TP0326, a Treponema pallidum β‐barrel assembly machinery A (BamA) orthologue and rare outer membrane protein

Abstract

SUMMARY Definitive identification of Treponema pallidum (Tp) rare outer membrane proteins (OMPs) has long eluded researchers. TP0326, the sole protein in Tp with sequence homology to a Gram-negative OMP, belongs to the BamA family of proteins essential for OM biogenesis. Structural modeling predicted that five polypeptide transport-associated (POTRA) domains comprise the N-terminus of TP0326, while the C-terminus forms an 18-stranded amphipathic β-barrel. Circular dichroism, heat-modifiability by SDS-PAGE, Triton X-114 phase partitioning and liposome incorporation supported these topological predictions and confirmed that the β-barrel is responsible for the native protein's amphiphilicity. Expression analyses revealed that native TP0326 is expressed at low abundance, while a protease-surface accessibility assay confirmed surface exposure. Size-exclusion chromatography and blue native polyacrylamide gel electrophoresis revealed a modular Bam complex in Tp considerably larger than that of E. coli. Non-orthologous ancillary factors and self-association of TP0326 via its β-barrel may both contribute to the Bam complex. Tp-infected rabbits mount a vigorous antibody response to both POTRA and β-barrel portions of TP0326, whereas humans with secondary syphilis respond predominantly to POTRA. The syphilis spirochete appears to have devised a stratagem for harnessing the Bam pathway while satisfying its need to limit surface antigenicity.

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

“…This method is advantageous because it not only preserves the integrity of the spirochete's fragile OM during immunolabeling but also allows for its controlled removal by detergent solubilization in order to expose periplasmic antigens. Because T. pallidum expresses BamA at a very low copy number (ϳ100 molecules per cell) (34), at the outset, we confirmed that our rat ␤-barrel and POTRA antisera could recognize their target antigens at nanogram levels with recombinant proteins as well as in T. pallidum lysates (see Fig. S2 in the supplemental material).…”

“…Among our highest ranked candidates was TP_0326, discovered in 2000 as TP_92 by Cameron et al (19) using a differential screening strategy to identify opsonic targets in T. pallidum. Recently, we confirmed the status of TP_0326 as a bona fide rare OMP (34) and, using recombinant constructs, established that it possesses the bipartite topology characteristic of members of the Omp85 superfamily (7). In the present study, we demonstrated by indirect immunofluorescence analysis (IFA) of T. pallidum that only the ␤-barrel of native TP_0326 contains surface-exposed epitopes.…”

“…Previously, we used biophysical methodologies to establish that recombinant TP_0326 possesses the characteristic bipartite topology (i.e., the 5-domain POTRA arm and ␤-barrel) of a BamA ortholog (4,34). We also demonstrated by proteinase K accessibility that native TP_0326 is surface exposed in T. pallidum (34).…”

“…Previously, we used biophysical methodologies to establish that recombinant TP_0326 possesses the characteristic bipartite topology (i.e., the 5-domain POTRA arm and ␤-barrel) of a BamA ortholog (4,34). We also demonstrated by proteinase K accessibility that native TP_0326 is surface exposed in T. pallidum (34). We began the present study by using our gel microdroplet immunofluorescence assay (29,45) to determine the cellular locations of the ␤-barrel and the POTRA arm in live treponemes.…”

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

“…The CD spectra of all MOMP preparations had minima near 215 nm, which is indicative of β‐sheet secondary protein structure49, 50 and consistent with MOMP being a β‐barrel protein. CD spectra of β‐barrel outer membrane proteins such as MBOMP from Entamoeba , OMP P66 from Borrelia burgdorferi and BamA from Treponema pallidum have been reported in the literature 51, 52, 53. The shape of the CD spectra of these β‐barrel proteins is diverse with minima varying between 210 nm and 220 nm.…”