title: 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.
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.
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University , Stony Brook, New York 11794, United States.
authors.forename: Megan H
authors.identifier: 0000-0002-9589-7118
authors.initials: MH
authors.lastname: Touchette
authors.affiliation: Department of Chemistry, Stony Brook University , Stony Brook, New York 11790, United States.
authors.forename: Erik R
authors.initials: ER
authors.lastname: Van Vlack
authors.affiliation: Department of Chemistry, Stony Brook University , Stony Brook, New York 11790, United States.
authors.forename: Lu
authors.initials: L
authors.lastname: Bai
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University , Stony Brook, New York 11794, United States.
authors.forename: Jia
authors.initials: J
authors.lastname: Kim
authors.affiliation: Department of Chemical Physiology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Armand B
authors.initials: AB
authors.lastname: Cognetta
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University , Stony Brook, New York 11794, United States.
authors.forename: Mary L
authors.initials: ML
authors.lastname: Previti
authors.affiliation: Department of Chemical Physiology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Keriann M
authors.initials: KM
authors.lastname: Backus
authors.affiliation: Department of Medicine, Stony Brook University , Stony Brook, New York 11794, United States.|Proteomics Center, Stony Brook University , Stony Brook, New York 11794, United States.
authors.forename: Dwight W
authors.initials: DW
authors.lastname: Martin
authors.affiliation: Department of Chemical Physiology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Benjamin F
authors.identifier: 0000-0001-5330-3492
authors.initials: BF
authors.lastname: Cravatt
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University , Stony Brook, New York 11794, United States.
authors.forename: Jessica C
authors.identifier: 0000-0001-7148-1073
authors.initials: JC
authors.lastname: Seeliger
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D015870:Gene Expression; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008958:Models, Molecular; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014774:Virulence
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D002352:Carrier Proteins; D008055:Lipids; D011994:Recombinant Proteins; D000217:Acyltransferases; C112580:antigen 85A, Mycobacterium tuberculosis
keywords: cell wall; lipid transport; lipoprotein; mycobacteria; mycolic acid; outer membrane; photo-cross-linking; protein interactions