# Genome mining and chemistry-driven discovery of a cell wall lipopeptide signature for Mycobacterium avium ssp. paratuberculosis ancestral lineage

John P Bannantine, Gilles Etienne, Anne Lemassu, Thierry Cochard, Christelle Ganneau, Sandrine Melo, Cyril Conde, Hedia Marrakchi, Sylvie Bay, Franck Biet

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John P Bannantine, Gilles Etienne, Anne Lemassu, Thierry Cochard, Christelle Ganneau, et al. Genome mining and chemistry-driven discovery of a cell wall lipopeptide signature for Mycobacterium avium ssp. paratuberculosis ancestral lineage. ACS Infectious Diseases, 2025, 11 (6), pp.1635-1647. 10.1021/acsinfecdis.5c00181. pasteur-05078870

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Genome mining and chemistry-driven discovery of a
cell wall lipopeptide signature for *Mycobacterium
avium* ssp. *paratuberculosis* ancestral lineage

John P. Bannantine¹, Gilles Etienne², Anne Lemassu², Thierry Cochard³, Christelle Ganneau⁴,
Sandrine Melo³, Cyril Conde³, Hedia Marrakchi², Sylvie Bay⁴*§ and Franck Biet³*§

¹USDA—Agricultural Research Service, National Animal Disease Center, Ames, Iowa, 50010,
USA

²Institut de Pharmacologie et de Biologie Structurale (IPBS) UMR-5089, CNRS / Université Toulouse, Toulouse, 31000, France

³INRAE, UMR ISP 1282, Université de Tours, Nouzilly, 37380, France

⁴Institut Pasteur, Université Paris Cité, CNRS UMR3523, Chimie des Biomolécules, Chem4Life,
Paris, 75015, France

§Co-senior authorship.

*E-mail: franck.biet@inrae.fr; sylvie.bay@pasteur.fr

**KEYWORDS:** *Mycobacterium avium* subsp. *paratuberculosis*, Johne's Disease, Non-Ribosomal
Peptide Synthetases, Lipopeptide, Diagnosis, GlycoPeptidoLipids
ABSTRACT:

*Mycobacterium avium* subsp. *paratuberculosis* (*Map*) causes Johne's disease (JD), a chronic infection responsible for considerable economic losses to dairy industries worldwide. Genetically clonal, *Map* has evolved into three distinct genetic lineages designated CII, for bovine strains, SI and SIII, for ovine strains. Previous studies have established that *Map* does not produce glycopeptidolipids, characteristic of the cell wall surface of mycobacteria belonging to the *M. avium* complex, but rather sugar-free lipopeptide compounds synthesized by non ribosomal peptide synthetases. In this study, we combined genomic, machine learning, (bio)chemical, and analytical approaches to identify the metabolites biosynthesized by NRPS in the most ancestral SI strains of *Map*. We have thus characterized a lipotripeptide (L3P-2) signature for the SI genetic lineage, demonstrating that the evolution of this *Map* subspecies has been accompanied by a diversification of the cell wall lipopeptides. Finally, L3P-2 shows promise for improved serological diagnosis of JD.
Non-ribosomal peptide synthetases (NRPS) are large multi-enzyme machineries that assemble numerous peptides of wide structural and functional diversity ¹. In mycobacteria, NRPS are present in biosynthetic gene clusters (BGCs) involved in the biosynthesis of cell wall compounds. The envelope of mycobacteria has a particularly unusual cell wall, containing an outer membrane consisting of an inner leaflet composed of a complex thick layer including mycolic acids, and an outer leaflet composed of a wide variety of extractable lipids, collectively forming the mycomembrane ². Among the lipids biosynthesized by NRPS are the GlycoPeptidoLipids (GPLs), present in many non-tuberculous mycobacteria (NTM) ³. GPLs are present in opportunistic pathogens such as *Mycobacterium chelonae*, *Mycobacterium abscessus* ⁴, ⁵ or *Mycobacterium avium* complex (MAC) ⁶ and they share a common lipopeptide structure consisting of a mixture of C28-30 3-hydroxy and 3-methoxy fatty acids amidated by a tripeptide-amino-alcohol core of D-Phe-D-allo-Thr-D-Ala-L-alaninol. In addition, the lipopeptide core is glycosylated on allo-Thr and alaninol, resulting in great structural heterogeneity varying by the length of the fatty acyl chain, the degree of hydroxylation or *O*-methylation of the glycosidic parts and the number or nature of substituting sugars, depending on the strain ⁵. GPLs account for up to 70% of parietal lipids and play an important role in cell wall integrity, mycobacterial virulence, biofilm formation and antibiotic resistance ⁴.

*Mycobacterium avium* subsp. *paratuberculosis* (*Map*) is a pathogen that has evolved from the ancestral *Mycobacterium avium* subsp. *hominissuis* (*Mah*) organisms belonging to the MAC ⁷. *Map* is the etiological agent of paratuberculosis, also known as Johne's disease (JD), one of the most common infectious diseases of livestock worldwide ⁸. It is associated with a high health impact and substantial socio-economic costs for governments, agricultural industries and taxpayers. Despite decades of costly surveillance and control campaigns, with expenditures in
excess of $250 million per year in the USA and similar figures in most industrialized countries, Johne's disease remains endemic and continues to progress in most countries. The prevalence of the disease remains very high: around 50% in European herds and above 80% in the USA ⁹⁻¹¹. The inability to control this disease, characterized by a very slow and chronic course, is due to significant gaps in our knowledge of its causative agent, but is also mainly due to ineffective prophylaxis resulting from the complex biology of this infectious disease. Infection is particularly slow to develop, and usually occurs via the fecal-oral route. It can present in a wide variety of sub-clinical and clinical forms over an incubation period that can range from 1 to 14 years. This chronic infection of the intestine, mainly targeting ileal Peyer's patches, results in discontinuous excretion of *Map*, making diagnosis and treatment of infected animals extremely difficult. For routine diagnosis, most analyses are carried out using ELISA serological assays, which suffer from low specificity and sensitivity, and for which the response of bovine antibodies to *Map* changes as the disease progresses ¹². It is not until the disease enters its clinical phase that a more consistent and predominant antibody response is observed. Vaccines do exist, but their efficacy is limited and they are not deployed in most countries to avoid interfering with the diagnosis of bovine tuberculosis.

Surprisingly, among MAC members, *Map* is the only species that does not produce GPL (Figure 1). Instead, this major cattle and sheep pathogen produces non-glycosylated lipopeptide compounds. Recent genomic studies have revealed that *Map* is subdivided into three distinct genetic lineages: subtype CII, also known as bovine type, and subtypes SI and SIII, also known as ovine type, depending on the host from which they are isolated ¹³. Interestingly, the study of BGCs has shown variability in *Map* NRPS genes which is linked to their belonging to different genetic lineages. Strains from the CII lineage, by far the most represented, have a 5-module NRPS
responsible for the biosynthesis of a pentapeptide core consisting of D-Phe-N-Methyl-L-Val-L-
Ile-L-Phe-L-Ala-OMe (L5P), whereas strains of the SIII line have a 3-module NRPS capable of
biosynthesizing L3P (named L3P-1 in this article) with a tripeptide core of D-Phe-N-Methyl-L-
Val-L-Ala-OMe, whose antigenic properties present specific features <sup>14, 15</sup>.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Cell surface lipids that distinguish MAC complex subspecies. Core gene phylogeny inferred from Roary analysis of 47 complete genome of *M. avium* sp. strains with RAxML and corrected for horizontal gene transfer by ClonalFrameML. Unrooted phylogeny visualized in iTOL without branch length. This enables visualization of the tree topology of the *M. avium* species members, *M. avium* subsp. *avium* (*Maa*) and *M. avium* subsp. *hominissuis* (*Mah*) designed i.e. *Non-Map* and *M. avium* subsp. *paratuberculosis* (*Map*). This tree can form 2 separated phylogroups according to the type of surface compound, Lipopeptide or GlycoPeptidoLipids. The</figcaption></figure>
most ancestral *Map* SI strains are circled in yellow. The asterisk indicates that the surface compounds of these strains were previously unknown and are the subject of this study.

In this study, we focused on the most ancestral *Map* strains, from the SI genetic lineage, an ecotype found mainly in Australia, New Zealand and Scotland. Despite the limited number of strains available due to their extremely slow growth, which makes them virtually unculturable, we have assembled a unique set of strains, sequenced and analyzed by genomics, machine learning and (bio)chemistry to identify for the first time the NRPS production of a lipopeptide core specific to this SI ecotype, present at the evolutionary interface between *Map* and the other members of the MAC. The analysis of these NRPSs and the exact characterization of their lipopeptide structure provide a comprehensive picture of the diversity of parietal lipopeptides biosynthesized in *Map*, and whose initial application could improve the serodiagnosis of Johne's disease.

## RESULTS

*In silico* analysis of the NRPS locus, NRP prediction and diversity.

To enable the identification of BGC encoding NRPS enzymes and regulatory transporters essential for production of the parietal metabolites GPL and LP, we employed genomic analyses and bioinformatic-genome mining methods. With antiSMASH 7.0 ¹⁶, ¹⁷, these machine-learning tools successfully enabled the discovery of a BGC locus from the fully assembled genome sequence of the SI Map 6756 strain ¹⁸ (Figure 2A). Bioinformatic predictions suggest that this locus includes an NRPS potentially responsible for the synthesis of a tripeptide that forms the core of a hitherto undescribed LP called L3P-2 (Figure 2B). The prediction of an epimerization domain strongly suggests that the alanine amino acid in position 1 is in the D configuration. A methyltransferase domain within the second module predicts that the valine amino acid in position
2 is *N*-methylated. Finally, the presence of a thioesterase domain implies the release of the mature oligopeptide from the NRPS machinery. Overall, bioinformatics tools predicted the production of a putative *N*-terminal acylated tripeptide whose sequence corresponds to D-Ala-*N*-Methyl-L-Val-L-Ala.

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

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

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

**Figure 2.** *In silico* analysis of the NRPS locus and prediction of the corresponding amino-acid sequence. (A) Genomic organization of the BGC locus in *Map* 6756 that includes the predicted NRPS candidate gene SLH60_08260 accompanied by enzymes, transport-related and regulator genes. (B) Prediction of the SLH60_08260 encoded NRPS modules and the structures of the three amino-acids loaded. Domains within each module are represented by circles and defined in the box at the right. (C) Predicted sequence and structural formula of the fully assembled lipotripeptide L3P-2 represented with a C20 fatty acid.
Surrounding the NRPS genes are several genes likely to be involved in L3P-2 synthesis, transport and regulation (Figure 2A). AntiSMASH was used to identify and compare NRPSs and their products deduced from the complete genomes of mycobacteria belonging to the MAC complex. Examination of the BGC loci in the available genomes of other SI-type strains occupying a position at the interface between *Map* and non-*Map* species revealed that this organization is conserved (Figure 3). This analysis, combined with phylogenetic analysis of the strains, revealed that the composition and size of NRPSs vary from one species to another within the MAC complex (Figure 3).

<figure><img src="image_6.png" /><figcaption>**Figure 3.** Diversity and specificity of Non-Ribosomal Peptide Synthases within *Map* and Non-*Map*. The phylogenetic tree was inferred from GBDP distances calculated from complete genomic sequences of 42 *M. avium* subspecies strains. *Map* C-type strains are shaded in blue, SIII strains</figcaption></figure>
in green, SI strains in yellow, *Maa* in light orange and *Mah* in pink. Bioinformatics analysis of NRPSs using the antiSMASH machine learning tool shows that within the *Map* strains BGCs have a single NRPS but differ in the number of modules (i.e 5-module (blue arrows) or 3-module (green arrows and yellow arrows)) whose deduced products are different. On the other hand, in Non-*Map* strains, 2 NRPSs with 2 modules (indicated by red arrows) positioned in tandem and identical in sequence are systematically found, and their deduced product, a GPL, is ubiquitous. The host from which these strains were isolated is shown.

In non-*Map* strains, GPLs are invariably synthesized by two tandemly organized NRPS of identical composition and size. In *Map*, on the other hand, each genetic lineage produces a different LP by a single NRPS whose composition and size vary from lineage to lineage. Surprisingly, the amino acid at position 1 in the peptide core of SI-type L3P-2 is predicted to be an alanine, in contrast to the phenylalanine systematically conserved in LPs and GPLs (Figure 2 and 3).

**Identification of the native LP produced from SI strains.**

This *in silico* prediction was used to guide biochemical and physicochemical analyses aimed at purifying and characterizing the native structure of this new SI lineage-specific metabolite (Workflow detailed in Figure S1). In agreement with the bioinformatic predictions, two-dimensional (2D) analytical TLC of the lipid extract from the SI *Map* strain 6756 shows a spot migrating between the synthetic standards L3P-1 and L5P, representing the respective lipopeptides associated with the SIII and CII *Map* strains¹⁴, ¹⁵ (Figure S2).

In an initial Matrix-Assisted Laser Desorption Ionization-Time Of Flight (MALDI-TOF) Mass Spectrometry (MS) analysis, we observed that the total lipid extract of SI strain 6756 showed
neither the L5P, characteristic of CII strains (expected major peak at *m/z* 940 amu) nor the L3P-1 characteristic of SIII strains (expected major peaks at *m/z* 680 and 708 amu) ([M+Na]⁺ ions), but instead two main peaks at *m/z* 604 and 632 amu (i.e. *m/z* 76 amu less than L3P-1), plus a minor peak at *m/z* 618 amu were noted (Figure 4A). This total lipid extract was then subjected to saponification with 1N NaOH. MALDI-TOF MS analysis showed a 14 amu down-shift of molecular ions at *m/z* 590, 604 and 618 amu, suggesting that *i*) the lipid is alkali resistant, as expected for an *N*-terminal lipopeptide and *ii*) hydrolysis of an *O*-methyl ester group occurred (probably from the *C*-terminus) (Figure S3), as observed for SIII L3P-1¹⁴. In fact, assuming the same stereochemistry in both S strains, the combined MS and bioinformatics data all converge towards the following L3P-2 as the putative SI LP structure: Lipid-CO-D-Ala-L-*N*-Methyl-L-Val-LAla-*OCH*₃ (Figure 2C).

<figure><img src="image_7.png" /><figcaption>**Figure 4.** SI-type *Map* produces L3P-2. MALDI-TOF MS spectra of chloroform/methanol-extracted lipids from SI-type *Map* 6756 (A), purified LP from 2D-TLC spot (B), (see Figure 5),</figcaption></figure>
and synthetic L3P-2 (C). The peaks at *m/z* 590/604/618/632/646 amu correspond to the [*M*+Na]⁺ adducts. The peak at *m/z* 620 amu corresponds to the [*M*+K]⁺ adduct.

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

**Figure 5.** Isolation of target LP by semi-preparative 2D-TLC. 2D-TLC of total SI-type *Map* 6756 lipids (1 mg spotted), was analyzed using chloroform/methanol (96:4 vol/vol) as first dimension and toluene/acetone (80:20 vol/vol) as second dimension, and chemically synthesized L3P-2 (25 µg) as standard. The TLC plate was sprayed with water. The asterisk indicates the position of the new putative LP, which was then scraped and eluted in chloroform/methanol 50:50 (vol/vol). The contrast of this image was improved with image processing software.

To enable complete formal identification of the LP biosynthesized by the SI strain, the predicted L3P-2 (with the C20 fatty acid taken as representative compound (Figure 2C)) was chemically synthesized as a reference compound to drive purification of the native lipopeptide and to confirm its identity. This synthetic L3P-2 was prepared by solid-phase peptide synthesis using Fmoc
chemistry and purified by silica gel chromatography, yielding 148 mg of lipopeptide (74% yield, > 99% purity by HPLC) (Figure S4).

To isolate and purify the native lipopeptide, a total lipid extract was first analyzed by semi-preparative 2D-TLC. One mg of total lipid extract from strain 6756 was loaded onto a silica plate with synthetic L3P-2 as a lateral marker. The spot of interest was detected by comparison with the synthetic L3P-2 and visualized by water spraying, an advantageous non-destructive method (Figure 5). It was scraped off and the LP was then eluted from the adsorbent layer in 50:50 (vol/vol) chloroform/methanol (see materials section).

The resulting MALDI-TOF MS spectrum clearly identifies the native LP as the *in silico* predicted L3P-2 at *m/z* 604/618/632/646 amu (Figure 4B) and the peak at *m/z* 604 amu ([M+Na]⁺ ion) matches that of the synthetic control with C20 fatty acid (Figure 4C). Additional peaks differing by 14 amu (i.e. one methylene unit) observed for the native antigen (Figures 4A and 4B) suggest the presence of different fatty acid chain lengths. In particular, the major compounds at *m/z* 604/632 amu can be attributed to L3P-2 with C20 and C22 acyl chains, respectively. Crude lipid extracts from other SI strains, notably strains M71/03, M99/03 and 21P, were analyzed by 2D TLC and MALDI-TOF MS; these extracts gave LP chromatographic spots and MS peaks similar to that of strain 6756 (Figure S5).

In order to obtain a batch of highly pure native L3P-2 in sufficient quantity for its full characterization by MALDI-TOF MS/MS, nuclear magnetic resonance (NMR) and gas chromatography (GC) MS, the crude lipid extract from each strain was subjected to orthogonal two-stage purification by solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) (Figure S6, 6, and S7). By taking advantage of л-л interactions, a biphenyl HPLC column enables highly efficient separation of the three lipopeptides, L3P-2, L3P-
1 and L5P, which have different amounts of Phe residues, as shown on the analytical HPLC MS chromatograms (Figure 6A). Overall, after loading of 37.5 mg of crude extracts from strain 6756 and 20 mg of crude extracts from strain M99/03 on the SPE column, the respective semi-purified LP were obtained (Figure 6B, S7B). The final HPLC purification step then resulted in 135 µg and 103 µg of the purified native antigens, respectively (Figure 6C, S7C), based on a calibration curve of the synthetic standard.

<figure><img src="image_9.png" /><figcaption>**Figure 6.** HPLC-MS chromatograms. HPLC-MS chromatograms showing the sequential purification of native L3P-2 from the SI-type *Map* 6756. Spectra shown from top to bottom are: synthesized standards (mixture of L3P-2, L3P-1 and L5P, 0.2 µg each, see assigned peaks) (A, black), semi-purified crude *Map* extract after SPE (B, green), purified native lipopeptide after HPLC (C, red). Y-axes indicate total ion current intensity.</figcaption></figure>

These two lipopeptides gave the same high resolution mass spectra as that of the synthetic L3P-2 control (peak at *m/z* 582 amu ([M+H]⁺ ion) (Figure S8). Both isolated lipopeptides from native source were also compared with the synthetic or native references L3P-2 and L3P-1 by MALDI-
TOF-MS/MS and NMR spectroscopy<sup>14</sup>. Purified L3P-2 (from SI strains 6756 and M99/03) and the synthetic control showed identical MS/MS fragmentation patterns. Fragment ions of the parental ion *m/z* 604 amu, corresponding to all possible cleavages of the peptide bond between the first *N*-terminal amino acid (probably Ala) and the *N*-Methyl-Val residue ([a2, b2, c2] and [x2, y2, z2]) and all possible cleavages of the peptide bond between the *N*-Methyl-Val residue and the Ala residue ([a3, b3, c3] and [x3, y3, z3]) (Figure 7A) were shared between synthetic and native L3P-2 (Figure 7B, Figure S9). Furthermore, all of these fragment ions [a, b, c] detected from synthetic and purified L3P-2 showed a downward shift of ~76 amu compared with the corresponding fragment ions [a, b, c] from the synthetic L3P-1. And all but one of these [x, y, z] fragment ions, detected from synthetic and purified L3P-2, showed the same value as the corresponding [x, y, z] fragment ions from synthetic L3P-1 (Figure 7B, S9 and S10).

<figure><img src="image_10.png" /><figcaption>**Figure 7.** Tandem MS spectra of purified native and synthetic L3P-2 show identical fragmentation patterns. L3P-2 purified from SI-type *Map* strains 6756 lipids was analyzed by MALDI-TOF</figcaption></figure>
MS/MS and compared to synthetic L3P-2 and L3P-1 from SIII-type *Map* S397. Structural formula of L3P-2 and typical fragmentation at the Ala-*N*-Methyl-Val ([a2, b2, c2] and [x2, y2, z2] fragment ions) and the *N*-Methyl-Val-Ala bonds ([a3, b3, c3] and [x3, y3, z3] fragment ions) (A). Ions originating from the fragmentation at the Ala-*N*-Methyl-Val and *N*-Methyl-Val-Ala bonds from the parental ion at *m/z* 604 amu (L3P-2) or *m/z* 680 amu (L3P-1) for a C20 fatty acyl chain (B). The corresponding spectra are shown in Figure S10.

These data suggest that the two *C*-terminal amino acids are identical in L3P-1 and L3P-2, and that the difference between the two lipopeptides lies in the first *N*-terminal amino acid, namely Phe in L3P-1 and probably Ala in L3P-2. In addition, L3P-2 from strain M99/03 presented a MS/MS pattern of fragmentation similar to that of strain 6756 and synthetic L3P-2 (Figure S9, S10). Finally, all these data are consistent with the structure proposed for L3P-2, namely an Ala-*N*-Methyl-Val-Ala tripeptide sequence with an *N*-terminal C20 fatty acid and a *C*-terminal methyl ester.

NMR spectroscopy was used to resolve fine structural detail of the synthetic L3P-2 and to confirm the structure of the native corresponding antigen. As the peptide's central Val residue is *N*-methylated, the sequence of amino acid residues could not be given by conventional TOCSY-NOESY NMR analysis. The one dimensional ¹H spectrum (Figure 8, upper spectrum) shows two characteristic signals of -NH- at proton chemical shift (δ<sub>H</sub>) greater than 6 ppm, three CHα (δ<sub>H</sub> 4.5-5 ppm), one *OCH<sub>3</sub>* (δ<sub>H</sub> 3.76 ppm), one *NCH<sub>3</sub>* (δ<sub>H</sub> 3.06 ppm), and characteristic signals of aliphatic acyl chains (δ<sub>H</sub> 0.8-2.2 ppm), confirming the lipopeptidic structure of L3P-2. ¹H-¹H COSY spectrum (Figure S11) allows the resolution of four spin systems corresponding to both alanines, valine and fatty acyl chain, whose characteristic proton chemical shifts are recorded in the table in Figure S12.
<figure><img src="image_11.png" /><figcaption>**Figure 8.** $^{1}$H- and $^{1}$H-$^{13}$C- NMR spectroscopy confirm the structure of the L3P-2. 2-D $^{1}$H-$^{13}$C HSQC (red cross peaks) and $^{1}$H-$^{13}$C HMBC (blue cross peaks) of synthetic L3P-2. $^{1}$H and $^{13}$C NMR data are indicated in red (aliphatic moiety) purple and pink (alanine residues) and blue (valine residue).</figcaption></figure>

The absence of -NH from the valine residue and the presence of -NCH₃ strongly suggests that this residue is *N*-methylated. The $^{1}$H-$^{13}$C HSQC spectrum (Figure 8, red cross peaks) identified the chemical shifts of the carbons, recorded in Figure S12. Intramolecular $^{1}$H-$^{13}$C HMBC analysis (Figure 8, blue cross peaks) identified the carbonyls of the fatty acyl and amino acid residues (Figure 8 and S12). The analysis of correlations between carbonyls and the -NH and -CHα of neighboring residues reveal the sequence of L3P-2: i) fatty acyl carbonyl (δC 173 ppm) correlates with -NH of the first alanine at δH 6.45 ppm and -CHα at δH 4.92 ppm, ii) the carbonyl of the first alanine residue (δC 175 ppm) correlates with -NCH₃ at δH 3.06 ppm and with valine -CHα at δH
4.53 ppm, *iii*) the carbonyl of the valine residue (δC 170 ppm) correlates with the -NH of the second alanine residue at δH 4.51 ppm, *iv*) the carbonyl of the second alanine residue (δC 173.6 ppm) correlates with -OCH₃ at δH 3.76 ppm. Taken together, these results confirm the Ala-Val-Ala sequence, *N*-fatty acylation on the *N*-terminal Ala residue, *N*-methylation on the Val residue, *O*-methylation on the C-terminal Ala residue, all of which validate the proposed structure of native L3P-2. Finally, the ¹H-¹H COSY spectra of purified lipids from *Map* 6756 and *Map* M99/03 and of synthetic L3P-2 all overlap (Figure S13), showing all the characteristic peaks and identical structures. These results, together with HPLC, MS and MS/MS data demonstrate the presence of L3P-2, and that only SI strains produce this previously unknown compound L3P-2.

Finally, to identify the amino acid configurations, the optical purity of individual amino acids in native L3P-2 was analyzed by GCMS after acidic hydrolysis of the lipopeptide using deuterated reagents. After comparison of retention times and mass spectra with those of relevant standards, the results unambiguously showed the presence of the enantiomeric forms of D-Ala (48%), L-Val (92.5%) and L-Ala (52%) in the LP from M99/03 strain (data not shown). Overall, the proposed structure of L3P-2 produced by *Map* type SI from the NRPS gene *SLH60_08260* sequence was confirmed: a peptide core in the form of D-Ala-*N*-Methyl-L-Val-L-Ala with a *C*-terminal methyl ester and a *N*-terminal fatty acid chain (predominantly C20 and C22 in the native form).

## Antigenicity of L3P-2 and diagnostic potential

Synthetic L3P-2 was assessed using well-defined sera from sheep naturally infected with *Map* type SI strains and sera from noninfected sheep (details in the method section) (Figure 9A). In receiver operating characteristic analysis (ROC analysis) of L3P-2, the area under the curve and its standard error were 0.9851 (95 % confidence interval, 0.9560 to 1.014) (Figure 9B). These
results confirm that L3P-2 has potential to discriminate *Map* infected and uninfected sheep at a population level. To determine specificity, we also investigated the recognition of L3P-2 by sera from animals infected with *Mah*, a very closely-related subspecies, and by field sera from cattle naturally infected with *Mycobacterium bovis* (*M bovis*) which causes bovine tuberculosis. L3P-2 reacted against *Map* positive sera but did not react significantly against sera from animals noninfected or infected with *Mah* or with *M. bovis* (Figure 9C).

<figure><img src="image_12.png" /><figcaption>**Figure 9.** Antigenicity of L3P-2. (A) ELISA using L3P-2 as the coating antigen to measure antibody response of sheep sera against L3P-2. The panel of sera tested included 16 negative controls (sheep not infected by *Map*) versus 21 *Map* positive sera (*Map+*). Serum samples were</figcaption></figure>
tested in triplicate. The statistical differences between the groups were determined using the non-
parametric Mann Whitney test. The horizontal bars indicate median, while shorter bars indicate
confidence interval. (B) ROC analysis of ELISA data. All results are expressed as individual OD
and were compared by ROC analysis. A equals the area under the receiver operating characteristic
curve. (C) L3P-2 used to discriminate *Map* infection from other mycobacterial infections. ELISA
were performed on plates coated with L3P-2. The panel of sera tested included 10 *Map* positive
sera (*Map*+), 13 *Map* negative sera (Controls), 3 *M. bovis* positive sera (*M. bovis*+) and 9 *Mah*
positive sera (*Mah*+). Results are shown as O.D.405 nm of means of individual samples tested in
triplicate. For each group Means ± SD are indicated as colored lines. The statistical analyses
between the groups were determined using the non-parametric Mann Whitney test. Significance
was determined when p value < 0.05. (*p* < 0.05; ****p < 0.0001). Not significantly different (ns).

Hence, we conclude that L3P-2 is antigenic and can be used in an ELISA-based format capable
of discriminating *Map*-infected animals, in particular sheep infected with SI strains.

DISCUSSION

The use of bioinformatic approaches revealed that the most ancestral *Map* strains (type SI) are
capable of biosynthesizing a specific LP whose structure could be confirmed by lipid extracts from
different strains and precisely determined by the combination of HPLC, MS and NMR analyses.
The chemically produced LP was recognized by serum from animals infected with SI strains.

The identification of new bacterial NRP metabolites, among which LP, should always be tackled
through a multidisciplinary strategy ¹⁹. This is particularly true for the *Map* SI type that represents
a real challenge as it is an extremely slow-growing strain and highly difficult to culture. Natural
SI antigens, including NRP compounds, are therefore very hard to purify from biomass and, at
best, can only be obtained in minute quantities. Nevertheless, the L3P-2 was isolated at 100 µg
scale from two SI *Map* strains. It is worth noting that, by contrast to the previous SIII study¹⁴, the
two-step purification of the natural SI *Map* extract allowed us to achieve a high final purity with
no contaminating peaks in the NMR spectrum.

In this context, the combination of both genome mining and synthetic chemistry was chosen as
a key asset for the unambiguous analytical identification of a new NRP from this SI *Map* strain.
Several studies have highlighted the value of total synthesis for the identification of metabolites,
and even its necessity for the precise assignment of their stereochemistry ¹⁹, ²⁰. Herein, we found
that bioinformatics prediction of LP structure from genomic data was fully confirmed by all
analyses that did rely on the synthetic L3P-2, used as a reference to guide purification and analyses
of the native NRP antigen. Future studies will be necessary for the validation of the function of the
*NRPS* identified by bioinformatic prediction.

NRPS are large, modular protein complexes containing several very similar domains, whose
extremely long genes are responsible for much of the fragmentation in microbial assemblages and
prevent any prediction of their secondary metabolite products without the aid of sophisticated
machine-learning software such as antiSMASH ¹⁷, ²¹. Advanced long read sequencing techniques
²² and the assembly of NRP BGCs into single contigs are crucial steps in the discovery of natural
products through genome mining ²³. These results add to our knowledge of LP diversity in *Map*,
and for the most ancestral SI-type strains at the interface between *Map* and non-*Map* members of
MAC.

In this study, we succeeded in assembling a unique complete genome panel for previously under-represented SI-type strains. The study extended to the genomes of members of the MAC complex, highlighting the unique feature of the *Map* subspecies to produce LP diversity in contrast to the
GPLs systematically described in other MAC members but also in NTM associated with human diseases ⁵. Given the high genetic similarity between the four members of the *M. avium* subspecies ¹³, and the fact that only the paratuberculosis subspecies produces LPs and causes Johne's disease, it is tempting to speculate that there is a link between LP production and animal intestinal infections. And even more so that L5P may have a predilection for bovine tissue while L3P variants may have a predilection for ovine tissues. Knockout mutagenesis and gene engineering will go a long way to answering these questions definitively with the caveat that these strains are virtually unculturable, and their replication time is measured in days.

The chemical characteristics of L3P-2, including long fatty acyl chains, may enable these molecules to cross the complex mycobacterial cell envelope and reveal a structure very close to that of SIII ¹⁴, which is not very surprising given their phylogenetic proximity ¹³, ²⁴. What is surprising, however, is that the first amino acid of the L3P-2 peptide is a D-Alanine residue, in contrast to the D-Phe residue found systematically in all other *Map* LPs, including L5P and L3P-1, but also in GPLs.

Previous studies have shown that *Map* has a highly closed genome characteristic of a clonal species ²⁵. Over the course of its evolution, this species has differentiated into 3 distinct genetic lineages (CII, SI and SIII). While there are very limited genetic signatures that can distinguish these lineages, including large sequence polymorphisms (LSP)²⁶, single nucleotide polymorphisms (SNPs)⁷ and insertion sequences (IS)²⁷, fewer studies have been able to decipher phenotypic differences between C and S strains ²⁸. And no studies have reported any phenotypic differences between SI and SIII strains.

Some notable differences between C and S strains include, time needed for bacterial isolation, which takes 6 to 8 weeks with C strains but for S strains the generation time is much longer ²⁹.
Iron storage under low-concentration conditions has only been observed in C-type strains, not S-type<sup>30</sup>. Some virulence factors such as adhesins show differences in structure and function between C and S strains<sup>31</sup>. Interestingly, macrophage infection studies revealed differences in virulence between C and S strains, suggesting peculiarities in wall lipid composition between S and C strains. Recently, still at the cell wall level, work has shown a differential production of a hallmark fatty acid, tuberculostearic acid, between C and S strains<sup>28</sup>. The results of this work not only provide a differential marker between strains C and S, which had been shown previously<sup>14</sup>, but also reveal for the first time the presence of a different cell wall compound in each of *Map*'s 3 genetic lineages (CII, SI and SIII). These results open up new avenues of research to understand the mechanisms of evolution and adaptation within *Map*.

Notably, the chemical synthesis of LP3-2 was essential not only to elucidate the natural SI *Map* metabolite but also to provide a sufficient amount for further testing. Our results show that L3P-2 is recognized by serum from sheep naturally infected with SI-type strains, illustrating its potential for paratuberculosis diagnostics. Although in routine use, the serological diagnostic assays available today lack specificity and sensitivity<sup>10</sup>, which may well explain the inability to control the disease worldwide. Existing JD ELISA assays all use a total mycobacterial lysate from strain *Mav* ST18, and *Mycobacterium phlei* lysate to preadsorb serum samples thereby reducing cross reactivity. However, the composition of these non-specific protein preparations are difficult to consistently and reproducibly generate and introduce another step in the procedure. This study demonstrates the added value of using synthetic *Map* antigens, which are easily accessible via organic chemistry, available in large quantities, and ultra-pure. Unlike the crude mycobacterial lysates just mentioned, the preparation of such high-quality antigens can be advantageously standardized paving the way for robust *Map*-specific immunological studies, which eliminate the
need for serum preadsorption. Studies are currently underway to further evaluate the diagnostic potential of L3P-2 for paratuberculosis in combination with other *Map* LPs <sup>32</sup> (Filed patent EP24305914.4).

Apart from the role of this compound, and LPs in *Map* more generally, in myco-membrane integrity and immunomodulatory properties, little is known about their biological functions. Indeed, lipopeptides are a diverse class of amphiphilic molecules containing a non-polar fatty acid chain and amino acid residues of varying length and hydrophobicity, which from NRPS exhibit a wide variety of bioactivities, ranging from antimicrobial to surfactant to cell signaling <sup>1, 33</sup>. For example, a family of lipo-tripeptides were described in *Xenorhabdus bovienii* to be involved in their symbiosis <sup>34</sup>. Interestingly, it has been shown that *Pseudomonas* lipopeptides can have amoebicidal properties <sup>35</sup>. Since *Map* can interact with amoebae <sup>36</sup> or even develop a symbiotic relationship, it would be interesting to study the role of L3P-2 and LPs on interactions with environmental hosts such as amoebae, which could explain the persistence and transmission of disease from the environment.

In *M. tuberculosis*, a lipodipeptide has been described as a new virulence factor, defined as a homeostatic zinc metallophore required for bacillus colonization <sup>37</sup>.

Studying the biological functions of these factors could, in the future, provide a better understanding of host-pathogen interactions and disease outcome.

## CONCLUSIONS

In this study, we have characterized a new lipopeptide named L3P-2, produced by the most ancestral *Map* strains, as a distinguishing feature setting them apart from the other *Map* genetic lineages and from other MAC strains. The diversity of lipopeptides revealed in *Map* raises the
question of their role in the pathogen evolution and provides opportunities for their potential
diagnostic applications.

METHODS

Genomes strains and culture.

Strains and genomes used in this study are described in Table S1. To broaden comparative
genomic analysis and provide a vision of MAC phylogeny, data set included 23 complete genomes
recently available for *Map* in NCBI, 17 complete genomes of *Mah* and 7 complete of *Maa*.

The *Map* isolates were propagated on slopes of Middlebrook 7H11 agar, 0.5% (vol/vol) glycerol, 10% (vol/vol) Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment medium (Becton, Dickinson, Oxford, Oxfordshire, UK), and 2 mg/mL of mycobactin J (ID-Vet, France) at 37°C. Mycobacteria were harvested in the early to mid-log phase of growth, and the cells were pelleted at room temperature for 10 min at 6,000 X g and stored at – 20°C until used for biochemical analysis.

Bioinformatic analysis.

Sequences were extracted from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) and BGCs
were identified using the prediction tool antiSMASH 7.0. ¹⁶. The NRPS domain analysis and
annotation, prediction of the core chemical structure of NRPSs were confirmed using the
antiSMASH. For the phylogeny analysis, the genome sequences were uploaded to the Type
(Strain) Genome Server (TYGS), available under https://tygs.dsmz.de, for a whole genome-based
taxonomic analysis ³⁸. For the phylogenomic inference, all pairwise comparisons among the set of genomes were conducted using GBDP and accurate intergenomic distances inferred under the algorithm 'trimming' and distance formula d5. 100 distance replicates were calculated each. Digital DDH values and confidence intervals were calculated using the recommended settings of the GGDC 4.0. Results were provided by the TYGS on 2024-03-18.

## Lipid extraction and 2D-TLC.

Lipids were extracted from the SI type *Map* cell pellets with chloroform/methanol (Fisher Scientific) (1:2 then 1:1 and 2:1 (vol/vol)). Lipid extracts were then washed three times with water, evaporated to dryness at 40°C and weighed (total process time under 45 min). For analytical purposes, 500 µg of crude extracts were loaded on 2D-TLC plates and eluted using chloroform/methanol (96:4 (vol/vol)) in the first dimension followed by toluene/acetone (80:20 (vol/vol)) in the second dimension. Synthetic L3P-2, or L3P-1 or L5P were deposited at 15 µg in chloroform and served as markers for each dimension. TLC plates were revealed by spraying 10% copper sulfate in 8% phosphoric acid, followed by charring.

Semi-preparative 2D-TLC were developed in the same way (1 mg spotted for the crude lipid extracts and 25 µg of the L3P-2 synthetic standard), but revealed by spraying water ³⁹. The spot of interest was scraped from the silica gel, eluted in chloroform/methanol 50:50 (vol/vol) and then washed three times with water.

## Synthetic lipopeptides production and characterization.

The L5P and L3P reference compounds were prepared as previously described¹⁴, ¹⁵. The control L3P-2 was synthesized on solid phase using the standard Fmoc chemistry protocol, as previously
described¹⁵. After cleavage from the resin, the crude product was purified on a silica gel column using dichloromethane/methanol (Sigma-Aldrich) as eluent (from 98:2 to 97:3 (vol/vol)), and 148 mg of the lipopeptide were obtained (yield 74%, HPLC purity > 99%). The synthetic L3P-2 was characterized by electrospray ionization MS (positive mode, Q-Tof Micro Waters) after direct infusion. It was analyzed by HPLC using an Agilent 1200 pump system with a UV detector at 210 nm, a Kinetex Biphenyl column (2.6 µm, 100 Å, 2.1 mm × 100 mm) (Phenomenex) at 30°C, and a gradient of methanol (A)/water (Milli-Q™) + 0.1% formic acid (FA) (Sigma-Aldrich) (B) over a period of 20 min, from 85 to 100% A, then 10 min at 100% A (0.3 mL/min). L3P-2 was also characterized by NMR (see analytical procedures below). All the results demonstrate both the identity and the purity of the compound.

MS: C₃₃H₆₃N₃O₅ m/z 582.4843 [M+H]⁺ (calcd 582.4841), 604.4659 [M+Na]⁺ (calcd 604.4660).

HPLC: Rt 10.4 min, purity > 99%.

## Purification by SPE and HPLC.

For the 6756 and the M99/03 strains, the crude lipid extract was first purified by SPE using SepPack silica columns (500 mg Waters, ~20 mg of extract/column) and a gradient of acetone (Sigma-Aldrich) in toluene (Sigma-Aldrich) from 0% to 30%, with incremental steps of 5% (3 column volumes for 0% and 5%, then 1 column volume for the following percentages). The resulting fractions were evaporated, resuspended in FA/H₂O (Sigma-Aldrich and / (Milli-Q™ respectively) 9:1 (vol/vol) and then analyzed by two methods: HPLC-MS (see details below) and 1D-TLC (0.2 mm silica gel 60 UV254) (Macherey-Nagel) in comparison to synthetic controls L3P-2, L3P-1 and L5P (1 µg of each). The TLC was eluted with toluene/acetone 80:20 (vol/vol) and stained with 10% copper sulfate in 8% phosphoric acid. The fractions that contain L3P-2 were
pooled (15%-20% acetone), evaporated, resolubilized in FA/H₂O 9:1 (vol/vol) and purified by HPLC using an Agilent 1200 pump system with a UV detector at 210 nm. A Kinetex Biphenyl column (2.6 µm, 100 Å, 4.6 mm × 150 mm) (Phenomenex) at 25°C was used, and a gradient of methanol (VWR) + 0.1% FA (A)/water + 0.1% FA + 5% MeOH (B) was applied over a period of 20 min, from 88 to 95% A (200-300 µL/injection, 1 mL/min). The fractions containing L3P-2 (retention time ~8.5 min) were pooled, evaporated and freeze-dried. A blank sample was also prepared using exactly the same consumables and protocol at each step, including fraction collect at the same time range. Quantification of the L3P-2 batches issued from the two SI-type strains was performed by integration of the peak absorbance areas at 210 nm, employing a calibration curve of the synthetic standard established in the range of 0.1 to 5 µg (see analytical method described above to characterize the synthetic L3P-2).

## Analytical procedures.

### HPLC-MS

The SPE fractions and the purified L3P-2 were analyzed using an Ultimate 3000 RS HPLC coupled with a UV diode array detector (210, 230, 254 nm) and with a Q Exactive mass spectrometer (Thermo Fisher Scientific). The MS instrument was equipped with an electrospray ionization source (H-ESI II Probe) and operated in the positive mode. The samples were cooled to 4 °C on the autosampler. A linear gradient was applied with methanol (Honeywell) + 0.1% FA (Fluka) (A)/water (Biosolve Chimie) + 0.1% FA (B) over a period of 20 min (85-100% A, 0.25 mL/min), using a Kinetex Biphenyl column (2.6 µm, 100 Å, 2.1 mm × 100 mm) (Phenomenex) at 25°C. Spectra were analyzed using the software XCalibur 4.2 and Freestyle 1.7/SP2 (Thermo
Fisher Scientific). Results were compared to the synthetic standards injected as a mixture of L3P-2, L3P-1 and L5P in FA/H₂O 9:1 (vol/vol) (3 µL at 0.07 mg/mL each).

Optical purity.

The amino acid optical purity was analyzed by GC-MS after hydrolysis of the lipopeptide in DCl 6N in D₂O, esterification with DCl in methanol and acylation using trifluoroacetic anhydride or pentafluoropropionic anhydride. (C.A.T. GmbH).

MALDI-TOF MS and MS/MS.

These analyses were conducted in the positive ionization and reflectron mode by accumulating 10 spectra of 250 laser shots, using the 5800 MALDI TOF/TOF Analyser (Applied Biosystems/Absciex) equipped with a Nd:Yag laser (349 nm wavelength) as previously described¹⁴.

NMR

For NMR analyses 1-D ¹H and 2-D experiments, compounds were dissolved in CDCl₃ 99.9% purity, Eurisotop, CEA Saclay, France). Experiments were conducted using a 600 MHz Bruker NMR spectrometer equipped with cryosonde at 295°K. ¹H chemical shifts are expressed in ppm using chloroform signal as internal reference (7.26 ppm). The cosygpmfqf, hsqcetgpsisp2.2 and hmbcetpgl3nd standard Bruker® sequences were used for bidimensional ¹H-¹H COSY (homonuclear Correlation Spectroscopy), ¹H-¹³C HSQC (Heteronuclear Single Quantum Coherence) and ¹H-¹³C HMBC (Heteronuclear Multiple Bond Correlation) experiments. The resulting spectra were processed with TopSpin Bruker software.
## L3P-2 ELISA.

Sera from sheep infected with *Map* type SI strains were kindly provided by Karen Stevenson, Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, EH26 0PZ, UK. The presence of *Map* antibodies in the sera was verified using IDEXX and ID-Vet commercial assays. Other sera controls from animals infected by closely related mycobacterial species such as *Mah* or *M. bovis* were included³².

ELISA experiments using synthetic antigen L3P-2 were adapted from previous study³². Briefly, L3P-2 antigen was diluted at 50 µg/mL in ethanol. Maxisorp microtiter plates (Nunc, Roskilde, Denmark) were coated with 50 µL of antigen preparation then air-dried for 18 hours at 37°C to allow the solvent to evaporate. The plates were washed three times with PBS supplemented with 0.1% (v/v) Tween 20 (PBST) (Sigma). After washing, the wells were blocked with 50 µL of Blocking Buffer (PBSTG: PBS - 0.05% Tween 20, 0.5% Gelatin) (Gibco) at room temperature for 2 hours. Fifty microliters of serum (diluted at 1:50 in PBSTG) were then added to each well and incubated for 1 hour 30 min at 37°C. Microtiter plates were washed five times with PBST and 50 µL of solution of Recombinant Protein G Peroxidase Conjugated (Thermo Scientific) diluted at 0.5 µg/mL in PBST, were added to each well and incubated for 1 hour at room temperature. After washing five times with PBST the reaction was visualized using 3,3',5,5'-tetramethylbenzidine (Sigma) and 0.5% v/v H₂O₂. The reaction was stopped with 1N sulfuric acid after 2 min of incubation in the dark and ODs were read at 405 nm on a GloMax® Discover GM3000 (Promega, Madison, USA).

The ELISA results of positive sera compared to negative control sera for each antigen were subjected to receiver-operator characteristic (ROC) curve analysis. This method estimates the
sensitivity and specificity of the ELISA, calculate likelihood ratios and provides an overall measure of assay accuracy as area under the ROC curve (AUC) to evaluate the assay performance ⁴⁰, ⁴¹. The optimal cut-off value is provided by ROC analysis as being associated with the maximal likelihood ratio. Statistical differences between groups of animals unexposed or experimentally infected either with Map or Mah or between groups of cattle naturally infected by *M. bovis* or *Map* was evaluated by Wilcoxon's matched pairs test. Differences were considered significant at p < 0.05. All statistical analyses were performed using statistical software (GRAPHPAD PRISM version6.07 for Windows, GraphPad software, San Diego, CA).

## ASSOCIATED CONTENT

### Supporting Information

*M. avium* complex strains and genome sequences used in this study (Table S1), Schematic representation of the workflow for identifying the lipopeptide from SI-type Map strain (Figure S1), 2D-TLC of total SI-type SI-type Map (Figure S2), MALDI-TOF MS analysis of Map 6756 total lipid extract after saponification (Figure S3), HPLC analysis of the synthetic L3P-2 (Figure S4), 2D-TLC and MALDI-TOF MS spectra of total lipids of SI-type *Map* strains (Figure S5), 1D-TLC analysis of the semi-purified crude extract of SI-type *Map* strain M99/03 (Fugure S6), HPLC-MS chromatograms showing the sequential purification of native L3P-2 from the SI-type *Map* M99/03 (Figure S7), High resolution product ion mass spectra of the L3P-2 (Figure S8), MALDI-TOF MS/MS analysis of purified native L3P-2 from SI-type Map strain M99/03 lipids (Figure S9). The native 6756 and M99/03 L3P-2 and the synthetic L3P-2 display identical MS/MS spectra (Figure S10). Characteristic 1H and 13C- NMR spectroscopy data for the synthetic L3P-2 (Figure S12) and ¹H-¹H COSY spectra of synthetic L3P-2 (Figure S13).
ACKNOWLEDGMENTS

We thank F. Bonhomme (Chimie Biologique Epigénétique, CNRS UMR3523, Chem4Life) for
his help in analytical work.

This study was funded by Institut Carnot Pasteur Microbes & Santé (ANR 21 CARN 0023-01)
and by Carnot France Futur Elevage (Grant number 00007952). S.B. and C.G. were supported by
the Institut Pasteur. F.B, T.C., C.C. and S.M. were supported by INRAE. J.B. was supported by
the USDA-Agricultural Research Service.

NMR experiments were performed on the PICT-Genotoul platform of Toulouse and funded by CNRS, Université de Toulouse-UPS, IBiSA (Infrastucture en Biologie Santé et Agronomie), European structural funds and the Occitanie region.

For the purpose of open access, the author has applied a CC-BY public copyright licence to any
Author Manuscript version arising from this submission.
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