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.
The legend to Fig. 6E should read as follows. "Immunoblot of unboiled, recombinant TprC N and TprC C separated by SDS-PAGE and then probed with rabbit anti-TprC Fl polyclonal antiserum (asterisk indicates TprC C trimer)." Page 2331: The legend to Fig. 7C should read as follows. "Reactivities of the anti-TprC Fl antiserum and IRS used for the opsonophagocytosis assays in Fig. 5 against TprC N and folded, unboiled TprC C. The immunoblot shown in Fig. 6E was stripped and reprobed with IRS. Bottom and top panels show the reactivity of TprC N and TprC C monomers with anti-TprC Fl antiserum (reproduced from Fig. 6E) and IRS, respectively."
IntroductionOuter membrane proteins (OMPs) play critical roles in disease pathogenesis and are vaccinogens. Topologic characterisation of surface-exposed β-barrels of Treponema pallidum (Tp) Nichols rare OMPs enabled a novel strategy to assess sequence diversity and evolution of Tp in geographically diverse locations.MethodsThrough early 2017, sequences encoding TprC (TP0117), TprD (TP0131), and BamA (TP0326) β-barrels were amplified from secondary syphilis patients from Cali (n=16) and swabs from patients in San Francisco (SF, n=6) and Czech Republic (CZ, n=9). Strains were assigned to the Nichols or SS14 clade based on tp0548 and/or tp0558 sequences.Results23 assignable CZ and Cali strains belonged to either the SS14 or Nichols clade (SS14 predominant), while all 6 SF strains belong to the SS14 clade. Sequence diversity at the three OMP loci was greatest in Cali, with evidence of recombination within tprC and bamA alleles, as well as between strains and clades at all 3 genetic loci. SF strains contained nearly identical sequences at all 3 genetic loci. The SS14 and Mexico A reference strains, both belonging to the SS14 clade, have identical tprDs (tprD2) but different tprC and bamA alleles. Mexico A tprCs were common at all three geographic locations, including Nichols clade strains from Cali. Mexico A bamAs were prevalent in Cali and SF, while CZ SS14 clades contained only SS14 bamAs. OMP sequences were obtained from all three loci in 7 of 8 Nichols clade strains. Of these 7, only 1 matched the Nichols reference strain, while the other 6 contained Mexico A alleles in at least 1 OMP locus. Of the 21 SS14 clade strains, 10 contained Mexico A alleles at all 3 loci; 2 contained Mexico A trpCs and Nichols bamAs; and 9 contained Mexico A tprCs and SS14 bamAs.ConclusionOMP loci are evolving independently within Tp. Recombination of OMP sequences appears to be occurring between Tp strains and clades within patients. Mexico A OMP alleles are circulating widely among Tp strains. These findings have major ramifications for syphilis vaccine development.
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