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Infection and Immunity, August 2005, p. 4853-4863, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.4853-4863.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Surfaceome of Leptospira spp.

Paul A. Cullen,1,2 Xiaoyi Xu,3 James Matsunaga,3,4 Yolanda Sanchez,3 Albert I. Ko,5,6 David A. Haake,3,4 and Ben Adler1,2*

Australian Bacterial Pathogenesis Program,1 Victorian Bioinformatics Consortium, Department of Microbiology, Monash University, VIC 3800 Australia,2 Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073,3 Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California 90095,4 Gonçalo Moniz Research Center, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Bahia, Brazil,5 Division of International Medicine and Infectious Diseases, Weil Medical College of Cornell University, New York, New York 100216

Received 31 January 2005/ Returned for modification 7 March 2005/ Accepted 17 March 2005

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 p31LipL45, 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 (<107 cells) offering the potential to analyze bacterial surface expression during infection.

* Corresponding author. Mailing address: Australian Bacterial Pathogenesis Program, Victorian Bioinformatics Consortium, Department of Microbiology, Monash University, VIC 3800 Australia. Phone: 61-3-9905-4815. Fax: 61-3-9905-4811. E-mail: Ben.Adler{at}med.monash.edu.au.

Editor: J. T. Barbieri

Infection and Immunity, August 2005, p. 4853-4863, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.4853-4863.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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