Surfaceome of <em>Leptospira</em> spp.

Paul Cullen

Xiaoyi Xu

James Matsunaga

Yolanda Sanchez

Albert Ko

David Haake

Ben Adler

Australian Bacterial Pathogenesis Program,^{Victorian Bioinformatics Consortium, Department of Microbiology, Monash University, VIC 3800 Australia,}^{Division of Infectious Diseases, 111F, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073,}^{Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, California 90095,}^{Gonçalo Moniz Research Center, Oswaldo Cruz Foundation, Brazilian Ministry of Health, Salvador, Bahia, Brazil,}^{Division of International Medicine and Infectious Diseases, Weil Medical College of Cornell University, New York, New York 10021}⁶

^{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:}ua.ude.hsanom.dem@reldA.neB.

Received 2005 Jan 31; Revised 2005 Mar 7; Accepted 2005 Mar 17.

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, {"type":"entrez-protein","attrs":{"text":"Q8F8Q0","term_id":"81471683","term_text":"Q8F8Q0"}}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^{cells) offering the potential to analyze bacterial surface expression during infection.}

Abstract

Leptospirosis is a zoonosis of global distribution caused by infection with one of more than 230 serovars belonging to pathogenic species of Leptospira (10a, 25). Immunity to infection is mediated principally by antibodies, which opsonize leptospires for phagocytosis by both neutrophils and macrophages (29, 39) and also mediate complement-dependent killing (1). Lipopolysaccharide (LPS) is the major component of the leptospiral cell surface (10a, 41). It is the target antigen for antibodies that are agglutinating, opsonic, and protective (3, 11, 23, 24). However, LPS-mediated immunity is restricted to serovars which are antigenically related.

The leptospiral outer membrane contains few integral transmembrane proteins, with the trimeric porin OmpL1 being the only such protein that has been identified and characterized (14, 37). However, the membrane contains numerous lipoproteins, which are anchored to the membrane through their N-terminal lipid moieties (9, 13). Some of these have been shown to stimulate partial immunity in animal models. LipL32 delivered by recombinant adenovirus partially protected gerbils from acute infection (6), while LipL41 showed synergistic immunoprotection with OmpL1; neither protein was protective when administered alone (18). Another outer membrane lipoprotein, LipL36, was shown to be expressed by leptospires growing in vitro but not within the mammalian host (4). Although only partial protection has been achieved to date, leptospiral outer membrane proteins constitute attractive vaccine candidates because they are well conserved across the pathogenic species of Leptospira (9, 13).

Clearly, the leptospiral surface is important when we consider the interaction of bacteria with host cells and tissues in the context of pathogenesis and immunity to infection. However, to date there have been no global studies undertaken to identify the components of the outer membrane that are exposed on the leptospiral cell surface. Such studies are critical because they reduce the number of proteins that need to be assessed as potential vaccine targets and highlight proteins that are likely to be involved directly in interactions with the host. For example, LipL36 was shown to be anchored to the inner leaflet of the outer membrane and therefore to be localized to the periplasm (13, 38, 41). Due to the different techniques utilized to assess surface exposure, there is no information regarding the relative exposure of the different leptospiral surface proteins, which is also of critical importance when proteins are selected to assess as potential vaccinogens.

The aim of the present study was therefore to identify all of the protein components of the leptospiral surface by labeling of viable leptospires, affinity capture of the labeled proteins, and their identification by mass spectrometry (MS). This process also allowed the relative surface exposure of leptospiral surface components to be approximated. In addition, several independent experiments were undertaken to verify the surface exposure of the major outer membrane protein (MOMP) LipL32.

Acknowledgments

Work conducted at Monash University was supported by a program grant (to B.A.) from the National Health and Medical Research Council, Canberra, Australia, and by the Victorian Bioinformatics Consortium funded by the Government of Victoria, Australia. The remaining portion of the study was supported by Public Health Service grants AI-34431 (to D.A.H.) and AI-01605 (to A.I.K.) from the National Institute of Allergy and Infectious Diseases; VA Medical Research Funds (to J.M. and D.A.H.); and grant 09224-7 from Biomanguinhos, the Oswaldo Cruz Foundation, Brazilian Ministry of Health (to A.I.K.).

We thank R. Hartskeerl for monoclonal antibody F71C2. We thank George Sachs and David Scott for providing access to the confocal microscope and assistance in its use. We gratefully acknowledge Mitra Mastali's technical contribution to this study.

Acknowledgments

Notes

Editor: J. T. Barbieri

Notes

Editor: J. T. Barbieri

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