title: Selective association of outer surface lipoproteins with the lipid rafts of Borrelia burgdorferi.
abstract: Borrelia burgdorferi contains unique cholesterol-glycolipid-rich lipid rafts that are associated with lipoproteins. These complexes suggest the existence of macromolecular structures that have not been reported for prokaryotes. Outer surface lipoproteins OspA, OspB, and OspC were studied for their participation in the formation of lipid rafts. Single-gene deletion mutants with deletions of ospA, ospB, and ospC and a spontaneous gene mutant, strain B313, which does not express OspA and OspB, were used to establish their structural roles in the lipid rafts. All mutant strains used in this study produced detergent-resistant membranes, a common characteristic of lipid rafts, and had similar lipid and protein slot blot profiles. Lipoproteins OspA and OspB but not OspC were shown to be associated with lipid rafts by transmission electron microscopy. When the ability to form lipid rafts in live B. burgdorferi spirochetes was measured by fluorescence resonance energy transfer (FRET), strain B313 showed a statistically significant lower level of segregation into ordered and disordered membrane domains than did the wild-type and the other single-deletion mutants. The transformation of a B313 strain with a shuttle plasmid containing ospA restored the phenotype shared by the wild type and the single-deletion mutants, demonstrating that OspA and OspB have redundant functions. In contrast, a transformed B313 overexpressing OspC neither rescued the FRET nor colocalized with the lipid rafts. Because these lipoproteins are expressed at different stages of the life cycle of B. burgdorferi, their selective association is likely to have an important role in the structure of prokaryotic lipid rafts and in the organism's adaptation to changing environments. IMPORTANCE Lipid rafts are cholesterol-rich clusters within the membranes of cells. Lipid rafts contain proteins that have functions in sensing the cell environment and transmitting signals. Although selective proteins are present in lipid rafts, little is known about their structural contribution to these domains. Borrelia burgdorferi, the agent of Lyme disease, has lipid rafts, which are novel structures in bacteria. Here, we have shown that the raft-associated lipoproteins OspA and OspB selectively contribute to lipid rafts. A similar but non-raft-associated lipoprotein, OspC, cannot substitute for the role of OspA and OspB. In this study, we have demonstrated that lipoprotein association with lipid rafts is selective, further suggesting a functional adaptation to different stages of the spirochete life cycle. The results of this study are of broader importance and can serve as a model for other bacteria that also possess cholesterol in their membranes and, therefore, may share lipid raft traits with Borrelia.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Toledo
authors.forename: Jameson T
authors.initials: JT
authors.lastname: Crowley
authors.forename: James L
authors.initials: JL
authors.lastname: Coleman
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: LaRocca
authors.forename: Salvatore
authors.initials: S
authors.lastname: Chiantia
authors.forename: Erwin
authors.initials: E
authors.lastname: London
authors.forename: Jorge L
authors.initials: JL
authors.lastname: Benach
mesh_terms: D000942:Antigens, Bacterial / Q000032:analysis* / Q000235:genetics; D000954:Antigens, Surface / Q000032:analysis* / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis* / Q000235:genetics; D001428:Bacterial Vaccines / Q000032:analysis* / Q000235:genetics; D025065:Borrelia burgdorferi / Q000737:chemistry*; D003902:Detergents / Q000378:metabolism; D017353:Gene Deletion; D005816:Genetic Complementation Test; D006801:Humans; D008074:Lipoproteins / Q000032:analysis* / Q000235:genetics; D021962:Membrane Microdomains / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission
chemical_list: D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D003902:Detergents; D008074:Lipoproteins; C072045:OspA protein; C079616:OspC protein; C077030:OspB protein, Borrelia burgdorferi