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

• Published in: ACS infectious diseases Vol. 3; no. 5; pp. 336 - 348
• Main Authors: Touchette, Megan H, Van Vlack, Erik R, Bai, Lu, Kim, Jia, Cognetta, Armand B, Previti, Mary L, Backus, Keriann M, Martin, Dwight W, Cravatt, Benjamin F, Seeliger, Jessica C
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 12.05.2017
• Subjects: Acyltransferases - chemistry; Acyltransferases - genetics; Acyltransferases - metabolism; Amino Acid Motifs; Antigens, Bacterial - chemistry; Antigens, Bacterial - genetics; Antigens, Bacterial - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Membrane - chemistry; Cell Membrane - metabolism; Cloning, Molecular; Gene Expression; Kinetics; Lipids - chemistry; Models, Molecular; Mycobacterium smegmatis - genetics; Mycobacterium smegmatis - metabolism; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - metabolism; Mycobacterium tuberculosis - pathogenicity; Plasmids - chemistry; Plasmids - metabolism; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Virulence; outer membrane; protein interactions; mycolic acid; lipoprotein; lipid transport; cell wall; mycobacteria; photo-cross-linking
• ISSN: 2373-8227; 2373-8227
• DOI: 10.1021/acsinfecdis.6b00179

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.