# Unveiling a novel MmpL-driven transport of outer membrane glycolipids in mycobacteria by direct channeling

Julie Couston, Valentin Wasselin, Christian Chalut, Pontus Gourdon, Mickaël Blaise

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## ► To cite this version:

Julie Couston, Valentin Wasselin, Christian Chalut, Pontus Gourdon, Mickaël Blaise. Unveiling a novel MmpL-driven transport of outer membrane glycolipids in mycobacteria by direct channeling. APPICOM, Nov 2024, Carry le Rouet, France. hal-05373652

HAL Id: hal-05373652

https://hal.science/hal-05373652v1

Submitted on 19 Nov 2025

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# Unveiling a novel MmpL-driven transport of outer membrane glycolipids in mycobacteria by direct channeling

Julie Couston¹, Valentin Wasselin², Christian Chalut², Pontus Gourdon³,⁴, Mickaël Blaise¹

¹Institut de Recherche en Infectiologie de Montpellier (IRIM), CNRS, University of Montpellier, France

²Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, University of Toulouse III - Paul Sabatier (UT3), France

³Department of Biomedical Sciences, University of Copenhagen, Denmark

⁴Department of Experimental Medical Science, Lund University, Sweden

## Background and model proposed

Opportunistic mycobacterial species still represent a **major threat for the human health**. One challenge in the design of new therapies lies in the presence of an **unusual thick and hydrophobic mycomembrane**, composed of a large variety of complex trehalose-based glycolipids. Among them, trehalose polyphosphate (TPP) is present in a broad range of nontuberculous mycobacteria [1] and involved in *M. abscessus* virulence [2,3]. Unlike TPP biosynthesis that is already well-described [1,4], the transport of the TPP precursor, DAT, from the cytoplasm to the mycomembrane by the RND superfamily of transporter member MmpL10 remains to be elucidated [1]. Interestingly, MmpL10 belongs to the **subgroup of MmpLs harboring a flexible "coiled-coil"-like extension** at its periplasmic domain.

Herein, we propose a novel MmpL-driven transport, studied through MmpL10, in which the presence of the coiled-coil extension drives the transporter's trimerization and allows the translocation of the substrates by a direct channeling mechanism.

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## 1- TPP transport to the mycomembrane

TLC of the total extensible lipids of the mycomembrane (CHCl3::MetOH, 9::1)

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AlphaFold prediction structure of MmpL10 from *M. smegmatis* and its **conserved residues involved in the proton motive force** (p.m.f.) of MmpL family of transporters.

MmpL10<sub>Msmeg</sub> and MmpL10<sub>Mab</sub>
share 74 % identity

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MmpL10 residues Asp260 and Asp894, involved in the p.m.f., are required for the presence of TPP at the mycomembrane

1. *M. smegmatis* mc²155 WT
2. PMM23 (ΔmmpL10)
3. PMM23 pMV361e (empty)
4. PMM23 pML10 (MmpL10-WT-HA)
5. PMM23 pML10_D260A (MmpL10-D260A-HA)
6. PMM23 pML10_Y261F (MmpL10-Y261F-HA)
7. PMM23 pML10_D894A (MmpL10-D894A-HA)
8. PMM23 pML10_Y895F (MmpL10-Y895-HA)

## 2- Culture, expression and purification of FL and truncated MmpL10

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When devoid of its extension, MmpL10-trunc is eluted as a **monomer**;

while a **3-fold increase in MW**
can be noticed for MmpL10-FL

## 3- Negative staining

Data collected on a JEOL 1400 Flash Transmission Electron Microscope (120 kV)

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MmpL10-FL

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MmpL10-trunc

Data processing on cryoSPARC

56 micrographs
11,999 particles extracted
5,833 particles selected for 2D classification

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The micrographs in negative stain attest that MmpL10 forms a trimer in solution, and that in absence of the extension the transporter is a monomer

## Conclusion and perspectives

These preliminary data are consistent with the hypothesis that the coiled-coil extension domain promotes MmpL10 assembly into a homotrimer, and which enables the translocation of DAT to the mycomembrane.

As most MmpL family members possess this extension, these preliminary data could be the basis to understand a wide range of lipids and glycolipids transport in actinobacteria.

A high-resolution structure in cryoEM is awaited though to confirm this hypothesis.