UCLA / VA Leptospirosis Research Laboratory
Characterization of Leptospiral Membrane Proteins.
Our laboratory has now identified and characterized the genes encoding twelve membrane proteins, six of which are located either partly or exclusively in the outer membrane. The trans-membrane and lipoprotein classes of membrane proteins are represented in both the outer and inner membranes (see Figure 1 and the Leptospiral Membrane Protein Website). Of the six leptospiral OMPs, there is experimental evidence for surface-exposure of OmpL1 and LipL41, while LipL36 appears to be a subsurface antigen (see Figure 1). Studies are ongoing to determine the degree of surface-exposure for the other three leptospiral OMPs: LipL32, P31LipL45, and LipL48.
The porin, OmpL1. OmpL1 is the first transmembrane outer membrane protein (OMP) to have been described in a pathogenic spirochete. OmpL1 was originally isolated in surface immunoprecipitation studies intended to identify proteins exposed on the leptospiral surface (Haake et al., 1991). The N-terminal amino acid sequence was obtained from the 33-kDa surface-immunoprecipitated protein and used to design oligonucleotide primers which allowed isolation of the ompL1 gene (Haake et al., 1993). Examination of the OmpL1 sequence revealed beta-sheet membrane spanning segments typical of gram-negative OMPs , consistent with its property of heat-modifiable electrophoretic mobility. Polyclonal antiserum generated by immunizing rabbits with a purified, recombinant His6-OmpL1 fusion protein was used to demonstrate OmpL1 surface exposure by immunoelectron microscopy. Subsequent structural studies found that OmpL1 is an integral membrane protein which is present in the native leptospiral membrane as trimers (Shang et al., 1995), another feature typical of gram-negative transmembrane OMPs. Purified, recombinant OmpL1 exhibits porin activity in planar lipid bilayer studies. We believe that research on OmpL1 may be relevant to transmembrane OMPs of other pathogenic spirochetes, a subject which is currently of great interest because proteins of this type are potential targets of a protective host immune response.
Figure 1. Leptospiral membrane protein architecture. The leptospiral surface is dominated by lipopolysaccharide (LPS). Some outer membrane (OM) proteins are surface-exposed (e.g. OmpL1 and LipL41) while others are not (e.g. LipL36). In spirochetes, the endoflagella (EF) are subsurface structures located in the periplasm. The peptidoglycan (PG) cell wall is more closely associated with the inner membrane (IM) than the OM. Known inner membrane proteins include signal peptidase (SP), penicillin binding proteins (PBPs), LipL31, and ImpL63. The heat-shock protein, GroEL, is largely cytoplasmic in location.
Leptospiral lipoproteins. Like other bacterial lipoproteins, leptospiral lipoproteins appear to be anchored to membranes by fatty acids that modify the amino-terminal cysteine. Our laboratory has isolated the genes encoding six different leptospiral lipoproteins (Table 1), five of which are located either exclusively or partly in the outer membrane. We designated the leptospiral lipoproteins based upon their apparent molecular mass. For example, our first leptospiral lipoprotein was designated LipL41 because it has a apparent molecular mass of 41-kDa.
LipL41 was initially identified in the same surface-immunoprecipitation studies that isolated OmpL1 (Haake et al., 1991). Subsequent surface-immunoprecipitation experiments confirmed that LipL41 is exposed on the leptospiral surface, while another outer membrane lipoprotein, LipL36, appears to be restricted to the periplasmic leaflet of the outer membrane (Shang et al., 1996). The most abundant proteins in spirochetes are lipoproteins, and Leptospira species are no exception to this rule. In collaboration with Richard Zuerner and Carole Bolin at the National Animal Disease Center in Ames, Iowa, we have demonstrated that the leptospiral major outer membrane protein is LipL32 (Haake et al., 2000). LipL32 and LipL41 appear to be expressed constitutively by all pathogenic Leptospira species under all environmental conditions, while LipL36 and a fourth lipoprotein OMP, LipL48, are subject to differential expression (Barnett et al., 1999). LipL36 and LipL48 are not expressed during infection, but are expressed in large amounts in culture-attenuated organisms. Genes encoding two additional lipoproteins have been isolated in this laboratory by Dr. James Matsunaga by screening a lambda TriplEx (Clontech) expression library constructed using partially-digested DNA from the virulent L. kirschneri with serum from a hyperimmunized rabbit. LipL45 is expressed in early-passage cultures isolated from hamsters infected with L. kirschneri, but not in high-passage cultures of the same strain. LipL45 is processed to a 31-kD growth phase regulated peripheral membrane protein, designed P31LipL45, which is expressed in both low- and high-passage cultures (Matsunaga et al., 2002). The second lipoprotein identified by Dr. Matsunaga is LipL31, which is located exclusively in the inner membrane. Several other leptospiral lipoproteins, LipL53, LipL59, and LipL71, are thought to exist on the basis of intrinsic labeling of L. kirschneri with tritiated palmitate.
Table 1: Confirmed and Potential Leptospiral Lipoproteins
|LipL31||F F A S C G D||-||?||IM||+|
|LipL32||S I T A C G A||+||+||OM||+|
|LipL36||A L T A C K S||+||+||OM||-|
|LipL41||F L G N C A A||+||+||OM & IM||+|
|LipL45||V F N A C K K||+||?||OM & IM*||+|
|LipL48||S F I N C K E||+||?||OM||-|
*The 31-kD form of LipL45 is a peripheral membrane protein associated with both membranes.
In each case, the deduced amino acid sequences reveal a signal peptide followed by a lipoprotein signal peptidase cleavage site (see Table 1). Although some of these lipoprotein signal peptidase cleavage sites are atypical when compared to E. coli lipoprotein signal peptidase cleavage sites (usually L-X-Y-C), they are consistent with those of lipoproteins from other spirochetes. There is good evidence for relaxing the lipobox requirements in spirochetes. In a review of spirochetal lipoproteins (Haake, 2000), 28 spirochetal protein sequences for which there is experimental evidence of lipid modification were compared allowing definition of the spirochetal "lipobox" as:
Several other lines of evidence also support the conclusion that leptospiral lipoproteins are modified by lipid at an N-terminal cysteine residue. Lipid modification appears to be responsible for the hydrophobicity of these proteins. Native LipL32, LipL36, LipL41, and LipL48 partition into the Triton X-114 detergent phase, while the corresponding His6 fusion proteins partition into the Triton X-114 aqueous phase. Intrinsic labeling of L. kirschneri with tritiated palmitate results in selective labeling of LipL32, LipL36, and LipL41. In the case of LipL32, acid treatment resulted in removal of the tritium label, consistent with hydrolysis of the linkage between palmitate and the amino-terminal cysteine (Haake et al., 2000). For LipL36 and LipL41, lipidation has been shown to be sensitive to globomycin, a selective inhibitor of lipoprotein signal peptidase.
Transmembrane inner membrane proteins (IMPs). We and others have described a number of leptospiral transmembrane IMPs, most of which are thought to be anchored to the inner membrane by hydrophobic alpha-helical transmembrane domains. Some of these proteins are presumed to be IMPs on the basis of strong homologies with IMPs of other bacteria. The gene encoding leptospiral LepB (signal peptidase) was isolated at the National Animal Disease Center using a monoclonal antibody, 1H8 (Trueba et al., 1995). Genes encoding a family of penicillin-binding proteins have been isolated at the Pasteur Institute (Pbp1A and Pbp3; Brenot et al., 2001) and in this laboratory (Pbp1B and Pbp1C). Rudy Hartskeerl, at the Royal Tropical Institute in the Netherlands has isolated the gene encoding leptospiral SecY (GenBank Accession #AF115283). The gene encoding ImpL63, isolated in this laboratory, has no homologues in GenBank, but has been determined to be an inner membrane component on the basis of localization studies described in the next section.
Please refer to the Leptospiral Membrane Protein Website for a tabular summary of leptospiral membrane proteins.
Fractionation of Leptospiral Membranes.
Localization of membrane components is an issue of fundamental importance in the characterization of membrane proteins. Determining whether a membrane protein is a component of the outer membrane is a major criterion in the identification of potential protective immunogens. Characterization of leptospiral membrane components has been greatly facilitated by development of highly sensitive antibody reagents (Leptospiral Membrane Protein Website). These tools have proven to be essential in validating leptospiral membrane fractionation procedures. The standard approach to leptospiral outer membrane isolation involves treatment of organisms with 1 M NaCl resulting in formation of salt-altered cells (SACs) in which the outer membrane balloons away from the protoplasmic cylinder. SACs are then treated with 0.04% SDS which releases the outer membrane from the protoplasmic cylinder (Auran et al., 1972). We have developed two additional methods of leptospiral outer membrane isolation, which complement the standard approach.
Triton detergent extraction studies. Treatment of spirochetes with nonionic detergents such as Triton X-100 and Triton X-114 is a rapid and straightforward method of outer membrane solubilization. Although concerns have been raised about contamination of detergent extracts with inner membrane components in the case of B. burgdorferi and T. pallidum, this approach appears to be selective for the leptospiral outer membrane. Treatment of L. kirschneri with Triton detergents results in partial to complete solubilization of leptospiral LPS, OmpL1, LipL32, LipL36, Lip41, LipL48, and P31LipL45. As an indication of their hydrophobicity, all of these outer membrane components also partition into the Triton X-114 detergent phase. In previously published studies, we have shown that leptospiral penicillin-binding proteins, GroEL, and endoflagella proteins are not solubilized by Triton detergents (Haake et al., 1991; Haake et al., 1996). More recently, we have also shown that LipL31 and ImpL63 are completely insoluble when intact L. kirschneri are extracted with Triton detergents. In control experiments, Triton X-100 is able to solubilize LipL31 and ImpL63 after sonication and lysozyme treatment of L. kirschneri. This result indicates that the failure to solubilize LipL31 and ImpL63 when intact organisms are treated with Triton is due to the integrity of the inner membrane/peptidoglycan cell wall, rather than an intrinsic insolubility of these proteins in Triton detergents.
Isolation of leptospiral outer membrane vesicles (OMVs). To avoid contamination of the outer membrane with cytoplasmic membrane proteins when using detergents, we have developed a novel approach to isolation of the leptospiral outer membrane in the form of OMVs. We found the leptospiral outer membrane can be isolated from SACs by alkaline plasmolysis without the use of any detergent. Treatment of SACs with hypertonic sucrose (10 mM Tris, pH 9), combined with vigorous vortexing, results in release of the outer membrane in the form of small unilamellar vesicles. The outer membrane vesicles (OMVs) are then purified away from the protoplasmic cylinders by isopycnic centrifugation on a 27-55% (w/v) sucrose gradient. Two populations of OMVs were found in the sucrose gradient, a heavier fraction (OMV-H) and a lighter fraction (OMV-L) with densities of 1.16 and 1.18 grams/ml, respectively. The protoplasmic material was found in the sucrose gradient at a density of 1.20 grams/ml. The OMVs were found to be free of protoplasmic cylinder components such as LipL31, ImpL63, flagella, and the GroEL heat-shock protein. The OMVs were found to contain leptospiral LPS, OmpL1, the lipoproteins LipL32, LipL36, LipL41, LipL48, P31LipL45 and a number of less well charac-terized proteins with molecular masses of 16-, 21-, 21.5-, 22-, 31-, 36-, 44-, 48-, 90-, and 116-kD.
Leptospiral Membrane Proteins Expressed During Infection.
The global distribution of pathogenic Leptospira species is a reflection of their ability to adapt to environmental conditions both inside and outside of mammalian hosts. Given what is known about the leptospiral life-cycle, it would not be surprising if certain membrane proteins were subject to differential expression. Outer membrane proteins that are expressed during mammalian infection would be of greater interest as targets for a protective immune response. Although, given the success of OspA immunization in prevention of B. burgdorferi infection, it should be kept in mind that it is possible for proteins that are not expressed during mammalian infection to be effective vaccinogens.
Immunohistochemistry studies. Using kidney tissue obtained from hamsters infected with L. kirschneri, immunohistochemistry studies have been performed to determine the expression and distribution of outer membrane antigens during renal infection (Barnett et al., 1999; Haake et al., 2000). Immunoblots performed with sera from hamsters challenged with host-derived L. kirschneri demonstrated reactivity with OmpL1, LipL32, and LipL41, but not LipL36 or ImpL63. Although LipL36 is a prominent outer membrane antigen of cultivated L. kirschneri, its expression also could not be detected in infected hamster kidney tissue by immunohistochemistry, indicating that expression of this protein is down-regulated in vivo. In contrast, LPS, OmpL1, and LipL41 were demonstrated on organisms colonizing the lumen of proximal convoluted renal tubules at both 10 and 28 days post infection. Table 1 summarizes the expression of additional leptospiral lipoproteins by L. kirschneri during renal infection of hamsters based on additional immunohistochemistry experiments.
Figure 2. Immunohistochemistry of serial sections of hamster kidney 28 days after challenge with L. kirschneri showing reactivity (arrow) to OmpL1 (left-hand panel), but not to LipL36 (right-hand panel).
Immune Response to Membrane Proteins in Leptospiral Infection. To develop a more comprehensive understanding of leptospiral membrane proteins we have used the humoral immune response to leptospiral infection as a reporter to identify which leptospiral membrane proteins are expressed during infection. Experiments in our laboratory show that sera from hamsters surviving experimental infection with host-derived L. kirschneri contain antibodies to OmpL1 and LipL41 (Barnett et al., 1999). More recently, we have been collaborating with Albert Ko (Salvador, Brazil) and Paul Levett (St. Michael, Barbados) in an effort to expand these observations to natural infection in humans. Previous 1D immunoblot studies had indicated the importance of 32- and 58-kDa antigens in the human immune response to leptospirosis. We showed these proteins to be LipL32 and GroEL respectively using one- and two-dimensional immunoblot analysis of leptospiral proteins was performed using convalescent sera from leptospirosis patients from Barbados and Brazil (Guerreiro et al., 2001). We also identified OmpL1, LipL41, and a number of other potentially important leptospiral protein antigens as targets of the humoral immune response during human leptospirosis by immunoblot (Guerreiro et al., 2001) and ELISA analysis (Flannery et al., 2001).
Immunoprotection Studies in Hamsters.
We have examined the immunoprotective capacity of the leptospiral porin, OmpL1, and the leptospiral outer membrane lipoprotein, LipL41, in the Golden Syrian hamster model of leptospirosis (Haake et al., 1999). Specialized expression plasmids were developed to facilitate expression of leptospiral proteins in Escherichia coli as the membrane-associated proteins, OmpL1-M and LipL41-M. Although OmpL1-M expression is highly toxic in E. coli, this was accomplished using the pMMB66-OmpL1 plasmid which has undetectable background expression without induction. LipL41-M expression and processing was enhanced by constructing the expression plasmid, pET15b-LipL41*, in which the lipoprotein signal peptidase cleavage site of LipL41 has been altered by site-specific mutagenesis from LGNC to LAGC, which is identical to that of E. coli murein lipoprotein. The resulting plasmid, pET15b-LipL41*, yields much more efficient expression and processing of LipL41 in E. coli. Active immunization of hamsters with E. coli membrane fractions containing a combination of OmpL1-M and LipL41-M was found to provide significant protection against homologous challenge with L. kirschneri serovar grippotyphosa. 28 d after intraperitoneal inoculation, survival in animals vaccinated with both proteins was 71% (95% CI, 53% to 89%), compared with only 25% (95% CI, 8% to 42%) in the control group (P < 0.001). On the basis of serological, histological, and microbiological assays, no evidence of infection was found in the vaccinated survivors. Interestingly, the protective effects of immunization with OmpL1-M and LipL41-M were synergistic, since significant levels of protection were not observed in animals immunized with the membrane-associated forms of either OmpL1 or LipL41 alone. In contrast to immunization with the membrane-associated forms of leptospiral proteins, hamsters immunized with detergent-solubilized His6-OmpL1 and His6-LipL41 fusion proteins, either alone or in combination, were not protected. These data indicate that the manner in which OmpL1 and LipL41 are formulated is an important determinant of immunoprotection. We have not tested whether immunization with OmpL1-M and LipL41-M is cross-protective against infection with heterologous species or serovars, but we have compared OMP sequences from ten leptospiral serovars representing five different species. A high level of amino acid sequence conservation was found with 98.9%, 97.3%, and 90.9% average amino acid sequence identity for LipL32, LipL41, and OmpL1, respectively.
Acknowledgements. The UCLA/VA Leptospirosis Research Laboratory is led jointly by Drs. David Haake and James Matsunaga, and supported by VA Medical Research Funds (to D.H. and J.M.) and NIH/NIAID Grant AI-34431 (to D.H.). The research accomplishments described on this website would not have been possible without the dedication and insight of Current and Former Members of the laboratory including Garlo Chao, Manjula Dundoo, Erin Hurley, Betty Liu, Carleen Martinich, Mary Mazel, Adam McCoy, Jay Preutz, Miguel Rocha, Yolanda Sanchez, Ellen Shang, Terry Summers, and Tracy Young. We are also grateful for the generosity and contributions of our Collaborators, including David Blanco, Cheryl Champion, Maurice Exner, Michael Lovett, James Miller, Jarlath Nally, and Liz Wagar at UCLA; Paul Levett at the Centers for Disease Control in Atlanta, Georgia; Isabelle Saint Girons and Catherine Werts at the Pasteur Institute in Paris, France; Richard Zuerner at the National Animal Disease Center in Ames, Iowa; Carole Bolin at Michigan State University; Jeanne & Dean Barnett at the University of Southern Indiana; Rudy Hartskeerl at the Royal Tropical Institute in Amstersdam, the Netherlands; Albert Ko at the Brazilian Ministry of Health in Salvador, Brazil; Michele Barocchi, Brendan Flannery, and Lee Riley at UC Berkeley; and last but not least, Ben Adler, Dieter Bulach, and Paul Cullen at Monash University in Melbourne, Australia.
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