Molecular characterization and cellular localization of TpLRR, a processed leucine-rich repeat protein of Treponema pallidum, the syphilis spirochete

Abstract

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...

Characterization of a Novel Leucine-Rich Repeat Protein Antigen from Group B Streptococci That Elicits Protective Immunity

“…1). A similar pattern of low-level homology was also observed between LrrG and another LRR protein, TpLRR (28% identity and 42% similarity overall), located in the outer membrane of Treponema pallidum (78). Through the use of various bioinformatic approaches as well as visual analysis of the entire amino acid sequence of LrrG, 15 full-length LRRs and 2 partial LRRs (Fig.…”

The Streptococcal Blr and Slr Proteins Define a Family of Surface Proteins with Leucine-Rich Repeats: Camouflaging by Other Surface Structures

“…This protein is not related to the Blr and Slr proteins studied here, and the repeats in LrrG lack the classical LRR consensus sequence LXXLXLXXNXL. However, LrrG is related to a group of proteins with repeats designated TpLRR, which have been identified for several bacterial pathogens (21,51). Of note, the LrrG protein is one of the proteins which Tettelin et al (58) concluded is not exposed on the surface of S. agalactiae, but LrrG was nevertheless reported to be a target for antibodies that protect against S. agalactiae infection (50).…”

Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane

“…First, because of the lack of a priori knowledge of T. pallidum outer membrane constituents and the paucity of monospecific antibodies directed against treponemal outer membrane proteins, it has not been possible to directly correlate intramembranous particles visualized by freeze-fracture electron microscopy with individual outer membrane-associated proteins visualized by SDS-PAGE. Second, because isolated outer membranes contain subsurface contaminants (9,60,67), the mere presence of a previously uncharacterized polypeptide in isolated outer membranes cannot be considered tantamount to its being a rare outer membrane protein. These caveats have necessitated the development of an algorithm to guard against prematurely designating polypeptides in isolated outer membranes as rare outer membrane proteins (57,67).…”

“…Second, because isolated outer membranes contain subsurface contaminants (9,60,67), the mere presence of a previously uncharacterized polypeptide in isolated outer membranes cannot be considered tantamount to its being a rare outer membrane protein. These caveats have necessitated the development of an algorithm to guard against prematurely designating polypeptides in isolated outer membranes as rare outer membrane proteins (57,67). The first step involves designation of highly enriched outer membrane-associated polypeptides as candidate outer membrane proteins.…”