TprC/D (Tp0117/131), a Trimeric, Pore-Forming Rare Outer Membrane Protein of Treponema pallidum, Has a Bipartite Domain Structure

Abstract

e Identification of Treponema pallidum rare outer membrane proteins (OMPs) has been a longstanding objective of syphilis researchers. We recently developed a consensus computational framework that employs a battery of cellular localization and topological prediction tools to generate ranked clusters of candidate rare OMPs (D. L. Cox et al., Infect. Immun. 78:5178 -5194, 2010). TP0117/TP0131 (TprC/D), a member of the T. pallidum repeat (Tpr) family, was a highly ranked candidate. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning, and liposome incorporation confirmed that full-length, recombinant TprC (TprC Fl ) forms a ␤-barrel capable of integrating into lipid bilayers. Moreover, TprC Fl increased efflux of terbium-dipicolinic acid complex from large unilamellar vesicles and migrated as a trimer by blue-native PAGE. We found that in T. pallidum, TprC is heat modifiable, trimeric, expressed in low abundance, and, based on proteinase K accessibility and opsonophagocytosis assays, surface exposed. From these collective data, we conclude that TprC is a bona fide rare OMP as well as a functional ortholog of Escherichia coli OmpF. We also discovered that TprC has a bipartite architecture consisting of a soluble N-terminal portion (TprC N ), presumably periplasmic and bound directly or indirectly to peptidoglycan, and a C-terminal ␤-barrel (TprC C ). Syphilitic rabbits generate antibodies exclusively against TprC C , while secondary syphilis patients fail to mount a detectable antibody response against either domain. The syphilis spirochete appears to have resolved a fundamental dilemma arising from its extracellular lifestyle, namely, how to enhance OM permeability without increasing its vulnerability to the antibody-mediated defenses of its natural human host.

A Homology Model Reveals Novel Structural Features and an Immunodominant Surface Loop/Opsonic Target in the Treponema pallidum BamA Ortholog TP_0326

“…Previously, we noted the heterogeneity of surface antibody binding within spirochete populations and proposed that it helps explain the duality of secondary syphilis (i.e., the parallel occurrence of bacterial clearance and immune evasion) (44). We contend that this phenomenon is reproduced ex vivo by the lack of internalization of sizable percentages of treponemes in opsonophagocytosis assays, as seen here and elsewhere (42,44,56), following incubation with IRS, patient sera, and antisera to surface-exposed regions of known OMPs. The punctate distribution of TP_0326 along the length of the spirochete, unaffected by removal of the OM, suggests that the mobility of the protein within the OM bilayer is restricted.…”

“…The conceptual breakthrough in our quest for T. pallidum rare OMPs was the use of a structural bioinformatics approach to mine the spirochete's genome for proteins predicted to adopt a ␤-barrel conformation; TP_0326/BamA, the only OMP in T. pallidum related to OMPs in Gram-negative bacteria, emerged as a top candidate in the putative T. pallidum OM proteome (29). The finding of BamA (29) and subsequently a BAM complex (34) implied the existence of other ␤-barrel OMPs which serve as substrates for the OM biogenesis machinery, a supposition confirmed by biophysical characterization and surface localization of the T. pallidum repeat C (TprC) subfamily of proteins (42,62). Because TP_0326 resides constitutively at the hostpathogen interface, one might anticipate that its involvement in the disease process extends beyond its role in OM biogenesis.…”

“…Rat polyclonal antiserum directed against the POTRA arm (29, 34) and FlaA (TP_0249) (36) were described previously. Antisera directed against the entire ␤-barrel, L3␤6, L4, L6 Ab , L7, and E. coli Skp were generated in 6-week-old female Sprague-Dawley rats as described previously (42). Polyclonal antibodies recognizing the POTRA arm and L4 were generated in rabbits by Rockland, Inc., according to their established protocol.…”

“…Opsonophagocytosis assays were performed as described previously (42,47). Live treponemes were preincubated with 10% heat-inactivated sera for 2 h at 37°C in CMRL medium; at the end of preincubation, the motility of treponemes was confirmed by dark-field microscopy.…”

The TP0796 Lipoprotein of Treponema pallidum Is a Bimetal-dependent FAD Pyrophosphatase with a Potential Role in Flavin Homeostasis

“…9). In this model, FAD diffuses into the periplasm via a putative outer membrane porin (77). Its relative abundance in human plasma makes FAD the likeliest candidate as an FMN source (78).…”