an average hydrodynamic diameter i.e. the effective diameter of a sphere having the same translational coefficient than the NPs. In the case of anisotropic nanoparticles, one can use a refined version of DLS named depolarized dynamic light Scattering (DDLS), which allows size and aspect ratio characterization of very small particles (down to few nanometers). [2] In this communication, we demonstrate the measurement capabilities of a new and patented commercial DDLS system developed by Cordouan Technologies (named Thetis™), by presenting experimental results on small gold nanorods. The DDLS results are compared with TEM ones to qualify the relevance and accuracy of the measurements by this non-invasive method.

Principle

The standard DLS system allows to determine the diameter of NPs by measuring their diffusion behavior (Brownian motion) by monitoring -at a given angle of observation  -the fluctuations of the light scattered by the NP suspensions. More precisely, the autocorrelation function (ACF) calculated from the light intensity fluctuations by baseline subtraction and square root gives access to a translational diffusion coefficient (Dt), which is related to the hydrodynamic radius of the particle by the well-known Stokes-Einstein equation. In the case of anisotropic particles, their dynamics in the liquid are strongly influenced by their size and shape and include both translational and rotational random motions, which are described by a translational diffusion coefficient (Dt) and a rotational diffusion coefficient (Dr), respectively.)

Using the properties of linearly polarized light it becomes possible to measure Dt and Dr and then in a second step, to deduce, the size and aspect ratio of the anisotropic particles by rather complex numerical inversion of theoretical formulas. [3] At first, on the Thetis apparatus described here, a Vertically polarized incident laser beam is focused onto the sample to be analyzed. The scattered light is then collected at different scattering angles (θ) using a compact motorized goniometer. Use of high quality bi-prism polarization splitter enables splitting the scattered beam towards two distinct detection channels set with crossed polarizers allowing the vertical polarized (V-V) and horizontal depolarized (V-H) scattered light beams to be measured separately and possibly simultaneously. In the case of anisotropic particles, the V-V eq.1 eq.2 and V-H detection channels lead to two different ACF, GVV and GVH, calculated from the respective scattered intensity fluctuations of IVV(t) and IVH(t).

Looking at the equations, [5] one can notice that both ACFs contain the same "mixed relaxation mode" combining the translational decay rate Γt and the rotational decay rate Γr.

eq.3 eq.4 = eq.5

=

eq.6

Thus, one method to determine Dt and Dr from DDLS measurements consists in plotting the two relaxation decay rates (the mixed mode and the pure translational mode) as a function of the square of the wave vector q 2 and to determine the slope of the linear fit, which theoretically should be the same for both curves. The wave vector q is defined by: eq. 7 With n the liquid phase refractive index, λ the laser wavelength, and θ the detection/scattering angle.

Figure 1

Experimental results

To evaluate Thetis instrument performances, three model suspensions of gold nanorods (NR) were used: Au-NR1 and Au-NR2 synthesized using protocol reported in literature, [4][5][6] and Au-NR3 commercial sample from NanoComposix (Product n° GRCN980batch n°ASP0002). A first estimate of the size and aspect ratio of these NPs was achieved by the analysis of transmission electron microscopy (TEM) images.

Then the samples were analyzed by DDLS for comparison purpose. In each case, the measurements were performed by varying the scattering angle θ from 30° to 150°, acquiring both V-V and V-H data for each detector position.

Figure 2 shows the normalized ACFs measured on sample Au-NR1 at seven different scattering angles for V-V ACFs (Fig. 2a) and V-H ACFs (Fig. 2b) respectively.

Figure 2a

Figure 2b Using the SBL (Sparse Baysian Learning) multimodal algorithm designed by Cordouan, the ProTheta ® software extracts the different decay rate values from each ACFs and plots them as a function of q 2 . Finally, using the model of De La Torre et al. for cylindrical shapes, [5] the hydrodynamic dimensions of Au-NR1/2/3 were calculated by inversion of the measured diffusion coefficients:

Au-NR1 Au-NR2 Au-NR3 The table above clearly shows that the particle size measurements obtained with the DDLS Thetis setup are consistent with those from the TEM analysis, especially for Au-NR2 nanorods for which both the length and the aspect ratio were accurately determined within the accuracy of the measurement.

Conclusion

These results highlight the relevance and robustness of the multi-angle DDLS method for characterizing the size and aspect ratio of anisotropic nanoparticles in suspension. The setup can also be used to discriminate in a simple way and without ambiguity anisotropic nanoparticles from a polymodal sample of isotropic particles.

Acknowledgements

We thank the New Aquitaine Region, "France 2030" and the M-Era.net programs for funding (2017 call, FMF project) for support in the industrialization of the Thetis instrument.