title: Surfaceome of Leptospira spp.
abstract: The identification of the subset of outer membrane proteins exposed on the surface of a bacterial cell (the surfaceome) is critical to understanding the interactions of bacteria with their environments and greatly narrows the search for protective antigens of extracellular pathogens. The surfaceome of Leptospira was investigated by biotin labeling of viable leptospires, affinity capture of the biotinylated proteins, two-dimensional gel electrophoresis, and mass spectrometry (MS). The leptospiral surfaceome was found to be predominantly made up of a small number of already characterized proteins, being in order of relative abundance on the cell surface: LipL32 > LipL21 > LipL41. Of these proteins, only LipL32 had not been previously identified as surface exposed. LipL32 surface exposure was subsequently verified by three independent approaches: surface immunofluorescence, whole-cell enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Three other proteins, Q8F8Q0 (a putative transmembrane outer membrane protein) and two proteins of 20 kDa and 55 kDa that could not be identified by MS, one of which demonstrated a high degree of labeling potentially representing an additional, as-yet-uncharacterized, surface-exposed protein. Minor labeling of p31(LipL45), GroEL, and FlaB1 was also observed. Expression of the surfaceome constituents remained unchanged under a range of conditions investigated, including temperature and the presence of serum or urine. Immunization of mice with affinity-captured surface components stimulated the production of antibodies that bound surface proteins from heterologous leptospiral strains. The surfaceomics approach is particularly amenable to protein expression profiling using small amounts of sample (<10(7) cells) offering the potential to analyze bacterial surface expression during infection.
authors.affiliation: Australian Bacterial Pathogenesis Program, Victorian Bioinformatics Consortium, Department of Microbiology, Monash University, VIC 3800 Australia.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Cullen
authors.forename: Xiaoyi
authors.initials: X
authors.lastname: Xu
authors.forename: James
authors.initials: J
authors.lastname: Matsunaga
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Sanchez
authors.forename: Albert I
authors.initials: AI
authors.lastname: Ko
authors.forename: David A
authors.initials: DA
authors.lastname: Haake
authors.forename: Ben
authors.initials: B
authors.lastname: Adler
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001710:Biotin; D002473:Cell Wall / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D007919:Leptospira / Q000378:metabolism* / Q000648:ultrastructure; D008074:Lipoproteins / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D016253:Microscopy, Immunoelectron
chemical_list: D001425:Bacterial Outer Membrane Proteins; C406589:LipL32 protein, Leptospira; D008074:Lipoproteins; D001710:Biotin