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Published on: September 26, 2025
A Screen for Protein-Protein Interactions in Live Mycobacteria Reveals a Functional Link between the
Megan H Touchette1, Erik R Van Vlack2, Lu Bai2
1Department of Pharmacological Sciences, Stony Brook University , Stony Brook, New York 11794, United States.
Abstract:
Outer membrane lipids in pathogenic mycobacteria are important for virulence and survival. Although the biosynthesis of these lipids has been extensively studied, mechanisms responsible for their assembly in the outer membrane are not understood. In the study of Gram-negative outer membrane assembly, protein-protein interactions define transport mechanisms, but analogous interactions have not been explored in mycobacteria. Here we identified interactions with the lipid transport protein LprG. Using site-specific photo-cross-linking in live mycobacteria, we mapped three major interaction interfaces within LprG. We went on to identify proteins that cross-link at the entrance to the lipid binding pocket, an area likely relevant to LprG transport function. We verified LprG site-specific interactions with two hits, the conserved lipoproteins LppK and LppI. We further showed that LprG interacts physically and functionally with the mycolyltransferase Ag85A, as loss of either protein leads to similar defects in cell growth and mycolylation. Overall, our results support a model in which protein interactions coordinate multiple pathways in outer membrane biogenesis and connect lipid biosynthesis to transport.
Insights
This study reveals how the lipid transport protein LprG interacts with other proteins, like LppK, LppI, and Ag85A, to assemble lipids in the mycobacterial outer membrane, crucial for survival.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Outer membrane lipids are vital for pathogenic mycobacteria's virulence and survival.
- Lipid biosynthesis is studied, but outer membrane assembly mechanisms remain unclear.
- Protein interactions are known to mediate outer membrane assembly in Gram-negative bacteria, but not in mycobacteria.
Purpose of the Study:
- To identify protein interactions involved in mycobacterial outer membrane lipid assembly.
- To explore the function of the lipid transport protein LprG in this process.
Main Methods:
- Site-specific photo-cross-linking in live mycobacteria to map LprG interaction interfaces.
- Identification and verification of interacting proteins, including lipoproteins and mycolyltransferases.
- Functional analysis of LprG and its interacting partners through genetic manipulation.
Main Results:
- Three major interaction interfaces within LprG were mapped.
- Proteins LppK, LppI, and Ag85A were identified as LprG interactors.
- LprG physically and functionally interacts with mycolyltransferase Ag85A, with shared defects in cell growth and mycolylation upon loss of either protein.
Conclusions:
- Protein-protein interactions play a key role in coordinating mycobacterial outer membrane biogenesis.
- LprG acts as a central hub, connecting lipid biosynthesis pathways to transport mechanisms.
- Understanding these interactions provides insights into mycobacterial survival and virulence.
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