A Screen for Protein–Protein Interactions in Live Mycobacteria Reveals a Functional Link between the Virulence-Associated Lipid Transporter LprG and the Mycolyltransferase Antigen 85A

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.