# Interactome characterisation of a protein involved in lipid transport across the mycobacterial membrane: an XL-AP-MS approach.

Marion Beaupere, Eve Randrianaridera, Emmanuelle Mouton-Barbosa, Alexandre Stella, Odile Burlet-Schiltz, Christian Chalut, Manuelle Ducoux-Petit

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## To cite this version:

Marion Beaupere, Eve Randrianaridera, Emmanuelle Mouton-Barbosa, Alexandre Stella, Odile Burlet-Schiltz, et al. Interactome characterisation of a protein involved in lipid transport across the mycobacterial membrane: an XL-AP-MS approach. SMAP, Sep 2024, Lille, France. hal-05404481

HAL Id: hal-05404481

https://hal.science/hal-05404481v1

Submitted on 8 Dec 2025

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# Interactome characterisation of a protein involved in lipid transport across the mycobacterial membrane : an XL-AP-MS approach

Marion Beaupere<fnref n="1" />, Eve Randrianaridera<fnref n="2" />, Emmanuelle Mouton-Barbosa<fnref n="1" />, Alexandre Stella<fnref n="1" />, Odile Burlet-Schiltz<fnref n="1" />, Christian Chalut<fnref n="1" />, Manuelle Ducoux-Petit<fnref n="1" />

## INTRODUCTION

**Context** : Mycobacteria are a large family of bacteria, including the well-known representatives *Mycobacterium tuberculosis* and *Mycobacterium leprae*, responsible for tuberculosis and leprosy, respectively. The genus name "Mycobacteria" is due to the presence of a specific mycomembrane mainly composed of long-chain fatty acids: mycolic acids. These mycolic acids are the support for the integrity of the mycomembrane, which contributes significantly to the virulence of the pathogenic mycobacteria and their ability to resist broad-spectrum antibiotics (Chiaradia *et al.* 2017). The biosynthesis of the mycomembrane is therefore a perfect target for new therapeutic research against diseases such as tuberculosis, especially the proteins involved in the lipid transport and their interactions. Unfortunately, these proteins remain poorly characterised (Bories *et al.* 2024).

**Objectives** : Deciphering the **interactome** of MmpL10, a protein known to be involved in the transport of lipid chains to the mycomembrane (Thouvenel *et al.* 2020), will improve the knowledge of the proteins partners responsible for membrane assembly and integrity. Experiments will be carried out on the non-pathogenic and fast-growing strain of *Mycobacterium smegmatis*.

**Strategy** : XL-AP-MSMS of MmpL10

XL : stabilisation of protein interactions within complexes including MmpL10 (bait) by *in vivo* formaldehyde cross-linking.

AP : affinity purification of the complexes including the recombinant Twin-Strep tag-MmpL10.

MSMS : differential LC-MSMS analysis to identify and quantify the partners of MmpL10 compared to the control condition.

**Results** : This approach allowed us to identify and quantify more than 1,500 proteins. Of these, 48 were significantly enriched with the bait compared to the control condition. These 48 proteins are then potential candidates involved in the lipid transport across the mycomembrane and could provide insights into understanding of the pathogenic mycomembrane virulence.

<figure><img src="image_2.png" /><figcaption>**Figure 1** : Schematic representation of mycobacterial membrane</figcaption></figure>

## STRATEGY

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## RESULTS

### Control VS MmpL10

**Identification:**

* 12 359 peptides

* 1 748 proteins

**Quantitation:**

* 1 558 proteins

**Statistical analysis:**

* 48 proteins significantly enriched with the bait

* Non-variant proteins

* Enriched proteins with the bait

* Enriched proteins in control condition

<figure><img src="image_4.png" /><figcaption>Log2 Fold Change Experimental condition/ Control condition</figcaption></figure>

## CONCLUSION AND PROSPECT

In this study, we used an XL-AP-MS approach to identify the interacting partners of a plasma membrane protein from *Mycobacterium smegmatis*. This strategy allowed us to identify and quantify 1,500 proteins in this strain, including 48 proteins of interest for our biological question. These proteins are potential partners of MmpL10, a transmembrane protein involved in the first step of lipid transport across the mycobacterial membrane.

Further studies on these proteins could lead to the identification of the whole chain of proteins involved in the biosynthesis and integrity of the mycomembrane, and thus, to a better understanding of the biosynthesis mechanisms. This could lead to new therapeutic developments against diseases such as tuberculosis.

## REFERENCES

Bories *et al.*. HadBD dehydratase from *Mycobacterium tuberculosis* fatty acid synthase type II: A singular structure for a unique function. Protein Science. 2024. https://doi.org/10.1002/pro.4964

Chiaradia *et al.*. Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane. Scientific Reports. 2017 October 9. https://doi.org/10.1038/s41598-017-12718-4

Thouvenel *et al.* The final assembly of trehalose polyphleates takes place within the outer layer of the mycobacterial cell envelope. J Biol Chem. 2020 Aug 7. doi: 10.1074/jbc.

Bouyssié *et al.* Proline: an efficient and user-friendly software suite for large-scale proteomics. Bioinformatics. 2020 May 1. doi: 10.1093/bioinformatics/btaa118.

## CONTACT

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Mail : marion.beaupere@ipbs.fr

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LinkedIn : www.linkedin.com/in/marion-beaupere

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www.researchgate.net/profile/Marion-Beaupere

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<footnote n="1">¹ Institut de Pharmacologie et de Biologie Structurale, CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex, France.</footnote>

<footnote n="2">² Institut de Science des Matériaux de Mulhouse, CNRS, 15 Rue Jean Starcky, 68057 Mulhouse Cedex, France.</footnote>