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.

Venereal syphilis is a chronic, multisystem infectious disorder caused by the spirochetal bacterium Treponema pallidum subsp. pallidum (T. pallidum). Like all spirochetes, T. pallidum is an elongated, highly motile organism that consists of a fragile outer membrane surrounding a periplasmic space, a peptidoglycan-cytoplasmic membrane (PG-CM) complex, and a protoplasmic cylinder (36). There is now a substantial body of evidence that the outer membranes of T. pallidum and enteric gram-negative bacteria differ considerably with respect to composition and molecular architecture (52, 57). One of the key differences concerns the relative abundance of proteins with membrane-spanning domains. Whereas outer membranes of gram-negative bacteria contain high densities of such polypeptides (48), freeze-fracture electron microscopy and cell fractionation studies have shown that they are sparse in T. pallidum, hence the designation rare outer membrane proteins (52,57,59,77).Based on the assumption that T. pallidum rare outer membrane proteins are important in disease pathogenesis, molecular characterization of these polypeptides has become a major objective of contemporary syphilis research. One strategy recently developed to accomplish this goal is to isolate T. pallidum outer membranes for partial amino acid sequencing of candidate rare outer membrane proteins (9, 60). Using this approach, Blanco et al. (6) identified a 31-kDa protein (Tromp1) in isolated outer membranes which formed ion-conducting channels in planar lipid bilayers. More recently, the same investigators reported that recombinant Tromp1 expressed in Escherichia coli is surface exposed and that the recombinant protein possesses porin-like properties (7). However, to date, no evidence for either outer membrane location or surface exposure of native Tromp1 within motile or intact treponemes has been presented (6, 7). Moreover, Tromp1 has extensive sequence homology with the periplasmic substratebinding proteins of known ATP-binding cassette (ABC) transporters and the tromp1 gene is transcriptionally linked to open reading frames which encode homologs for ABC transporter components which, in gram-negative bacteria, are cytoplasmic membrane associated (30,33). The apparent discrepancy between the studies of Blanco and coworkers (6, 7) and these newer genetic data prompted efforts to clarify the physicochemical properties and cellular location of Tromp1 in T. pallidum. Here we present evidence that Tromp1, rather than being an outer membrane-spanning protein, actually is anchored by an uncleaved signal sequence to the T. pallidum cytoplasmic membrane.

Automated Edman degradation was used to obtain N-terminal and internal amino acid sequences from a 26-kDa protein in isolated Treponema pallidum outer membranes (OMs). The resulting sequences enabled us to PCR amplify from T. pallidum DNA a 275-bp fragment of the corresponding gene. The complete nucleotide sequence of the gene was determined from fragments amplified by long-distance PCR. Primer extension verified the assigned translational start of the open reading frame (ORF) and putative upstream promoter elements. The ORF encoded a highly basic (pI 9.6) 26-kDa protein which contained an N-terminal 25-aminoacid leader peptide terminated by a signal peptidase I cleavage site. The mature protein contained seven tandemly spaced copies (as well as an eighth incomplete copy) of a leucine-rich repeat (LRR), a motif previously identified in a number of prokaryotic and eukaryotic proteins. Accordingly, the polypeptide was designated T. pallidum leucine-rich repeat protein (TpLRR). Although Triton X-114 phase partitioning showed that TpLRR was hydrophilic, cell localization studies showed that most of the antigen was associated with the peptidoglycan-cytoplasmic membrane complex rather than being freely soluble in the periplasmic space. Immunoblot studies showed that syphilis patients develop a weak antibody response to the antigen. Lastly, the lrr T. pallidum gene was mapped to a 60-kb SfiI-SpeI fragment of the T. pallidum chromosome which also contains the rrnA and flaA genes. The function(s) of TpLRR is currently unknown; however, protein-protein and/or protein-lipid interactions mediated by its LRR motifs may facilitate interactions between components of the T. pallidum cell envelope.Syphilis is a sexually transmitted, multistage infection caused by the noncultivatable spirochetal pathogen Treponema pallidum (26). Like all spirochetes, T. pallidum is a highly motile bacterium in which an outer membrane (OM) surrounds the periplasmic space, peptidoglycan (PG)-cytoplasmic membrane (CM) complex, and protoplasmic cylinder (PC); within the periplasmic space are endoflagella, the organelles of motility (19). Compared with conventional gram-negative bacteria, we know comparatively little about the structure and composition of the T. pallidum cell envelope and how its diverse components promote survival of the syphilis spirochete in the demanding milieu of the mammalian host. Studies from a number of laboratories have provided compelling evidence that OMs of T. pallidum and gram-negative bacteria differ markedly with respect to physical properties, composition, and molecular architecture (6,29,32,34,42). While the paucity of surfaceexposed proteins appears to explain, at least in part, how this extracellular pathogen so successfully evades host immune responses during persistent infection, it also raises intriguing and fundamental questions regarding the mechanisms by which the bacterium acquires nutrients from its environment (32). In contrast to the protein-deficient OM, the vast majority of T. pallidum cell envelope con...