Characterization of the Leptospiral Outer Membrane and Description of Three Novel Leptospiral Membrane Proteins

Identification and characterization of outer membrane (OM) components are essential in the development of a molecular understanding of bacterial structure and function. Development of techniques for isolation of the OM from Leptospira species and other spirochetes has been difficult because of their unique architecture and a lack of sensitive markers for the cytoplasmic membrane. Like enteric gram-negative bacteria, spirochetes have both an OM and a cytoplasmic membrane, separated by a periplasmic space. However, spirochetal architecture differs significantly from that of gram-negative bacteria in that the peptidoglycan layer of spirochetes is associated with the cytoplasmic membrane rather than the OM (22, 24). For this reason, techniques developed for isolation of the gram-negative OM from components of the underlying cell wall and cytoplasmic membrane are unlikely to be useful in spirochetes. Several approaches have been used in the isolation of the leptospiral OM, including sodium dodecyl sulfate (SDS) treatment of salt-altered cells (2, 7, 25, 28), Sarkosyl (27), and Triton X-114 extraction and phase partitioning (20, 45). On the basis of these approaches, three classes of leptospiral OM proteins (OMPs) have been described: transmembrane, lipoprotein, and peripheral membrane proteins. OmpL1 was the first leptospiral OMP to be described (16). The structure of OmpL1 is predicted to contain at least 10 β-sheet transmembrane segments, which probably accounts for its heat-modifiable electrophoretic mobility. Its transmembrane structure is also supported by evidence that purified OmpL1 creates porin channels in the planar lipid bilayer assay (36). A second class of leptospiral OMPs are the lipoproteins that are anchored to the OM by fatty acids attached to an amino-terminal cysteine (15). Analysis of the detergent phase of Triton X-114 extracts of Leptospira kirschneri intrinsically labeled with tritiated palmitate indicates that the leptospiral OM contains at least five lipoproteins (37). The genes encoding three leptospiral OM lipoproteins, LipL32, LipL36, and LipL41, have been described (17, 18, 37). LipL32 is known to be the most prominent protein in the leptospiral protein profile and is an immunodominant antigen during human leptospirosis (10, 14, 17). LipL41 is a surface-exposed lipoprotein that provides synergistic immunoprotection with OmpL1 (19, 37). Recently, the peripheral membrane protein P31LipL45 was described, which represents a third type of leptospiral OMP (J. Matsunaga, T. A. Young, J. Croda, J. Lima, A. I. Ko, and D. A. Haake, 101st Gen. Meet. Am. Soc. Microbiol. 2001, abstr. D-229, p. 324, 2001). P31LipL45 is exported as a 45-kDa lipoprotein and processed to a 31-kDa C-terminal form that is associated with the OM. Evidence that P31LipL45 is a peripheral OMP, rather than an integral OMP, comes from studies showing that it is released from membranes by urea and that it partitions into the Triton X-114 detergent and aqueous phases. In the absence of a detergent-independent technique for isolation of the leptospiral OM, it has not been possible to confirm that proteins, such as lipoproteins, are actually OM components or rather cytoplasmic membrane proteins selectively extracted by detergents. An example of the aberrant behavior of spirochetal cytoplasmic membrane lipoproteins in detergent extraction methods is TpN47 of Treponema pallidum, which is readily extracted by Triton X-114 (32) yet has been shown to be virtually absent from T. pallidum OM vesicles (OMVs) isolated and purified by isopycnic sucrose gradient ultracentrifugation (4, 34). Another caveat about detergent-based methods of isolating spirochetal OMs was brought to light by the finding that LipL32 is selectively degraded during Triton X-114 phase partitioning (17, 45). Notably lacking from previous studies of the leptospiral OM are controls to assess contamination of the OM fraction with cytoplasmic membrane components. We have developed antisera to two leptospiral cytoplasmic membrane proteins that serve as sensitive markers of the cytoplasmic membrane. Leptospiral lipopolysaccharide (LPS) serves as a specific marker for the OM. By using these cytoplasmic membrane and OM detection tools, we have developed a method for isolation of the leptospiral OM in the form of membrane vesicles. Our method is a modification of techniques that use buffers that destabilize the spirochetal OM and facilitate its release in the form of OMVs (4, 5, 33, 34, 38). These OM isolation techniques take advantage of the structural lability of the spirochetal OM to produce OMVs that can be separated from protoplasmic-cylinder (PC)components by isopycnic sucrose gradient density ultracentrifugation. We believe that the method presented here provides the most accurate assessment of a protein's representation in the leptospiral OM.