There is now substantial evidence that Borrelia burgdorferi , the Lyme disease spirochete, undergoes major alterations in antigenic composition as it cycles between its arthropod and mammalian hosts. In this report, we cultivated B. burgdorferi 297 within dialysis membrane chambers implanted into the peritoneal cavities of rats to induce antigenic changes similar to those which occur during mammalian infection. Chamber-grown spirochetes, which remained fully virulent, did not express either outer surface protein A or Lp6.6, lipoproteins known to be downregulated after mammalian infection. However, they did, express p21, a well characterized outer surface protein E homologue, which is selectively expressed during infection. SDS-PAGE, two-dimensional gel electrophoresis, and immunoblot analysis revealed that chamber-grown borreliae also expressed uncharacterized proteins not expressed by in vitro-cultivated spirochetes; reactivity with sera from mice chronically infected with B. burgdorferi 297 confirmed that many of these novel proteins are selectively expressed during experimental murine infection. Finally, we used differential display RT-PCR to identify transcripts of other differentially expressed B. burgdorferi genes. One gene ( 2.9-7lpB ) identified with this technique belongs to a family of genes located on homologous 32-and 18-kb circular plasmids. The lipoprotein encoded by 2.9-7lpB was shown to be selectively expressed by chambergrown spirochetes and by spirochetes during experimental infection. Cultivation of B. burgdorferi in rat peritoneal implants represents a novel system for studying Lyme disease spirochetes in a mammalian host-adapted state. ( J. Clin. Invest. 1998. 101:2240-2250.) Key words: host-adapted • chamber implants • ticks • Borrelia burgdorferi • outer surface protein

The outer membrane of Treponema pallidum, the noncultivable agent of venereal syphilis, contains a paucity of protein(s) which has yet to be definitively identified. In contrast, the outer membranes of gram-negative bacteria contain abundant immunogenic membrane-spanning ␤-barrel proteins mainly involved in nutrient transport. The absence of orthologs of gram-negative porins and outer membrane nutrient-specific transporters in the T. pallidum genome predicts that nutrient transport across the outer membrane must differ fundamentally in T. pallidum and gram-negative bacteria. Here we describe a T. pallidum outer membrane protein (TP0453) that, in contrast to all integral outer membrane proteins of known structure, lacks extensive ␤-sheet structure and does not traverse the outer membrane to become surface exposed. TP0453 is a lipoprotein with an amphiphilic polypeptide containing multiple membrane-inserting, amphipathic ␣-helices. Insertion of the recombinant, nonlipidated protein into artificial membranes results in bilayer destabilization and enhanced permeability. Our findings lead us to hypothesize that TP0453 is a novel type of bacterial outer membrane protein which may render the T. pallidum outer membrane permeable to nutrients while remaining inaccessible to antibody.

Previous freeze-fracture electron microscopy (EM) studies have shown that the outer membrane (OM) of Treponema pallidum contains sparse transmembrane proteins. One strategy for molecular characterization of these rare OM proteins involves isolation of T. pallidum OMs. Here we describe a simple and extremely gentle method for OM isolation based upon isopycnic sucrose density gradient ultracentrifugation of treponemes following plasmolysis in 20% sucrose. Evidence that T. pallidum OMs were isolated included (i) the extremely low protein/lipid ratio of the putative OM fraction, (ii) a paucity of antigenic and/or biochemical markers for periplasmic, cytoplasmic membrane, and cytosolic compartments, and (iii) freeze-fracture EM demonstrating that the putative OMs contained intramembranous particles highly similar in size and density to those in native T. pallidum OMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the OMs contained a relatively small number of treponemal proteins, including several which did not appear to correspond to previously characterized T. pallidum antigens. Interestingly, these candidate rare OM proteins reacted poorly with syphilitic sera as determined by both conventional immunoblotting and enhanced chemiluminescence. Compared with whole cells, T. pallidum OMs were deficient in cardiolipin, the major lipoidal antigen reactive with antibodies in syphilitic sera. Also noteworthy was that other lipoidal constituents of OMs, including the recently discovered glycolipids, did not react with human syphilitic sera. These latter observations suggest that the poor antigenicity of virulent T. pallidum is a function of both the lipid composition and the low protein content of its OM.

Freeze-fracture and deep-etch electron microscopy were used to investigate the molecular architecture of the Treponema paUlidum outer membrane (OM). Freeze-fracture electron microscopy of treponemes freshly harvested from rabbit testes revealed that the intramembranous particles (IMPs) in both the concave and convex OM leaflets were distributed into alternating areas of relatively high and low particle dei1ity; in many OM fractures, IMPs formed rows that ran either parallel to or obliquely across the fracture faces. Statistical analysis (runs test) confirmed that the IMPs were nonrandomly distributed in both OM leaflets. Examination of deep-etched specimens revealed that the particles observed in freeze-fractured OMs also were surface exposed. Combined analysis of deep-etched and cross-fractured treponemes revealed that the OM particles were located in regions of the OM away from the endoflagella and closely apposed to the cytoplasmic membrane-peptidoglycan complex. When treponemes were incubated for extended periods with heat-inactivated immune rabbit syphilitic serum, no alteration in the distribution of OM IMPs was detected. In further experiments, approximately 1:1 mixtures of T. pallidum and Escherichia coli or separate suspensions of the nonpathogenic Treponema phagedenis biotype Reiter were fixed at 34°C or after cooling to 0°C (to induce lateral phase separations that would aggregate IMPs). Only particles in the T. paUlidum OM failed to aggregate in cells fixed at the lower temperature. The combined data suggest that the mobility of T. palidum rare OM proteins is limited, perhaps as a result of interactions between their periplasmic domains and components of the peptidoglycan-cytoplasmic membrane complex.Treponema pallidum, the spirochetal bacterium which causes venereal syphilis, has a remarkable ability to evade the vigorous cellular and humoral immune responses that it evokes in mammalian hosts (3,17,18,25). One strategy for elucidating the pathogen's extraordinary immunoevasiveness has been to investigate the molecular architecture of its outer membrane and to determine the precise cellular locations of its major immunogens. Prior freeze-fracture and deep-etch electron microscopy (EM) revealed that the T. pallidum outer membrane contains a paucity of surface-exposed protein immunogens (visualized by freeze fracture as rare intramembranous particles [IMPs] which may represent a single protein or protein oligomer) (40, 50). In contrast, evidence now exists that the bacterium's major membrane immunogens, molecules previously described as surface exposed (20,21,35,46), are associated with the cytoplasmic membrane via lipids at their N termini (1,10,12,38,39,43,45). Inasmuch as the polypeptide moieties of the lipoproteins presumably are extrinsic to the lipid bilayer (12), these molecules may not be visualized in freeze-fractured T. pallidum membranes.The discovery of the rare outer membrane protein(s) has raised fundamental questions concerning the role(s) of these molecules in syphilis pathogenesis and ...