Surface immunolabeling and consensus computational framework to identify candidate rare outer membrane proteins of Treponema pallidum

Abstract

Treponema pallidum reacts poorly with the antibodies present in rabbit and human syphilitic sera, a property attributed to the paucity of proteins in its outer membrane. To better understand the basis for the syphilis spirochete's "stealth pathogenicity," we used a dual-label, 3-step amplified assay in which treponemes encapsulated in gel microdroplets were probed with syphilitic sera in parallel with anti-FlaA antibodies. A small (approximately 5 to 10%) but reproducible fraction of intact treponemes bound IgG and/or IgM antibodies. Three lines of evidence supported the notion that the surface antigens were likely β-barrel-forming outer membrane proteins (OMPs): (i) surface labeling with anti-lipoidal (VDRL) antibodies was not observed, (ii) immunoblot analysis confirmed prior results showing that T. pallidum glycolipids are not immunoreactive, and (iii) labeling of intact organisms was not appreciably affected by proteinase K (PK) treatment. With this method, we also demonstrate that TprK (TP0897), an extensively studied candidate OMP, and TP0136, a lipoprotein recently reported to be surface exposed, are both periplasmic. Consistent with the immunolabeling studies, TprK was also found to lack amphiphilicity, a characteristic property of β-barrel-forming proteins. Using a consensus computational framework that combined subcellular localization and β-barrel structural prediction tools, we generated ranked groups of candidate rare OMPs, the predicted T. pallidum outer membrane proteome (OMPeome), which we postulate includes the surface-exposed molecules detected by our enhanced gel microdroplet assay. In addition to underscoring the syphilis spirochete's remarkably poor surface antigenicity, our findings help to explain the complex and shifting balance between pathogen and host defenses that characterizes syphilitic infection.

FIG. 1.

Current model for the molecular architecture of the T. pallidum cell envelope. Embedded within the OM are TP0326 (TP92), a BamA ortholog (; D. C. Desrosiers, M. A. D. Cummings, C. E. Cameron, and J. D. Radolf, unpublished), and other, as yet unidentified membrane-spanning proteins predicted to form β-barrels, represented schematically by a prototypical OMP, the E. coli porin OmpC (7). Anchored to the inner leaflet of the OM is the pore-forming lipoprotein TP0453 (56), the crystal structure of which was recently solved (A. Luthra, G. Zhu, D. C. Desrosiers, V. Mulay, A. P. Heuck, F. B. Romano, I. Barcena-Uribarri, R. Benz, M. G. Malkowski, and J. D. Radolf, unpublished data). Within the periplasmic space, but below the peptidoglycan (PG) (64), are shown some structurally characterized ABC transporter substrate-binding proteins, TP0655 (PotD) (82), TP0319 (PnrA) (38), MetQ (38a), TP0163 (TroA) (75, 76), and TP0034 (ZnuA) (40), associated with their corresponding inner membrane (IM) permeases. The locations of the CM and the CM proteins, the positioning of the flagellar filaments in relation to the PG, and the scale of the diagram are based upon data obtained by cryoelectron tomography of vitreously frozen treponemes (64, 77a).

FIG. 2.

Computational framework for prediction of the T. pallidum OMPeome. The numerals indicate the numbers of proteins identified by each OMP prediction tool and subsequent filters. Following the filter steps, the remaining candidates were grouped based on the number of OM localization/β-barrel topology programs that identified them and then analyzed for the presence of a cleaved signal sequence.

FIG. 3.

Surface labeling of T. pallidum with IRS. (A) Representative micrographs from seven independent experiments. DF, dark field. Scale bar = 2.5 μm. (B) Dot plot of key results. In this and subsequent plots, each dot presents the percentage of organisms in a particular labeling category from one experiment; the lines represent mean percentages. Complete results for all immunolabeling experiments are presented in Table 2.

FIG. 4.

Surface labeling of T. pallidum with pooled HSS. (A) Representative micrographs from seven independent experiments. Scale bar = 2.5 μm. (B) Dot plot of key results.

FIG. 5.

Variability of surface labeling of T. pallidum by IRS (A) and HSS (B). Scale bar = 2.5 μm.

FIG. 6.

Cytoplasmic membrane association of T. pallidum lipoidal antigens recognized by anti-VDRL antiserum. (A) Representative micrographs from three independent experiments. Scale bar = 2.5 μm. (B) Dot plot of key results.

FIG. 7.

Periplasmic location of TprK. (A) Representative micrographs from three independent experiments. Scale bar = 2.5 μm. (B) Dot plot of key results.

FIG. 8.

TP0136 is cytoplasmic membrane associated. (A) Representative micrographs from three independent experiments. Scale bar = 2.5 μm. (B) Dot plot of key results.

FIG. 9.

TprK lacks the amphiphilic character of β-barrel-forming proteins. (A) Freshly harvested T. pallidum, solubilized in 2% Triton X-114, was phase partitioned and subjected to immunoblot analysis with monospecific antisera directed against TrpK, TP0326, thioredoxin (Trx), and TroA. Lanes: whole cells (WC), aqueous phase (Aq), detergent-enriched phase (Det), and insoluble material (Ins). (B) Purified recombinant E. coli BamA and OmpG were phase partitioned and stained with Gel-code Blue following SDS-PAGE.

FIG. 10.

(A) β-Barrel structure of TprC (TP0117) predicted by TMBpro (108). Depicted is a cartoon representation of the model in which β-strands, loops, and α-helices are shown in yellow, green, and red, respectively. (B) Amino acid sequence alignment of TprC and TprK generated by MacVector using a PAM matrix. Identical residues are shown in boxes; above the aligned sequences are the β-sheets predicted for TprC by TMBPro.