# A dTDP-L-Rhamnose 4-epimerase required for glycopeptidolipid biosynthesis in Mycobacterium abscessus

John Jairo Aguilera-Correa, Fangyu Wei, Louis-David Leclercq, Yara Tasrini, Edukondalu Mullapudi, Wassim Daher, Kazuki Nakajima, Stéphane Canaan, Jean-Louis Herrmann, Matthias Wilmanns, et al.

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John Jairo Aguilera-Correa, Fangyu Wei, Louis-David Leclercq, Yara Tasrini, Edukondalu Mullapudi, et al.. A dTDP-L-Rhamnose 4-epimerase required for glycopeptidolipid biosynthesis in Mycobacterium abscessus. Journal of Biological Chemistry, 2024, pp.107852. 10.1016/j.jbc.2024.107852. inserm-04720824

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A dTDP-L-Rhamnose 4-epimerase required for glycopeptidolipid biosynthesis in *Mycobacterium abscessus*

John Jairo Aguilera-Correa, Fangyu Wei, Louis-David Leclercq, Yara Tasrini, Edukondalu Mullapudi, Wassim Daher, Kazuki Nakajima, Stéphane Canaan, Jean-Louis Herrmann, Matthias Wilmanns, Yann Guérardel, Liuqing Wen, Laurent Kremer

PII: S0021-9258(24)02354-8

DOI: https://doi.org/10.1016/j.jbc.2024.107852

Reference: JBC 107852

To appear in: *Journal of Biological Chemistry*

Received Date: 12 July 2024

Revised Date: 11 September 2024

Please cite this article as: Aguilera-Correa JJ, Wei F, Leclercq LD, Tasrini Y, Mullapudi E, Daher W, Nakajima K, Canaan S, Herrmann JL, Wilmanns M, Guérardel Y, Wen L, Kremer L, A dTDP-L-Rhamnose 4-epimerase required for glycopeptidolipid biosynthesis in *Mycobacterium abscessus*, *Journal of Biological Chemistry* (2024), doi: https://doi.org/10.1016/j.jbc.2024.107852.

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© 2024 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.
A dTDP-L-Rhamnose 4-epimerase required for glycopeptidolipid biosynthesis
in *Mycobacterium abscessus*

John Jairo Aguilera-Correa¹, Fangyu Wei³,§, Louis-David Leclercq⁴,⁵,§, Yara Tasrini¹,§, Edukondalu Mullapudi⁶, Wassim Daher¹,², Kazuki Nakajima⁷, Stéphane Canaan⁸, Jean-Louis Herrmann⁵, Matthias Wilmanns⁶,⁹, Yann Guérardel⁴,⁷, Liuqing Wen³, and Laurent Kremer¹,²*

¹Centre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, 1919 route de Mende, 34293, Montpellier, France.

²INSERM, IRIM, 34293 Montpellier, France.

³Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Chinese Academy of Sciences, 555 Zuchongzhi Rd, 201203, Shanghai, China

⁴Univ. Lille, CNRS, UMR 8576 - UGSF - Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France.

⁵Université Paris-Saclay, UVSQ, Inserm, Infection et inflammation, 78180, Montigny-Le-Bretonneux, France.

⁶European Molecular Biology Laboratory, Hamburg Unit, Notkestrasse 85, 22607 Hamburg, Germany.

⁷Institute for Glyco-core Research (iGCORE), Gifu University, Gifu, Japan

⁸Aix-Marseille Univ, CNRS, LISM, IMM FR3479, Marseille, France.

⁹University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.

⁵Equal contribution

*Corresponding author: Laurent Kremer; Tel: (+33) 4 34 35 94 47; E-mail: laurent.kremer@irim.cnrs.fr

**Running title:** The epimerase is required for glycopeptidolipid synthesis

**Keywords:** *Mycobacterium abscessus*, cell wall, glycopeptidolipid, Rhamnose, 6-deoxy-Talose, epimerase, morphotype, virulence, zebrafish.
32 **ABSTRACT**

33 *Mycobacterium abscessus* causes severe lung infections in cystic fibrosis patients and exhibits 34 smooth (S) or rough (R) morphotypes. Disruption of glycopeptidolipid (GPL) production results in 35 the S-to-R transition but the underlying molecular mechanisms of this transition remain 36 incompletely understood. Herein, we characterized MAB_4111c in relation to GPL synthesis and 37 investigated the effects of MAB_4111c deletion in *M. abscessus* pathogenicity. An enzymatic 38 assay indicated that MAB_4111c, also designated Tle for *Talose epimerase*, is converting dTDP-39 L-Rhamnose into dTDP-6-deoxy-L-Talose. A *tle* deletion mutant was constructed in the S variant 40 of *M. abscessus* and relative areas of Rhamnose and 6-deoxy-Talose and their methylated forms 41 expressed as ratios of total monosaccharides, showed an altered GPL profile lacking 6-deoxy-42 Talose. Thus, Tle provides dTDP-6-deoxy-L-Talose, subsequently used by the glycosyltransferase 43 Gtf1 to transfer 6-deoxy-Talose to the GPL backbone. Strikingly, the *tle* mutant exhibited a R 44 morphotype, showed impaired sliding motility and biofilm formation, and these phenotypes 45 were rescued upon functional complementation. Moreover, deletion of *tle* in *M. abscessus* 46 results in increased pathogenicity and killing in zebrafish embryos. Together, our results 47 underscore the importance of the dTDP-L-Rhamnose 4-epimerase activity in GPL biosynthesis 48 and in influencing *M. abscessus* virulence. 49
50 **INTRODUCTION**
51

52 *M. abscessus* is an emerging opportunistic pathogen causing a variety of human infections that 53 usually involve the skin and subjacent soft tissues, but also severe lung infections in patients with 54 various chronic lung disorders, such as bronchiectasis and chronic obstructive pulmonary disease 55 or cystic fibrosis (1, 2). This mycobacterium is intrinsically resistant to β-lactams, tetracyclines, 56 aminoglycosides, and macrolides, which are routinely used to treat Gram-negative and Gram- 57 positive bacterial infections (3), rendering treatments extremely challenging (4). Like other non- 58 tuberculous mycobacteria (NTMs), *M. abscessus* has shown the ability to develop biofilms, 59 structured bacterial aggregates enclosed in a self-produced polymeric matrix where multiple and 60 complex socio-microbiological relationships reign, in the environment, water distribution 61 systems (5, 6), hospital equipment (5, 6), human tissues, e.g., lungs (7), and medical devices (8, 62 63 these morphotypes rely mainly on the production of high (in S) or low (in R) levels of 64 glycopeptidolipids (GPLs), respectively (10–12). Genome sequencing identified multiple indels in 65 the *gpl* locus of the R strain as compared to the S strain (13) mainly in *mps1* and *mps2*, encoding 66 non-ribosomal peptide synthases responsible for the biosynthesis of the GPL peptide backbone, 67 and in *mmpL4a/mmpL4b*, encoding membrane proteins involved in GPL transport (13–15). Thus, 68 these genetic changes in the *gpl* locus result in the irreversible loss of the GPL and are directly 69 responsible for the R colony morphology (13).

70 GPLs are found in the outer leaflet of numerous NTMs (16). These lipids are pivotal for 71 aggregation, biofilm development, motility, interaction with host cells and pathogenesis (16–22). 72 Both S and R forms are isolated from sputums of patients with chronic lung infections but while 73 the S form is thought to represent the environmental variant of *M. abscessus* (which infects and 74 colonizes the lungs), the R form emerges from the S form within the infected host (18, 23, 24). 75 Several studies support the enhanced pathogenesis of the R form over the S form, the former 76 being associated with more persisting and aggressive infections in addition to the decline of the 77 lung functions (18, 25–28). In the context of infection with *M. abscessus*, the zebrafish model has 78 deserved considerable interest as it was used to help defining the interactions between *M. 79 abscessus* and the innate immune response (27, 29). In particular, zebrafish embryos are killed 80 by the *M. abscessus* R infection, as evidenced by intense bacterial proliferation, production of 81 serpentine cords, abscesses and inflammation (27, 30). These features are in agreement with
epidemiological surveys, which support the prominence of the R variant in patients with severe lung infections and chronic airway colonization in cystic fibrosis patients (18, 24). Overall, this highlights the clinical relevance of morphological distinctions between S and R morphotypes and importance to understand the S-to-R transition at a molecular level (13).

The general GPL structure consists of a mixture of 3-hydroxy and 3-methoxy C28-30 fatty acids amidated by a tripeptide-amino-alcohol (D-Phe-D-*allo*-Thr-D-Ala-L-alaninol) (10). This lipopeptide core is glycosylated on the *allo*-Thr linked with a 6-deoxy-α-L-Talose (6-d-Tal) and on the alaninol linked with an α-L-Rhamnose (Rha) (10). Some GPLs can be di-glycosylated containing a 3,4-di-*O*-acetylated 6-d-Tal and a 3,4-di-*O*-methylated or 2,3,4-tri-*O*-methylated Rha (31–34). We previously demonstrated that the glycosyltransferases Gtf1, Gtf2 and Gtf3, encoded within the *gpl* locus, transfer the 6-d-Tal, the first Rha and the second Rha to the lipopeptide core, respectively (35). Importantly, in a *gtf1* deletion mutant, the synthesis of GPL derivatives lacking 6-d-Tal leads to a R morphotype, mycobacterial cording and increased virulence and pathogenicity in zebrafish (35). This illustrates the importance of Gtf1 in modelling the GPL structure and in influencing *M. abscessus* virulence, although the functional and structural characterization of this enzyme remains to be established. In addition, the enzyme/pathway that provides dTDP-6-d-Tal, the substrate used by Gtf1 to talosylate GPL, remains unknown.

In mycobacteria, L-Rha is synthesized *via* the essential Rml pathway that generates dTDP-L-Rha from Glc-1-phosphate (36). This pathway is composed of four enzymes - RmlA (MAB_4113), RmlB (MAB_3779), RmlC (MAB_3780), and RmlD (MAB_3613c) - encoded by four genes that are not in close vicinity in the *M. abscessus* genome, except for *rmlB* and *rmlC*. Besides being a component of GPLs, Rha can be found in other glycolipids, such as phenolic glycolipids (37) and is present in all mycobacteria as being part of the linker disaccharide (Rha-N-acetyl-glucosaminyl-phosphate) unit that attaches the cell wall arabinogalactan (AG) to the peptidoglycan (PG) layer (36). In contrast to Rha, 6-d-Tal is an infrequent deoxyhexose found only in bacteria, essentially in the cell wall and capsules (38). The dTDP-6-d-Tal is the activated form of 6-d-Tal and it is synthesized from Glc-1-phosphate and dTTP partially or entirely *via* the Rml pathway. In *Haemophilus actinomycetemcomitans* NCTC9710 serotype C (partial synthesis *via* the Rml pathway), the first three enzymes, RmlA-C, are identical to those required to synthesize dTDP-L-Rha. The fourth enzyme, a dTDP-6-deoxy-L-talose 4-dehydrogenase (TII) is also a dTDP-4-keto-6-deoxy-L-mannose reductase such as RmlD, but the stereoselectivity of TII regulates dTDP-4-keto-6-deoxy-L-mannose, which is reduced to dTDP-6-deoxy-L-talose (39). In *Burkholderia*
thailandensis (complete synthesis via the Rml pathway), a dTDP-L-Rha 4-epimerase, WbiB, converts dTDP-L-Rha into dTDP-6-d-Tal (40). Interestingly, in M. abscessus, a paralogous rmlB gene (MAB_4111c) is present in the gpl locus (31) and in silico predictions suggest that MAB_4111c encodes a putative epimerase/dehydratase (41).

Herein, we focused on MAB_4111c to further advance our understanding of the synthesis of the dTDP-6-d-Tal substrate of Gtf1 and the presence of 6-d-Tal in the GPL of M. abscessus. We also aimed to study the consequences of MAB_4111c deletion on bacteriological features and disease pathology in this emerging pathogen in zebrafish. Bioinformatic and genetic studies were also performed to determine the distribution and prevalence of MAB_4111c orthologous genes in other NTMs.
124 **RESULTS**

125

126 **MAB_4111c encodes a preditive dTDP-L-Rhamnose 4-epimerase**

127 The S variant of *M. abscessus* synthesizes both diglycosylated GPL (GPL-2a) and triglycosylated
128 GPL (GPL-3), containing one and two partially *O*-methylated Rha residues, respectively, as well as
129 a di-*O*-acetyl 6-d-Tal residue **(Figure 1A)**. While most proteins encoded by the *gpl* cluster and
130 involved in the biosynthesis and transport of GPL have been characterized to date, the enzyme
131 catalyzing the interconversion between dTDP-Rha and dTDP-6-d-Tal in mycobacteria remains
132 enigmatic. *MAB_4111c* is located between *gtf3*, encoding the rhamnosyltransferase which adds
133 the second Rha to generate GPL-3 (35), and *atf2*, encoding an acetyltransferase which acetylates
134 the 6-d-Tal (34) **(Figure 1B)**. Bioinformatics analysis suggests that MAB_4111c may code for an
135 enzyme with epimerase activity, in agreement with a BLAST analysis which showed that
136 MAB_4111c shares 54% identity with WbiB, a dTDP-Rha 4-epimerase in *Burkholderia*
137 *thailandensis*, the first enzyme reported to catalyze the interconversion between dTDP-Rha and
138 dTDP-6-d-Tal (40). This analysis also shows that the putative catalytic Tyr residue (Y181) as well
139 as other residues participating in the binding of the NADH cofactor and dTDP-Rha substrate are
140 fully conserved in MAB_4111c **(Figure 1C)**.

141

142 **MAB_4111c converts dTDP-Rhamnose into dTDP-6-deoxy-Talose**

143 To experimentally validate the function of MAB_4111c as a dTDP-Rha 4-epimerase, a His-tagged
144 version of the protein was produced in *Escherichia coli*. Initial attempts to express and purify
145 soluble MAB_4111c for biochemical and structural analyses yielded insufficient protein
146 quantities. Thus, we generated a MAB_4111c-enriched soluble fraction obtained after partial
147 purification on Ni²⁺-coated beads, which was assayed by incubation with NADP⁺ and dTDP-Rha
148 and monitored for the production of dTDP-6-d-Tal **(Figure 2A)**. The HPLC profile shows the
149 presence of a weak signal with a retention time (RT) similar to the one of the dTDP-6-d-Tal
150 standard **(Figure 2B, upper panel)**. The nature of the observed weak signal was assessed by
151 LC/MS analysis in multiple reaction monitoring (MRM) mode **(Figure 2C)**. As shown in the upper
152 panel, the use of specific precursor ion [M-H]⁻ at m/z 547.1, characterizes of dTDP-dHex, showed
153 two signals with RT similar to dTDP-Rha and dTDP-6-d-Tal. Fragmentation of the minor signal
154 produced a MS/MS spectrum identical to standard dTDP-6-d-Tal, demonstrating that the minor
155 signal corresponds to dTDP-6-d-Tal **(Figure 2C, lower panel)**. Finally, NMR analysis of the total
reaction mixture showed signals characteristic of dTDP-6-dTal, along with signals attributed to the substrate dTDP-Rha, further demonstrating that MAB_4111c transforms dTDP-Rha into dTDP-6-d-Tal (Figure 2D). Specifically, weak proton signals at 5.17, 4.05, 3.82, and 3.75 ppm are characteristic signals of dTDP-6-d-Tal. According to the NMR spectrum of the dTDP-6-d-Tal standard, proton signals were elucidated as H1 (δ5.17 ppm), H2 (δ4.05 ppm), H3 (δ3.82 ppm) and H5 (δ3.75 ppm) of 6-d-Tal. The signal of H-4 proton (δ3.65 ppm) in 6-d-Tal is overlapped with H-4 proton signal of Rha. Kinetic analysis indicated that MAB_4111c could convert dTDP-Rha into dTDP-6-d-Tal with a 3-4% conversion rate within an hour, reaching a 7% conversion rate after 300 min of incubation (Figure 2E). To address the specificity of the reaction, a similar assay was set up by replacing dTDP-Rha by dTDP-Glc in the reaction mixture, where no dTDP-6-d-Tal was detected (Figure 2B, lower panel). In addition, we also assayed the reverse reaction using dTDP-6-d-Tal as a substrate and observed the formation of dTDP-Rha after 60 min of reaction (data not shown), thus confirming that MAB_4111c catalyzes both reactions.

Overall, these results suggest that MAB_4111c is an epimerase that specifically converts dTDP-Rha into dTDP-6-d-Tal *in vitro*. Given its activity, we propose to rename the protein Tle for *Talose epimerase*, based on a previous nomenclature used to designate a similar enzyme in *Acinetobacter baumannii* (42).

Structural prediction of Tle

Since we could not produce high yields of Tle for structural analysis, we employed AlphaFold 3 for structural predictions (43). We first screened the Protein Data Bank (PDB) for experimentally determined epimerase structures, most of which are dimeric. Sequence identity between Tle and available epimerase structures ranged from approximately 23-32%. Despite low sequence identity, epimerases typically exhibit high structural homology. Using AlphaFold 3, we generated a dimeric structure of Tle in the presence of NAD+. The prediction covered more than 90% of the structure with a very high predicted local distance difference test (pLDDT) score (Figure 3A). The oligomeric state prediction yielded predicted template modelling (pTM) and interface predicted template modelling (ipTM) values of 0.91 and 0.92, respectively, indicating that the predicted structure can be used with confidence for further analysis. The structure reveals a classic Short-Chain Dehydrogenase/Reductase (SDR) fold (44) (Figure 3B). The dimeric interface is formed by two α-helices from each protomer (residues 88-110 and 180-199), creating a four-α-helix bundle (Figure 3B). The overall architecture includes an N-terminal NAD+ binding domain, a typical
Rosmann fold and a C-terminal substrate-binding domain (Figure 3B). Comparison of the AlphaFold-generated Tle structure with the known *Pseudomonas aeruginosa* UDP-N-acetylglucosamine 4-epimerase WbpP (PDB code:1SB8) epimerase structure yielded a root mean square deviation (RMSD) of approximately 1.37 Å (Figure 3B), suggesting a high degree of structural similarity. WbpP was used because it had one of the best RMSD scores in our analysis. Notably, Tle includes an extended secondary structure element from residues 128 to 165, characterized by an additional α-helix, long loop and a pair of antiparallel β-strands. This feature contrasts with WbpP, which has a shorter loop in the corresponding region, highlighting a distinctive structural variation in Tle (Figure 3B).

We next compared the AlphaFold-predicted structure of Tle with the known WbpP structure to identify the key residues involved in catalysis. The binding site for NAD+, is highly conserved among SDR family enzymes (44). Structural alignment with WbpP confirmed that majority of the NAD+ binding residues are conserved in Tle (Figure S1A). However, some differences were noted: V30, H32, and H36 in Tle correspond to N48, A50, and G52 in WbpP (Figure S1B). The catalytic triad, which is typically represented as SYK in the SDR family (44–46) is conserved in Tle as S121, Y181, and K185 (Figure S1B). No activity was observed when replacing the wild-type enzyme by the Tle-Y181A mutant in the assay (Figure 2B and 2D), thus indicating that introducing a Y181A change leads to a catalytically inactive enzyme. We also assessed the activity of Tle mutated proteins in which the conserved residues S121, N210, R249 (Figure 1C and S1B) were substituted by alanines. As for the His-tagged wild-type protein, the different mutated variants were enriched by affinity chromatography on Ni2+ beads and the amount of each protein visualized by Western blotting using anti-His antibodies (Figure S2A). The relative concentration of each protein was determined by comparing the grayscale of the bands using ImageJ and adjusted so that the same quantity of each protein was added to the assay in the presence of NADP+ and dTDP-6-d-Tal. Figure S2B shows that none of these mutated proteins catalysed the formation of dTDP-6-d-Tal, confirming the importance of S121, N210, and R249 in the activity of Tle.

Overall, these data validate the AlphaFold model and supports the structural similarity between WbpP and Tle.

**Bacteriological characterization of a tle deletion mutant in M. abscessus**

To investigate the contribution and biological functions of the dTDP-L-Rha 4-epimerase in M. abscessus, *tle* was deleted in the GPL-producing S variant of M. abscessus, using a unmarked
deletion method (47) (Figure S3A). PCR/sequencing performed on the parental and mutant strain (Δ*tle*) using the primers listed in Table S1 confirmed the proper genotype of the mutant (Figure S3B). Genetic complementation of the mutant was done through specific integration at the *attB* chromosomal site (48) of a copy of *tle* fused to a GFP-tag at the 3'-end and placed under the control of the endogenous promoter. Probing the crude lysates from the various strains using anti-GFP antibodies revealed a single band of the expected size, corresponding to the Tle-GFP fusion protein, validating complementation of the mutant (Figure S3C).

A previous work had shown that deletion of *gtf1* in *M. abscessus* S was associated with R morphotype on agar plates (35). Observation of individual colonies showed that *Δtle* formed R, corded colonies (Figure 4A). Complementation of *Δtle* (*Δtle::C*), rescued the S morphotype (Figure 4A). The growth of all strains in liquid culture describes a logistic growth (R²>0.9 for all of them) (Figure 4B and Table S4). The growth rate of *Δtle* was higher than that of the R strain (p<0.0001), while the one of *Δtle::C* was similar to the growth rate of the parental S strain. This indicates that deletion of *tle* slightly impacts on the replication rate of *M. abscessus* in planktonic culture. However, additional transmission electron microscopy observations failed to show significant changes regarding the size and ultrastructural organization of the cell wall in *Δtle* (Figure S4). Partitioning of mycobacterial pellets between hexadecane and aqueous buffer is used as a quantitative marker of cell surface hydrophobicity in mycobacteria to associate low pathogenicity with reduced hydrophobicity (49). To address whether deletion of *tle* affects surface hydrophobicity, aqueous hexadecane-buffer partitioning was applied to the S, R, *Δtle*, and *Δtle::C* strains. *Δtle* appeared more hydrophobic than the parental S strain, in agreement with its R morphotype while the complemented strain displayed a hydrophobicity level similar to the parental S progenitor, correlating with its smoother morphological aspect (Figure 4C). Consistent with the ability of the *M. abscessus* S variants to slide from the centre towards the periphery of the plates (11, 28), *Δtle::C*, but neither R nor *Δtle*, showed a higher sliding motility (Figure 4D and 4E). Since sliding is dependent on the bacterial surface properties and interactions with the substrate, this suggests that *Δtle* is likely defective in surface-associated cell wall components. The *Δtle* colony biofilm was intermediate between the S and R biofilms (Figure 4F), being broader and higher than the S biofilm (Figure 4F, 4G and S5). In addition, the number of CFUs per membrane (Figure 4H), the colony volume (Figure 4I) and density of the biofilm (Figure 4J) were very similar between the R and *Δtle* strains. *Δtle::C* showed no difference with the S progenitor.
We next investigated whether *t/e* deletion affects drug susceptibility of *M. abscessus* towards a large panel of clinically used antibiotics. *Δt/e* showed only very slight changes in its antibiotype (Table S5), for instance a slight increase against amikacin (MIC of 16 µg/mL for the wild-type strain and 32 µg/mL for *Δt/e*), tigecycline (MIC of 0.5 µg/mL for the wild-type strain to 1 µg/mL for *Δt/e*), and a slight reduction against imipenem (MIC of 64 µg/mL for the wild-type to 32 µg/mL for *Δt/e*), clarithromycin (MIC of 8 µg/mL for the wild-type strain and 4 µg/mL for *Δt/e*) or rifabutin (MIC of 64 µg/mL for the wild-type strain and 32 µg/mL for *Δt/e*).

### Deletion of *t/e* leads to the production of GPL lacking 6-d-Talose

We next evaluated the impact of *t/e* deletion on the glycolipid content extracted from lyophilized mycobacteria. Analysis of the apolar lipid fraction by TLC shows that trehalose 6,6'-dimycolate (TDM) remains equally produced in all tested strains (Figure 5A). Analysis of the polar lipid fractions indicates that, except in the R strain lacking GPLs (33, 35), GPLs are produced at high levels in all strains (Figure 5A, left panel). However, while the parental S strain and *Δt/e::C* displayed an identical GPL profile, *Δt/e* exhibited lower migrating GPLs, resembling to those produced by *Δgtf1* (Figure 5A right panel and S6A), a previously characterized mutant lacking the Gtf1 glycosyltransferase, which adds 6-d-Tal to the peptide backbone (35). Mild alkaline hydrolysis deacetylated polar lipids from S and *Δt/e::C*, as shown by the lower R~f~ of NaOH-treated compared to untreated ones GPL (Figure 5B right panel). In contrast, saponification did not influence the GPL pattern of *Δt/e* and *Δgtf1* (Figure 5B right panel and S6C), suggesting that they are not acetylated, as previously demonstrated for *Δgtf1* (35). Mass spectrometry analysis of native polar lipids from S and *Δt/e::C* showed ions at *m/z* 1258/1286 and 1404/1432 (Figure 5C), previously identified as GPL-2a and GPL-3 (Figure 1A) (33, 34). After saponification, M-84 u.m.a ions at *m/z* 1173/1201 and 1319/1347 were detected, corresponding to dGPL-2a and dGPL-3, respectively, in agreement with the loss of the two acetyl groups substituting 6-d-Tal (Figure 5C and S6C-D). MS spectra of native and saponified polar lipids isolated from *Δt/e* and *Δgtf1*, all showed an intense ion at *m/z* 1187 accompanied with 1013/1041 pattern (Figure 5C and S6C-D), previously attributed to the unnatural isomers GPL-2b and GPL-1b, which are devoid of 6-d-Tal (35). The absence of the 6-d-Tal was further confirmed by the monosaccharide composition analysis of the polar lipids, which demonstrates the lack of 6-d-Tal in *Δt/e* and *Δgtf1* and its restoration in *Δt/e::C* (Figure 5D and S6E).
284 Distribution of *tle* orthologs in NTMs and other bacteria

285 We identified and analyzed 107 proteins orthologus to Tle considering the inclusion and exclusion
286 criteria described in the Materials and Methods. The neighbour-joining phylogenetic tree showed
287 two clear clades related to mycobacterial genes and other bacterial species. Orthologous were
288 found in at least 26 bacterial species belonging to the phyla Actinomycetota (genera
289 Nocardioides, Pseudonocardia, Desertimonas, Glaciibater, Curtobacterium, and Cryobacterium)
290 and Pseudomodota (genera Burkholderia, Paraburkholderia, Xanthomonas, Rhizobium, and
291 Chitinimonas). Seventy-nine mycobacterial species, excluding M. abscessus, possess an
292 orthologous gene to *tle* based on amino acid sequence analysis (Figure 6A). These included 42
293 slowly-growing mycobacteria, one intermediately-growing mycobacterium and 36 rapidly-
294 growing mycobacteria. This phylogenetic analysis indicates that Tle-related proteins can be found
295 in bacteria but are over-represented in mycobacteria. It also suggests that these orthologs may
296 have descended from a common ancestor and, therefore, that they can be viewed as orthologous
297 members of the same protein family.

298 In mycobacteria, 6-d-Tal has been only described in GPLs so far. We next investigated the
299 glycolipid profile, focusing on GPL from a set of slow-growing and rapidly-growing NTM clinical
300 isolates, which are representative strains present in the phylogenetic tree (Figure 6A). TLC
301 analysis of the apolar lipid fraction of all species from a broad panel showed an intense band
302 around R<sub>f</sub> 0.43 that was attributed to TDM (Figure S7A). TLC analysis of polar lipids in
303 chloroform/methanol/water (65:25:4, v/v/v) also showed phospho-*myo*-inositol mannosides
304 (PIMs) in all species (Figure S7B). Subsequent TLC analysis using chloroform/methanol/water
305 (90:10:1, v/v/v) as solvent revealed a wider lipid diversity, including GPL-like compouds that were
306 differentially expressed (Figure 6B). Overall, of the fifteen strains tested, ten strains (marked +)
307 showed bands corresponding to GPL, whereas five (marked –) did not. Some strains showed
308 similar TLC patterns such as M. avium and M. gilvum or M. genavense and M. lentiflavum,
309 suggesting that these pairs of strains have close GPL profiles. The other GPL-positive strains (M.
310 abscessus, M. aurum, M. goodii, M. simiae, M. moriokaense and M. paraffinicum) showed distinct
311 TLC patterns, indicating a large structural variability in their GPL content. As a control, we checked
312 that the identified GPL-related compounds were still detected as lower R<sub>f</sub> bands following
313 saponification. In contrast, all bands disappeared in M. branderi, M. chelonae, M. mageritense,
314 M. malmoense and M. scrofulaceum further confirming the absence of GPLs in these species
315 (Figure S8). We further established by GM/FID and GC/MS the monosaccharides composition of
the native polar lipid fractions from all the species in order to confirm the species-specific expression of GPLs (Figure 6C). All ten identified GPL-positive species expressed a wide range of deoxyhexose residues, including 6-d-Tal and its methylated-analogues (2-O-methyl 6-d-Tal, 3-O-methyl 6-d-Tal), and Rha with methylated analogues (3-O-methyl Rha, 3,4 di-O-methyl Rha, 2,3,4 tri-O-methyl Rha) in different ratios. The 6-d-Tal or its methylated forms were detected in all strains, whereas Rha or its methylated forms were detected in all strains except *M. paraffinicum* that exclusively expressed 6-d-Tal. The presence of 3-O-methyl 6-d-Tal in *M. genavense*, *M. lentiflavum*, *M. simiae* and *M. moriokaense* has already been observed in previous studies, in agreement with the present work. In contrast, the polar lipids of three of the five GPL-negative strains (*M. branderi*, *M. chelonae*, *M. mageritense*) did not show any traces of deoxyhexoses. Finally, *M. malmoense* and *M. scrofulaceum* showed low quantities of 6-d-Tal, Rha and 3-O-methyl Rha, suggesting that these two strains may synthetise deoxyhexose independently of GPL. Altogether, these results indicate the exclusive presence of 6-dTal in all GPL-like molecules in mycobacteria, albeit with traces found in *M. scrofulaceum*.

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### Deletion of *tle* in *M. abscessus* results in increased pathogenicity in zebrafish

*M. abscessus* S and R variants have very different infection outcomes when injected in zebrafish embryos (27, 30). While R leads to acute infection and high embryo mortality, S is less virulent and more associated with chronic persistent infection. Therefore, because we observed a R morphotype upon knocking-out *tle*, we wanted to use the zebrafish model to evaluate the effect of this mutation on the infection outcome. To do so, 30 hpf embryos were infected with approximately 250 CFU of either R, S, $\Delta tle$, $\Delta tle::C$ (all strains expressing a red fluorescent marker) or the PBS control, and monitored for embryo survival, bacterial burden and whole embryo imaging as outlined in Figure 7A. As expected, mortality of embryos injected with $\Delta tle$ was much higher than the S wild-type progenitor (10% and 42%, respectively) and more like the R morphotype (40%) at 12 dpi (Figure 7B). Complementation was only partial, similar to what was observed *in vitro*, with 21% of embryo mortality. In agreement with the enhanced embryo mortality, bacterial burden was significantly higher in embryos infected with $\Delta tle$, to levels close to the R strain, compared to S progenitor while infection with $\Delta tle::C$ led to bacterial loads comparable to those found with the S strain (Figure 7C). These results were confirmed by embryo imaging, where we can clearly see very small infection foci in embryos injected with S and the

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348 to the R, with a higher number of infected foci as well as a bigger size.
349 DISCUSSION

350
351 6-deoxy-Talose is a rare deoxyhexose found in Gram-negative, Gram-positive bacteria and
352 mycobacteria as a component of the cell wall and capsule structures (10, 35, 38, 42). The
353 activated nucleotide sugar form of 6-d-Tal is dTDP-6-d-Tal, which is synthesised from Glc-1-
354 phosphate and dTTP via the Rml pathway. Until now, the origin of the dTDP-6-d-Tal used by Gtf1
355 for talosylation of GPL remained unknown. Based on the structural predictions with validated
356 epimerases and the development of an enzymatic assay, we demonstrate here that
357 MAB_4111c/Tle catalyses the epimerisation of dTDP-Rha to dTDP-6-d-Tal, an activity lost when
358 the catalytic Y181 or substrate-binding residues are replaced with alanine. Since the
359 epimerization to TDP-6-d-Tal appears only partial in vitro, it is possible that in vivo, the activity of
360 Tle is coupled with and/or regulated by the cognate glycosyltransferase Gtf1, previously shown
361 to attach the talose residue to the GPL peptidic backbone (35).

362
363 Genomic analyses further showed that *tle* orthologues are widely distributed in actinomycetes,
364 and was found in approximately 42% of all mycobacterial species for which a genome sequence
365 is available (52). Even though GPL from various species appear structurally different, our
366 compositional analysis indicates that all contain 6-d-Tal (or 2-*O*-methyl-6-d-Tal or 3-*O*-methyl-6-
367 d-Tal) and that these species all possess a *tle* gene. Conversely, a few species failed to produce
368 GPLs despite the presence of *tle*, although it is very likely that these strains (at least some of
369 them) do not produce GPL due to the presence of mutation in the *gpl* biosynthetic and transport
370 gene cluster, as demonstrated previously in *M. abscessus* (13). *M. avium* represents another
371 typical example of an NTM producing GPL which encodes a *tle* gene. Interestingly, the lack of 6-
372 d-Tal attached to the *allo*-threonine in a R variant of *M. avium* MAC 104 has been previously
373 reported (53), and the genetic lesion responsible for the loss of 6-dTal in this strain corresponded
374 with the deletion of a genomic region containing *gtfA*, an ortholog of *gtf1* (54). This further
375 supports the hypothesis that the loss of 6-d-Tal conditioning a R morphotype is not specific to *M.
376 abscessus* but can be extrapolated to other NTMs.

376
377 To validate the structure-function relationship between Tle and 6-d-Tal-containing GPL, we
378 generated a *tle* unmarked deletion in the *M. abscessus* S variant. A thorough lipid analysis clearly
379 emphasized the absence of 6-d-Tal in the GPL of the mutant strain, while the presence of Rha
380 residues remained intact. Previously, we characterized a *gtf1* mutant, which, similarly to the *tle*
381 mutant, produced GPL lacking 6-d-Tal. Gtf1 uses dTDP-6-d-Tal produced by Tle, as a substrate to
glycosylate the D-*allo*-Thr residue of GPL. In addition to the loss of 6-d-Tal, both $\Delta gtf1$ and $\Delta tle$ share in common other phenotypes. First, deletion of *tle* produces a virtually R morphotype, although it appeared creamy on plates, while deletion of *gtf1* generated colonies with a R and dry texture. Whereas the vast majority of clinical isolates of the R morphotype have mutations in the biosynthetic genes *mps1*, *mps2*, or transporter genes *mmpL4a*, *mmpL4b*, *mmpS4* (15), GPL glycosylation can also lead to colony morphological changes (10). This study shows that enzymes involved in the production of nucleotide-activated sugars can also lead to these morphological changes. However, it remains to be established whether mutations in *tle* are responsible for the acquisition of an R morphotype occurring during infection in the host. Second, while GPL are present in the outermost mycobacterial layer (10), their presence/absence or modifications in their structures can profoundly alter the surface properties of *M. abscessus*. Sliding motility may play an important role in surface colonisation by mycobacteria in the environment and in the host (35). The *tle* mutant showed considerably reduced sliding motility. Previous studies between S and R strains underscored the correlation between low GPL production and enhanced hydrophobicity with virulence (2, 10). As expected for a R variant, we found that $\Delta tle$ was more hydrophobic than the parental S variant, as reported previously for the *gf1* mutant (35). Third, the development of biofilms appears crucial for *M. abscessus* infections (55, 56). The deletion of *tle* altered the biofilm development of *M. abscessus*. Although most of the measured parameters (number of bacteria, colony volume and density) indicated that the *tle* mutant behaved similarly to the R morphotype, its colony shape was significantly different from that of the S and R morphotypes, as the colony height of the *tle* mutant was greater and colony diameter was less wide than that of the reference R strain. This emphasizes the importance of 6-d-Tal for shaping *M. abscessus* colony-biofilms. Herein, we also took advantage of using the zebrafish model of infection as a reliable and manageable model for revealing and comparing the difference in virulence between the parental S and the $\Delta tle$ strains (27, 57). Our results are consistent with the previous findings obtained with $\Delta gtf1$ (35) and indicate that, in the presence of the sole innate immunity, the loss of 6-d-Tal in GPL translates into increased pathogenesis and lethal infection in zebrafish embryos.

Overall, this study also emphasizes the biosynthetic interconnection between the arabinogalactan and GPL pathways in *M. abscessus*, as depicted in **Figure 8**. The mycobacterial cell wall consists of a mycolic acid layer that is bound to peptidoglycan *via* the polysaccharide arabinogalactan. Arabinogalactan is covalently bound to the peptidoglycan *via* the $\alpha$-L-
rhamnopyranosyl-(1→3)-α-D-*N*-acetyglucosaminosyl-1-phosphate unit (58). On the one hand, the donor dTDP-Rha, used by the rhamnosyltransferase WbbL to produce the rhamnosyl-containing linker unit (59), is synthesized by four enzymes (RmlA-D) beginning with dTPP and Glc-1-phosphate. TDP-Rha biosynthesis is essential for the growth of mycobacteria (60), prompting to the development of dTDP-Rha inhibitors for new tuberculosis therapeutics (61). On the other hand, the donor dTDP-Rha is also used by the rhamosyltransferases Gtf2 and Gtf3, which add the first and second Rha to the GPL biosynthetic intermediates. That dTDP-Rha stands on the crossroad between the arabinogalactan and GPL pathways is further substantiated by its direct use by Tle, which converts dTDP-Rha into dTDP-6-d-Tal, which is itself used by Gtf1 to talosylate the rhamnosylated GPL intermediates. However, while arabinogalactan (and disaccharide linker unit) is fundamental to the structural integrity of the cell wall and required for mycobacterial viability, this is not the case for the GPLs, which are lacking in the R strains of *M. abscessus*. However, the loss of GPL has important consequences on *M. abscessus* pathogenesis and infection outcome, as shown in various animal models (26, 27, 30, 35) and in humans (18, 24).
427 EXPERIMENTAL PROCEDURES

428

429 Mycobacterial strains, growth conditions, and reagents

430 *M. abscessus* CIP104536T (S) and CIP104536T (R) strains used in this study were previously sequenced and their genomic differences identified and characterized (13, 62). All bacterial strains and plasmids used for this study are listed in **Table S2** and **Table S3**, respectively.
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432 Mycobacteria were grown in Middlebrook 7H9 broth (BD, New Jersey, USA) supplemented with 0.025% tyloxapol and 10% oleic acid-albumin-dextrose-catalase enrichment (OADC) (7H9<sup>OADC</sup>) or
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434 on Middlebrook 7H10 agar (BD) containing 10% OADC (7H10<sup>OADC</sup>) at 37 °C, with antibiotics when
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436 required. A Bio-Rad Gene pulser (25 µF, 2500 V, 800 Ω) was used to transform electrocompetent
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438 mycobacteria. After transformation, strains carrying the pMV306 (48) derivatives were selected
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440 on 250 µg/mL kanamycin.

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440 Expression of Tle in E. coli

441 *MAB_4111c* (*tle*) was PCR-amplified using *M. abscessus* genomic DNA using the specific primers
442 (**Table S1**, primers 9-10) and Q5 polymerase. The amplicons were cloned into pET30 restricted
443 with KpnI and EcoRI, enabling to introduce the genes in frame with the six-histidine tag. The
444 resulting pET30-*tle* was used for site-directed mutagenesis of specific amino acids involved in the
445 active site (Y181, primers 11-12) or the substrate-binding sites (S121, primers 13-14; N210,
446 primers 15-16; R249, primers 17-18) using the QuikChange II Site-Directed Mutagenesis Kit
447 (Agilent) and based on structural similarity with the *B. thailandensis* dTDP-L-Rha 4-epimerase
448 (UniProt ID: Q2SYH7 · WBIB_BURTA). The pET30 constructs containing the native or mutated *tle*
449 genes were used to transform *Escherichia coli* strain BL21 Star DE3 (Life Technologies). Cultures
450 were grown in terrific broth medium containing 50 µg/mL kanamycin at 37 °C under agitation
451 until an optical density at 600 nm (OD600) of 0.7-0.9 was achieved. Cultures were placed at 4 °C
452 for 30 min before addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and an
453 overnight incubation at 16 °C under agitation. Bacteria were collected by centrifugation and
454 pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 8, 400 mM NaCl, 5% glycerol,
455 supplemented with 10 mg/mL of NP-40, 78 µg/mL of dithiothreitol, 1 pill of cOmplete tablets,
456 Mini EDTA-free, EASYpac per 50 mL, 2.175 mg/mL of imidazole and 1mg/mL of lysozyme)
457 previously chilled on ice. Bacteria were lysed by sonication and the lysate clarified by
458 centrifugation (12,000 rpm at 4°C for 30 min), incubated overnight at 4 °C with nickel beads (Ni
Sepharose 6 Fast Flow, GE Healthcare), washed using washing buffer I (50 mM Tris-HCl pH 8, 350 mM NaCl, 5% glycerol, 78 µg/mL of dithiothreitol, 1 pill of cOmplete tablets, Mini EDTA-free, EASYpac per 50 mL, and 1.3 mg/mL of imidazole) and in washing buffer II (50 mM Tris-HCl pH 8, 200 mM NaCl, 5% glycerol, 78 µg/mL of dithiothreitol, 1 pill of cOmple tablets, Mini EDTA-free, EASYpac per 50 mL and 1.3 mg/mL of imidazole). Proteins were eluted in elution buffer (50 mM Tris pH=7, 100 mM NaCl, and 5% glycerol, and 17 mg/mL of imidazole) and then dialyzed overnight at 4 °C in dialysis buffer (50 mM Tris-HCl pH7, 200 mM NaCl, and 5% glycerol).

466

Enzymatic activity assay

A 50 µL reaction mixture (in 50 mM of PBS buffer pH 7.5) containing 10 mM of dTDP-L-Rha, 1 mM of NADP⁺, and Nickel-beads eluted fractions enriched with the various MAB_4111c protein variants (0.42 µg/µL MAB_4111c, 0.31 µg/µL MAB_4111c-Y181A, 0.86 µg/µL MAB_4111c-S121A, 0.70 µg/µL MAB_4111c-N210A, 0.75 µg/µL MAB_4111c-R249A, and 0.69 µg/µL MAB_4111c-R249A/N210A/S121A) was incubated at 37 °C for 1 hr. The reaction was stopped by diluting the reaction with cooled buffer of acetonitrile/100 mM aqueous ammonium acetate pH 4.5 (60% acetonitrile). As a control reaction, a similar assay was performed by replacing dTDP-L-Rha by 10 mM of dTDP-D-Glc. The diluted solution was analyzed by HPLC equipped with UV detector at 254 nm using ZIC®-cHILIC column. The column was eluted at 40 °C with acetonitrile/100 mM aqueous ammonium acetate pH 4.5 (60% acetonitrile) at a flow rate of 0.6 mL/min. The enzyme activity was determined by the conversion of the product (Yield = [dTDP-6-deoxy-L-Tal]/([dTDP-6-deoxy-L-Tal] +[dTDP-L-Rha]). To analyse the activity by NMR, a reaction mixture consisting of 500 µL deuterium oxide(D₂O) containing 10 mg of dTDP-L-Rha, 5 mg of NADP⁺, 5 mg of disodium hydrogen phosphate (Na₂HPO₄), and enriched MAB_4111c protein variants (0.14 µg/µL MAB_4111c, 0.13 µg/µL MAB_4111c-Y181A) was incubated at 37 °C for 5 hrs.

For the time-curve analysis, a 100 µL reaction mixture (in 50 mM of PBS buffer, pH 7.5) containing 10 mM of dTDP-L-Rha, 1 mM of NADP⁺, and 0.42 µg/µL of MAB_4111c was incubated at 37 °C for different time point. The reaction was stopped by diluting the reaction with cooled buffer of acetonitrile/100 mM aqueous ammonium acetate pH 4.5 (60% acetonitrile). The diluted solution was analyzed by HPLC equipped with UV detector at 254 nm using ZIC®-cHILIC column. The column was eluted at 40 °C with acetonitrile/100 mM aqueous ammonium acetate pH 4.5 (60% acetonitrile) at a flow rate of 0.6 mL/min. All dTDP-activated sugar nucleotides were produced enzymatically, as reported previously (63).

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LC-MS/MS analysis was performed on a triple quadrupole mass spectrometer LCMS8060XS (Shimadzu, Kyoto, Japan) coupled with a Nexcera system (Shimadzu, Kyoto, Japan). Chromatography was performed on a ZIC-cHILIC column as previously described (2.1 mm i.d. x 150 mm, 1.7 µm; SeQuant, Sweden) (64, 65). The mobile phases were: (A) 20 mM acetate buffer (pH 4.5) containing 90 % acetonitrile, and (B) the same buffer. The elution gradient was as follows: 12-18 % linear gradient of buffer B for 12 min; 18-25 % linear gradient of buffer B for 5 min; 25-55 % buffer B for 5 min; and 55 % buffer B for 5 min. The flow rate was maintained at 0.2 mL/min. Analysis of dTDP-rhamnose and dTDP-d-Tal was performed in the multiple reaction monitoring mode using the specific precursor ion [M-H]⁻ at *m/z* 547.1 and product ion pairs at *m/z* 321.1. Product ion scanning was also conducted by MS/MS analysis of the precursor ion [M-H]⁻ at *m/z* 547.1.

Structural modeling of Tle using AlphaFold 3

The dimeric structure of Tle was predicted using AlphaFold 3 (43). Two identical sequences of Tle, each comprising 353 amino acids, along with two copies of the NAD⁺ ligand, were input into the AlphaFold 3 web server (https://alphafoldserver.com/). The server produced five model outputs. The model with the highest predicted local-distance difference test (pLDDT) score was selected for further analysis. The pLDDT score ranges from 0 to 100, where 0 indicates the lowest confidence and 100 the highest confidence in the structural prediction. Additionally, for multimeric targets, AlphaFold 3 provides predicted template modelling (pTM) and interface predicted template modelling (ipTM) scores, both ranging from 0 to 1, indicating the confidence in the predicted oligomeric state of the protein.

Deletion of *tle* and complementation in *M. abscessus*

The deletion mutant was generated in the S morphotype of the reference strain CIP104536T (62) using the suicide vector pUX1-*katG* by double homologous recombination (47). Briefly, the upstream and downstream gene regions were PCR-amplified using the primers listed in Table S1 and ligated into the Pacl/Nhel-linearized pUX1-*katG*. After transformation, bacteria were selected on 7H10<sup>OADC</sup> supplemented with 250 µg/mL kanamycin with a visual screening of red fluorescent colonies, which have undergone the first homologous recombination. The second homologous recombination event was induced by isoniazid counter-selection and selected on 7H10<sup>OADC</sup> with 50 µg/mL isoniazid and screening for non-fluorescent colonies and susceptibility
523 to kanamycin (35, 47). Complementation was performed by introducing a complementation
524 plasmid generated using the integrative pMV306 (Table S3). Genes in fusion with a super folder
525 green fluorescent protein (sfGFP)-tagging sequence were amplified by PCR under the control of
526 the endogenous promoter. Proper gene deletion and all plasmids were verified by PCR and DNA
527 sequencing.

528
529

530 Fifty millilitres of cultures were grown in minimal salt medium (1 g/L KH2PO4, 500 mg/L NaCl, 2 531 g/L Na2HPO4, 1 g/L NH4Cl, 200 µM CaCl2, 2 mM MgSO4, 5% glycerol, and 0.02% tyloxapol). The 532 bacterial pellet was washed twice with PBS supplemented with 0.025% tyloxapol (v/v) and 533 resuspended in PBS supplemented with cOmplete™ protease inhibitors cocktail (Roche, Sigma 534 Aldrich) and disrupted by bead beating using 1-mm diameter glass beads and a Mixer Mill MM 535 301 (Retsch, Germany) for two pulses of 3 min at 30 Hz. Protein concentration was determined 536 using the BCA Protein Assay Reagent kit (Thermo Fisher Scientific), according to the 537 manufacturer's instructions. Equal amounts of proteins (20 µg) were separated by 12% SDS- 538 PAGE, transferred onto a nitrocellulose membrane, probed for 1 hr with either mouse anti-GFP 539 (dilution 1:1000; Sigma) (antibodies-online GmbH, Aachen, Germany) or mouse anti-Antigen 85 540 (dilution 1:20; loading control) antibodies. Membranes were washed with PBS supplemented 541 with 0.02% Tween20 (w/v) and incubated for 45 min with goat anti-mouse antibody conjugated 542 to HRP (dilution 1:5000; Abcam). Bands were revealed using a SuperSignal West Femto 543 (ThermoFisher Scientific) and a ChemiDoc MP system (Bio-Rad laboratories). For detection of the 544 MAB_4111c variants produced in E. coli, the membrane was probed with anti-His mouse 545 monoclonal antibodies (dilution 1:1000) and incubated with HRP-labeled goat anti-mouse 546 IgG(H+L) (dilution 1:2000). 547

548
549

550 Growth curves were established based on cultures grown for 72 hrs and diluted in 7H9OADC to
551 reach an initial OD595nm of 0.05 and dispensed in flat-bottom 96-well microtiter plates (200 µL per
552 well). Plates were statically incubated at 37 °C and measurements were taken on a daily basis
553 using a spectrophotometer multimode microplate reader (Tecan Spark 10M, Tecan Group Ltd.,
Switzerland).
Colony morphology was assessed from 72 hrs cultures in 7H9<sup>OADC</sup> and streaked on 7H10<sup>OADC</sup>.

Plates were incubated at 37 °C for 5 days and photographed using a Zeiss microscope equipped
with a Zeiss Plan Neo Fluor Z13/0.25 FWD objective and an Axiocam503 monochrome camera
(Zeiss, Jena, Germany). Images were processed using ZEN 2 (Blue Edition).

M. abscessus biofilm formation was performed following a modification of the colony-biofilm model previously described (66). Briefly, M. abscessus cultures were grown in Middlebrook 7H9<sup>OADC</sup>, 0.2% glycerol and tyloxapol (0.025%) at 37 °C and 80 rpm for 72 hrs. Cultures were centrifuged at 3,500 rpm for 5 min, washed two times with PBS and then diluted to an OD<sub>600nm</sub> of 0.5 (~1.5×10<sup>8</sup> CFU/mL). White black, polycarbonate membranes (diameter, 13 mm; pore size 0.2 µm, Whatman, Merck, Darmstadt, Germany) were placed on 7H10<sup>OADC</sup> and inoculated with 10 µL of the bacterial suspension. The membrane-supported biofilms were statically incubated for 5 days at 37 °C. Afterwards, the membrane-supported biofilms were imaged and processed using a ZEISS Axio Zoom V16 binocular equipped with a lighting device (Zeiss HXP 200C Zeiss, Germany at 7× zoom, detecting the grey level. On biofilm fluorescence pictures, a straight line was drawn (covering all the diameter of the biofilm-colony) and grey-level intensity (GLI) of each pixel as retrieved to draw GLI spectra using ZEN 2 software (Blue Edition). The colony volume was calculated by using the half volume of an ellipsoid: Colony volume = 2/3 ×π×GLIM×(CD/2)<sup>2</sup>, where GLIM is the GLI maximum and CD is the colony diameter in pixels. After taking pictures, each membrane-supported biofilm was processed for quantifying the number of CFU/membrane. Colony-biofilm density was estimated as a coefficient resulting of CFU/membrane and the colony volume.

Sliding motility of each strain was evaluated using a modified methodology previously described (28). For each strain, 10 µl of 24 hrs cultures (OD<sub>600nm</sub> = 0.9–1.0) were dropped in the centre of well from a 6-well containing 7H9 broth with 0.3% agar and without adding any carbon source. The sliding distance was evaluated and measured after incubation for 10 days at 37 °C under humidified conditions. This experiment was performed by using three biological replicates and two technical replicates.

581

582 Hydrophobicity assay

Mycobacterial surface hydrophobicity was determined following the methodologies developed previously (49). Each strain was seeded on 7H9<sup>OADC</sup> at 37 °C for 72 hrs, and then some colonies were resuspended and sonicated in 5 mL of 0.9% NaCl in PYREX® 16 × 100 mm disposable round-
bottom threaded culture tubes (Corning) before measuring the OD<sub>595nm</sub> (A<sub>0</sub>) of 200 µL in triplicate was measured using spectrophotometer multimode microplate reader (Tecan Spark 10M). Subsequently, 2 mL of hexadecane (Sigma Aldrich) was added to glass tubes, and the mixtures were vortexed for 30 s. After phase separation (~20 min), the OD<sub>595nm</sub> of 200 µL in triplicate was measured using a multimode microplate reader. Subsequently, the aqueous phase (A<sub>1</sub>) was measured again and compared to the organic phase. The percentage of cell surface hydrophobicity (H) of each strain immersed in the organic hexadecane phase was calculated using the equation H (%) = [(A<sub>0</sub> - A<sub>1</sub>)/A<sub>0</sub>] × 100. This experiment was performed by using three biological replicates and three technical replicates.

### Transmission electron microscopy

72-hrs cultures were washed with PBS, immersed overnight at 4 °C in PHEM buffer containing 2.5% glutaraldehyde (pH 7.4), rinsed in PHEM buffer and post-fixed in 0.5% osmium acid and 0.8% potassium ferricyanide trihydrate for 2 hrs at room temperature and in the dark. After washing twice with PHEM buffer, mycobacteria were dehydrated in a graded series of ethanol solutions (30-100%) and embedded in EmBed 812 using an Automated Microwave Tissue Processor for electron microscopy (Leica). Seventy nanometre sections were cut at different levels of each block/stem using a microtome (Leica-Reichert Ultracut E, Leica), counterstained in 70% ethanol with 1.5% uranyl acetate and lead citrate and observed using a Tecnai F20 TEM at 120KV.

### Drug susceptibility testing

Antimicrobial susceptibility was tested using broth microdilution following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (67). For this purpose, 96-well RAPMYCOI Sensititre™ titration plates (Thermo Fisher Scientific, Massachusetts, USA) were used following the manufacturer's recommendations.

### Phylogenetic analysis of *tle* orthologous genes

Ortholog genes of *tle* in the genus *Mycobacterium* were found using BLASTp (68) against the NCBI non-redundant (nr) database by limiting the search to mycobacteria (taxid:85007) with a full bacterial scientific name registered and extending the maximum target sequences up to 1,000. In case of having more than one time the same mycobacterial species name, only the one with
the highest identity percentage was considered. All the proteins related to the Rml pathway were excluded from the analysis. Orthologs of *tle* in other bacteria (non-mycobacteria) were found in a manner similar to that described above but excluding the search to Mycobacteria (taxid:85007) and limiting the maximum target sequences to 100. The Molecular Evolutionary Genetics Analysis (MEGA) version 11 software (69) was used to generate a ClustalW multiple sequence alignment of all the sequence and to construct Neighbour-Joining tree. The obtained tree was redrawn using TreeViewer Version 2.1.0 (70) and coloured with Inkscape (71). To structurally validate the findings from the phylogenetic tree, we analysed the presence of GPL and their glycosidic composition in 14 clinical NTM isolates identified with Genotype Mycobacterium CM or AS (Hain Lifescience, Nehren, Germany) from the Department of Clinical Microbiology of the University Hospital Fundación Jiménez Díaz (Madrid, Spain).

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## 630 Lipid extraction and analysis

Rapid-growing and slow-growing mycobacteria were grown on 7H10<sup>OADC</sup> agar plates for 72 hrs and 7H9<sup>OADC</sup> broth for at least 14 days. After growing, pellets were collected and lyophilized, then 25 to 50 mg of bacterial pellets were subjected to apolar lipid extractions using petroleum ether (72). GPL were extracted from the polar fraction using chloroform/methanol/0.3% NaCl (9/10/3, v/v/v) and then with chloroform/methanol/0.3% NaCl (5:10:4, v/v/v). The combined solvent extracts were mixed for 5 min with chloroform and 0.3% NaCl (1:1, v/v) and centrifuged at 3,000 g for 5 min to separate the lower organic phase from the aqueous phase. The upper aqueous layer was discarded, and the lower organic phase was evaporated under a stream of nitrogen and resuspended in chloroform/methanol (2:1, v/v) for TLC analysis or itol-acetates derivation.

Thin-layer chromatography (TLC) analysis. Between 50-150 µg of each extract were spotted along 5 mm lane on Silica gel 60 F254 plates (Merck). GPL were separated at 4 °C using chloroform/methanol/water (90:10:1, v/v/v). More polar lipids were separated using chloroform/methanol/water (65:25:4, v/v/v). Glycolipids were revealed by spraying the plates with orcinol in 20% sulfuric acid and charring.

Glycolipids saponification. Five hundred µg of the polar lipid fraction were dried under nitrogen. Then, 1 mL of 0.1 M sodium hydroxide in chloroform/methanol (1:1, v/v) was added, and heated overnight at 37 °C. One mL of butanol and 1 mL of water were added and the mixture vortexed for 1 min. After centrifugation for 30 s, the upper butanolic phase was dried under a nitrogen stream and dissolved in 300 µL of chloroform/methanol (2:1, v/v).
Itol-acetates derivation. After addition of 1 mL of 4 M TFA the sample was heated for 4 hrs at 100 °C, dried and desiccated overnight. The monosaccharides were reduced for 4 hrs at room temperature in 500 µL NaBH₄ 10 mg/mL in 2 M NH₄ and stopped with concentrated glacial acetic acid. Samples were dried at 55 °C under a nitrogen stream by co-distillation with methanol five times, dessicated overnight. Samples were peracetylated in 500 µL anhydride acetic for 4 hrs at 80 °C. The reaction products were washed five times with chloroform/water (1:1, v/v). The chloroform-rich phase was then filtered, dried, and dissolved in 500 µL chloroform. For GC-FID, 1 µL of itol-acetate derivatives was injected in splitless mode with an automatic sampler on a Solgel 1 MS 30 m × 0.25 mm × 0.25 µm capillary column with the following gradient temperature: 120 to 230 °C at 3 °C/min, then to 270 °C at 10 °C/min. Compounds were detected either with a flame ionization detector or a single quadrupole on a HP7820 or 5975N (Agilent Technologies). Previously determined retention times of differentially methylated Rha and unmodified 6-d-Tal were used for identification (33) while electronic impact mass spectrometry was used for methylated 6-d-Tal determination.

MALDI-TOF mass spectrometry. Before spotting 1 µL on the MALDI plate, 10 µL of 20 mg/mL dihydroxybenzoïc acid (DHB) in chloroform/methanol (1:2, v/v) were mixed with 5 µL of the sample extract in chloroform/methanol (2:1, v/v). MS positive and negative spectra were acquired on an Axima Resonance (Shimadzu, Kyoto, Japan) in reflectron mode.

Zebrafish maintenance

Zebrafish (Danio rerio) were kept and handled in compliance with the guidelines of the European Union for handling laboratory animals and approved by the Direction Sanitaire et Vétérinaire de l'Hérault for the ZEFIX-CRBM zebrafish facility (Montpellier) (registration number C-34-172-39). All experiments were approved by Le Ministère de l'Enseignement Supérieur et de la Recherche under the reference APAFIS#24406-2020022815234677 V3. Eggs were obtained by natural spawning, quickly bleached, and incubated at 28.5 °C in Petri dishes containing E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄). The transgenic reporter line Tg(mpx:eGFP)il14 (73) was used in this study.

Zebrafish infection

At 24 hrs post-fertilization (hpf), embryos were enzymatically dechorionated using 1mg/mL of pronase (stock of 5mg/mL diluted in E3) and placed in 60 mm Petri dishes containing E3 at 28.5
682 °C. Microinjection was performed as previously described (74). Embryos were injected in the
683 caudal vein with ±250 CFU of either the R, S, SΔtle, carrying the pMV306-mScarlet, SΔtle::C
684 carrying the pTEC27-tdTomato or the PBS control. Injected embryos were then rinsed twice and
685 transferred individually in 48-well plates containing E3 and randomized for survival assays and
686 CFU counts. Inoculum was checked by microinjecting a PBS drop and plating it on 7H10OADC. For
687 survival assay, embryos were monitored every 24 hrs for 12 days, and marked as dead in the
688 absence of a heartbeat. Bacterial burden was determined at 2dpi, 3dpi and 5dpi as following:
689 embryos were individually manually disrupted with pellet pestles in 50 µL of PBS supplemented
690 with 0.025% of tyloxapol. Lysates were sonicated for 30 s after adding 50 µL of PBS supplemented
691 with 2% of Triton-X-100. Serial dilutions were plated on LB agar plates containing rifampicin (15
692 µg/mL) and kanamycin (250 µg/mL) and incubated for 4 days at 37 °C.
693

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**Zebrafish microscopy**

For zebrafish imaging, embryos were anesthetized with 0.02% buffered MS222 and immobilized
with 0.5% low melting agarose in a lateral position. Images were acquired with an EVOS™ M7000
Imaging System at 3dpi and 5dpi with the Olympus™ 2X Objective PlanApo 0.08 NA and the EVOS
10x Objective 0.3NA using the EVOS™ Light Cube, GFP 2.0. After imaging, embryos were rinsed
and transferred back the 48-well plate with fresh E3. Image processing was done using ZEISS
ZEN3.7 software.

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**Statistical analyses**

All analyses were performed using R commander (75–76) and/or GraphPad Prism version 9.0.0
for Windows (GraphPad Software). The normality of data was evaluated using a Shapiro-Wilk
test. Descriptive data are cited as median and interquartile range in case of non-normal
distribution for each of the variables were calculated. A non-parametric Kruskal-Wallis test was
used to compare more than three groups and a Wilcoxon test was used to compare two
groups. Zebrafish survival assays are represented in Kaplan-Meier graphs and analysed with a Log
rank test. For determination of bacterial counts, CFU were log₁₀ transformed and the significance
between multiple selected groups was determined using one-way ANOVA with Šidák's Multiple
Comparisons test after validating the normality of the data.
714 **DATA AVAILABILITY**

715 All data are contained within the manuscript and the Supporting Information. The raw data can
716 be share upon request.

717

718 **SUPPORTING INFORMATION**

719 This article contains supporting information.

720

721 **ACKNOWLEDGMENTS**

722 We are grateful to M. Plays, P. Richard, and C. Hamela for zebrafish husbandry; Pr J. Esteban from
723 the Fundación Jiménez Diaz University Hospital (Madrid, Spain) for his generous gift of NTM
724 clinical isolates; and Chantal Cazevieille (Intitut des Neurosciences de Montpellier, Montpellier,
725 France) for her technical assistance during the TEM imaging. We are grateful to the PAGes-P3M
726 core facility (PLBS US 41 - UAR 2014) for providing the scientific and technical environment
727 conducive to achieving this work.

728

729 **FUNDING**

730 This project has been funded by the French National Research Agency grants 19-CE15-0012-01
731 (SUNLIVE) and 20-CE44-0019 (ILLome), by the Fondation pour la Recherche Médicale (Equipe
732 FRM EQU202103012588) and by JSPS Core-to-Core Program No. JPSCCA202000007.

733

734 **CONFLICT OF INTEREST**

735 The authors report there are no competing interests to declare.

736

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936 67. Fröberg, G., Maurer, F. P., Chryssanthou, E., Fernström, L., Benmansour, H., Boarbi, S., Mengshoel, A. T., Keller, P. M., Viveiros, M., Machado, D., Fitzgibbon, M. M., Mok, S., Werngren, J., Cirillo, D. M., Alcaide, F., Hyyryläinen, H.-L., Aubry, A., Andres, S., Nadarajan, D., Svensson, E., Turnidge, J., Giske, C. G., Kahlmeter, G., Cambau, E., Van Ingen, J., and Schön, T. (2023) Towards clinical breakpoints for non-tuberculous mycobacteria - Determination of epidemiological cut off values for the *Mycobacterium avium* complex and *Mycobacterium abscessus* using broth microdilution. *Clin. Microbiol. Infect.* **29**, 758-764

943 68. Altschul, S. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. *Nucleic Acids Res.* **25**, 3389-3402

945 69. Tamura, K., Stecher, G., and Kumar, S. (2021) MEGA11: Molecular evolutionary genetics analysis Version 11. *Mol. Biol. Evol.* **38**, 3022-3027

947 70. Bianchini, G., and Sánchez-Baracaldo, P. (2024) TreeViewer: Flexible, modular software to visualise and manipulate phylogenetic trees. *Ecol. Evol.* **14**, e10873

949 71. Developers, I. W. Draw Freely, Inkscape. https://inkscape.org/

950 72. Besra, G. S. (1998) Preparation of cell-wall fractions from mycobacteria. *Methods Mol. Biol.* **101**, 91-107

952 73. Renshaw, S. A., Loynes, C. A., Trushell, D. M. I., Elworthy, S., Ingham, P. W., and Whyte, M. K. B. (2006) A transgenic zebrafish model of neutrophilic inflammation. *Blood.* **108**, 3976-3978
954 74. Boudehen, Y.-M., Tasrini, Y., Aguilera-Correa, J. J., Alcaraz, M., and Kremer, L. (2023) Silencing essential gene expression in *Mycobacterium abscessus* during infection. *Microbiol. Spectr.* **11**, e0283623.

957 75. Using the R Commander, https://socialsciences.mcmaster.ca/jfox/Books/RCommander/

958 76. Fox, J. (2005) The R Commander: A Basic-Statistics Graphical User Interface to R. *J. Stat. Softw.* **14**, 1–42
960 FIGURE LEGENDS

961
962 **Figure 1.** MAB_4111c belongs to the GPL biosynthetic cluster and is a predicted epimerase. (A)
963
964
965 Structure of diglycosylated (GPL-2a) and triglycosylated (GPL-3) glycopeptidolipids. The green,
966
967 blue and yellow shadings represent the lipid, peptide and glycosydic moieties of the GPL. Di-O-
968
969 acetyl 6-d-Tal is drawn in red. (B) *M. abscessus* GPL locus encoding enzymes involved in the
970
971 synthesis, modification, and transport of GPL. MAB_4111c is indicated in bold. (C) Alignment of
972
973 the protein sequence of dTDP-L-Rha 4-epimerase from the *B. thailandensis* (ATCC 700388)
974
975 (wbiB_BURTA) with that of MAB_4111c from *M. abscessus*. The orange and blue shadings
976
977 represent the non-conserved and conserved and amino acids, respectively. The red, green and
978
979 blue asterisks indicate residues involved in the putative NADH-binding site, the substrate-binding
980
981 site and the catalytic residue reported in *B. thailandensis*, respectively.

982
983
984
985
986
987
988
989
990
991 **Figure 2.** Enzymatic activity of MAB_4111c. (A) Reaction catalyzed by MAB_4111c. (B) **Upper panel:** HPLC profile of the standards and products formed during the enzymatic reaction in the presence of MAB_4111c or MAB_4111c_Y181A with dTDP-Rha and NADP⁺. The incubation was performed for 60 min. The product of the reaction is labeled with a red box. **Lower panel:** HPLC profile of standard dTDP-Glc, standard dTDP-6-d-Tal and dTDP-Glc and NADP⁺ incubated with MAB_4111c for 60 min. (C) **Upper panel:** LC/MS profile of the enzymatic reaction mixture in MRM mode using the specific precursor ion [M-H]⁻ at *m/z* 547.1. **Lower panel:** MS² fragmentation spectra of standard dTDP-6-d-Tal and the enzymatic reaction product (*). (D) ¹H NMR spectra of dTDP-L-Rha and NADP⁺; MAB_4111c with dTDP-L-Rha and NADP⁺ after 120 min; MAB_4111c_Y181A with dTDP-L-Rha and NADP⁺ after 120 min; dTDP-6-d-Tal. The characteristic peaks of dTDP-6-d-Tal are indicated with red arrows. (E) Kinetic analysis of the reaction catalyzed by MAB_4111c in the presence of dTDP-L-Rha and NADP⁺.

992
993
994
995
996
997
998
999
1000
1001
1002
1003
1004
1005
1006
1007
1008
1009
1010
1011
**Figure 3.** Structural Prediction of Tle. (A) Cartoon representation of the Alphafold 3 predicted dimeric structure of Tle. The structure is colored according to the pIDTT score range (blue=high confidence, cyan=confidence, yellow=low confidence, orange=very low confidence). (B) **Left panel:** cartoon representation of the predicted Tle homodimer, with each chain colored in green and cyan. NAD is depicted as a stick model. **Right panel:** Alignment of the monomeric structure of Tle with the WbpP crystal structure (Tle in cyan and WbpP in magenta).
**Figure 4.** Deletion of *tle* in *M. abscessus* alters colony morphology, hydrophobicity, sliding motility and biofilm-colony development. (A) Knock-out of *tle* is associated with a switch from a S to a R-like morphotype. The scale bar represents 1 mm. (B) The deletion of *tle* is associated with a higher growth rate. The experiment was performed using two biological replicates with six technical replicates per biological replicate. Deletion of *tle* modifies hydrophobicity (C) and the sliding motility (D-E). The hydrophobicity experiment was performed using three biological replicates with three technical replicates per biological replicate. The sliding motility experiment was performed using three biological replicates with two technical replicates per biological replicate. The black scale bar indicates 5 mm. (F) Representative 2- and 2.5-dimensional pictures of colony-biofilms on biofilm-supporting membranes after 5 days of incubation. The absence of *tle* alters the biofilm-colony growth (F) (the scale bar represents 2 mm) and the biofilm-colony profile (G) (green, dark grey, red and blue dots represent the profiles of S, R, Δ*tle* and Δ*tle*::C, respectively). The CFUs per membrane are shown in (H), the colony volume in (I), and the colony density in (J). This experiment was performed using five biological replicates. The error bar denotes the interquartile range. \*: p<0.05, \*: p<0.01; \*: p<0.001.

**Figure 5.** Structural analysis of the GPL content in Δ*tle*. (A) TLC analysis representative from three independent experiments of native apolar (left) and polar (right) lipid extracts. Lipids were separated using chloroform/methanol/water (90:10:1, v/v/v) followed by spraying with orcinol and charring. GPL-1b and GPL-2b previously described (35) from Δ*tle* display a lower R<sub>f</sub> than wild-type GPL-2a and GPL-3, which are restored in Δ*tle*::C. TDM: trehalose dimycolate; DAT: Di-O-acyl trehalose. (B) TLC analysis of native (NaOH -) and saponified (NaOH +) polar fractions. Lipids were separated using chloroform/methanol/water (90:10:1, v/v/v) followed by spraying with orcinol and charring. The R<sub>f</sub> of GPL-2a and GPL-3 from Δ*tle* are not modified as it is the case for S and Δ*tle*::C. (C) MALDI-TOF positive spectra of native polar lipid fraction (black, NaOH -) from the S and Δ*tle*::C strains show ions for GPL-2a at *m/z* 1257.9/1285.9 and for GPL-3 at *m/z* 1403.9/1432. Spectra from saponified polar lipid fraction (red, NaOH +) show a 84 u.m.a decrease, to 1173.4/1201.4 and 1319.3/1347.4, corresponding to deacetylated dGPL-2a and dGPL-3, as previously described (34). Conversely, ions for GPL-1b at *m/z* 1013.8/1041.8 and from GPL-2b at 1187.9 were intense in native and saponified polar extracts from Δ*tle*. (D) Relative proportion of each monosaccharides was determined in the GPL-enriched saponified polar lipid fraction. 6-d-Tal could only be detected in S and Δ*tle*::C.
**Figure 6. Distribution of *tle* orthologues in NTM and correlation with the presence of 6-d-Tal.**

(A) Phylogenetic tree reconstructed using already available *tle* sequences. Non-mycobacterial species and mycobacterial species are represented in pink and blue areas, respectively. Green, yellow and red dots label slowly-growing, intermediate-growing and rapid-growing mycobacteria, respectively. (B) TLC analysis of the polar lipid fraction of a selected panel of NTM clinical strains, representative from two independent experiments. Lipids were separated using chloroform/methanol/water (90:10:1, v/v/v) followed by spraying with orcinol and charring. +: presence of GPL-like components, confirmed by their detection after saponification (Figure S8). -: absence of GPL-like components. (C) Deoxyhexoses composition analysis of polar lipid fractions separated with GC and GC-MS. Relative area of identified monosaccharides (n=4) show Rha residues in green and 6-d-Tal in blue. Identification of 6-d-Tal and Rha was confirmed by the retention time and fragmentation pattern of standards while *O*-methylated Rha was determined previously (33, 35). Identification of 2- and 3-*O*-methyl 6-d-Tal was deduced from the retention time and fragmentation pattern. Relative areas of Rha and 6-d-Tal and their methylated forms are expressed as ratios of total monosaccharides.

**Figure 7. Increased virulence and pathogenesis of *Δtle* in a zebrafish embryo model of infection.**

(A) Schematic overview of *in vivo* experiments. (B) Survival of infected embryos with the R strain (275±27 CFU), the S strain (280±36 CFU), *Δtle* (264±33 CFU) and *Δtle::C* (281±47 CFU). The graph shows the result of 4 pooled independent experiments. Embryos were marked dead in the absence of a heartbeat. Statistical analysis was performed using Log-rank (Mantel-Cox) test. ****: p<0.0001; **: p<0.01, ns: non-significant. (C) Bacterial burden from infected embryos at 2, 3 and 5dpi. The graph shows the result of 3 pooled independent experiments. n=5 embryos per time-point per condition. CFU counts were Log10 transformed. Statistical analysis was performed per time point using ordinary one-way ANOVA with Šídák's multiple comparisons test, after verifying the normality of the data. *: p<0.05; **: p<0.01; ***: p<0.001 ****: p<0.0001. (D) Imaging of infected embryos at 3dpi. One zoomed representative image of the head is shown per condition. Scale bar, 0.2 mm.

**Figure 8. The biosynthetic interconnections between the arabinogalactan and glycopeptidolipid pathways in *M. abscessus*.**

*dTDP-L-Rha* is generated by the successive action of the Rml pathway enzymes, consisting of RmlA (MAB_4113), RmlB (MAB_3779), RmlC
1056 (MAB_3780) and RmlD (MAB_3613c). This dTDP-L-Rha is used by WbbL (MAB_3612c) to produce
1057 the Rha-N-acetyl-glucosaminyl-phosphate disccharide unit that cross-links arabinogalactan to
1058 peptidoglycan. In addition, dTDP-L-Rha is also used by Gtf2 (MAB_4104) and Gtf3 (MAB_4112c)
1059 for GPL rhamnosylation (blue arrows). dTDP-6-d-Tal, produced from dTDP-L-Rha by the dTDP-
1060 Rha-4-epimerase (MAB_4111c/Tle) serves as the substrate for the talosyltransferase Gtf1 (green
1061 arrows), which adds one 6-d-Tal unit to mono- or di-rhamnosylated GPL precursors. The
1062 glycosylated GPLs (GPL-2a and GPL-3) are then transported across the inner membrane by the
1063 MmpL4 complex (MAB_4116c, MAB_4115c, MAB_4117c) and inserted into the outer leaflet of
1064 the mycomembrane in the *M. abscessus* S variant.
<caption>Figure 1</caption>

A

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

B

<figure><img src="image_5.png" /></figure>

C

WBIB_BURTA/1-363 1 MSDVNASLVDGKKILVTGGAGFIGCAISERLAARASRYVVMDNLH 45
MAB_4111c/1-353 1 MLISGGAGFIGSALSNRLIQAGYDVAVMDVLH 32

WBIB_BURTA/1-363 46 PQIHANAVRPVALHEKAELVVADVTDAGAWDALLSDFQPEIIHLAAETGTGQSLT 101
MAB_4111c/1-353 33 PQVHA - RGRAIDLPASVRLEFTGDVTHAPDWDAVLRLFEPSQVVHLAAETGTAQSLS 87

WBIB_BURTA/1-363 102 EASRHALVNVVGTTRLTDAVKHGIAVEHILLTSSRAVYGEGAWQKADGTIVYPGQ 157
MAB_4111c/1-353 88 EATRHGSVNVVGTQQLLDALSRSALVPDQLVLASSRAVYGEGAWQSG-AEVFYPRP 142

WBIB_BURTA/1-363 158 RGRAQLIEAAQWDFPGMTMLPSRADRTEPRPTSVYGATKLAQEHVLRAWSLA 208
MAB_4111c/1-353 143 RSHAQLLAGEWDPQPAGASATPLSSCADRTEPRPTNIYAGTKLAQEHLLASWAAA 198

WBIB_BURTA/1-363 209 TKTPLSELRLQNVYGPGQSLTNSYTGIVALFSRLAREKKVIPLYEDGNVTORDFVSI 264
MAB_4111c/1-353 199 HDTNLSVLRLQNVYGPGQSLTNSYTGIVTLFARLAREKQALEVYEDGRIVORDFVFI 254

WBIB_BURTA/1-363 265 DDVADAIVATLAREPEALSLF DIGSGQATSILDMARIIAAHYGAPEPQVNGAFRDG 320
MAB_4111c/1-353 255 DDVVDALFAAVHTPATERRTL DIGSGTATTIHELARKVADICAIAPEPVVVGKFRDG 310

WBIB_BURTA/1-363 321 DVRAACDISESLANLGWKPQWSLERGIGELQTWIAQELDRKN 363
MAB_4111c/1-353 311 DVRAASCDIAPAVEGLGWSPKWTLLEDGLHALLEWIDKDCPNRF 353
<caption>Figure 2</caption>

Journal Pre-proof

<figure><img src="image_6.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_10.png" /></figure>
<figure><img src="image_11.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_12.png" /></figure>
<figure><img src="image_13.png" /><figcaption>Figure 5</figcaption></figure>
<caption>Figure 6</caption>

A

<figure><img src="image_14.png" /></figure>

B

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

C

<figure><img src="image_18.png" /></figure>
<caption>Figure 7</caption>

<figure><img src="image_19.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_21.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>S</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>R</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>S Δtle</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>S Δtle::C</figcaption></figure>

<figure><img src="image_26.png" /></figure>
<figure><img src="image_27.png" /><figcaption>Figure 8</figcaption></figure>

**Legend**

● L-rhamnose

■ 6-deoxyL-talose

ζ N-acetyl-glucosaminyl-phosphate
**Declaration of interests**

[x] The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

[x] The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for *Journal name* and was not involved in the editorial review or the decision to publish this article.

[ ] The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

<figure><img src="image_28.png" /></figure>