scholarly journals O01.2 Pyrococcus furiosus thioredoxin as a platform to express extracellular loops of treponema pallidum outer membrane proteins

2021 ◽  
Author(s):  
J Montezuma Rusca ◽  
K Delgado ◽  
A Luthra ◽  
C LaVake ◽  
M LeDoyt ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Pyrococcus Furiosus ◽  
Outer Membrane Proteins ◽  
Treponema Pallidum ◽  
Extracellular Loops

scholarly journals Tight Turns of Outer Membrane Proteins: An Analysis of Sequence, Structure, and Hydrogen Bonding

2018 ◽  
Author(s):  
Meghan Whitney Franklin ◽  
Joanna S.G. Slusky
Keyword(s):  
Hydrogen Bonding ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Soluble Protein ◽  
Structure Prediction ◽  
Outer Membrane Proteins ◽  
Sequence Structure ◽  
Structural Class ◽  
Future Design ◽  
Extracellular Loops

I.AbstractAs a structural class, tight turns can control molecular recognition, enzymatic activity, and nucleation of folding. They have been extensively characterized in soluble proteins but have not been characterized in outer membrane proteins (OMPs), where they also support critical functions. We clustered the 4-6 residue tight turns of 110 OMPs to characterize the phi/psi angles, sequence, and hydrogen bonding of these structures. We find significant differences between reports of soluble protein tight turns and OMP tight turns. Since OMP strands are less twisted than soluble strands they favor different turn structures types. Moreover, the membrane localization of OMPs yields different sequence hallmarks for their tight turns relative to soluble protein turns. We also characterize the differences in phi/psi angles, sequence, and hydrogen bonding between OMP extracellular loops and OMP periplasmic turns. As previously noted, the extracellular loops tend to be much longer than the periplasmic turns. We find that this difference in length is due to the broader distribution of lengths of the extracellular loops not a large difference in the median length. Extracellular loops also tend to have more charged residues as predicted by the charge-out rule. Finally, in all OMP tight turns, hydrogen bonding between the sidechain and backbone two to four residues away plays an important role. These bonds preferentially use an Asp, Asn, Ser or Thr residue in a beta or pro phi/psi conformation. We anticipate that this study will be applicable to future design and structure prediction of OMPs.

scholarly journals Surface Immunolabeling and Consensus Computational Framework To Identify Candidate Rare Outer Membrane Proteins of Treponema pallidum

2010 ◽  
Vol 78 (12) ◽  
pp. 5178-5194 ◽  
Author(s):  
David L. Cox ◽  
Amit Luthra ◽  
Star Dunham-Ems ◽  
Daniel C. Desrosiers ◽  
Juan C. Salazar ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Treponema Pallidum ◽  
Immunoblot Analysis ◽  
Proteinase K ◽  
Membrane Proteome ◽  
Computational Framework ◽  
Shifting Balance ◽  
Dual Label

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.

scholarly journals Assessment of cell-surface exposure and vaccinogenic potentials of Treponema pallidum candidate outer membrane proteins

2007 ◽  
Vol 9 (11) ◽  
pp. 1267-1275 ◽  
Author(s):  
Farol L. Tomson ◽  
Patrick G. Conley ◽  
Michael V. Norgard ◽  
Kayla E. Hagman
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Treponema Pallidum

scholarly journals Correlation of Immunity in Experimental Syphilis with Serum-Mediated Aggregation of Treponema pallidum Rare Outer Membrane Proteins

1999 ◽  
Vol 67 (7) ◽  
pp. 3631-3636 ◽  
Author(s):  
Michael A. Lewinski ◽  
James N. Miller ◽  
Michael A. Lovett ◽  
David R. Blanco
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Treponema Pallidum ◽  
Freeze Fracture ◽  
Normal Serum ◽  
Acquired Immunity ◽  
Freeze Fracture Electron Microscopy ◽  
Membrane Spanning ◽  
Number Of Particles

ABSTRACT We have previously shown by freeze-fracture electron microscopy that serum from infection-immune syphilitic rabbits aggregates the low-density membrane-spanning Treponema pallidum rare outer membrane proteins (TROMPs). The purpose of this study was to determine if a relationship could be demonstrated between acquired immunity in experimental rabbit syphilis, serum complement-dependent treponemicidal antibody, and antibody directed against TROMPs as measured by the aggregation of TROMP particles. Three groups of T. pallidum-infected rabbits were treated curatively with penicillin at 9 days, 30 days, and 6 months postinfection to generate various degrees of immunity to challenge reinfection. Sera from rabbits completely susceptible to localized and disseminated reinfection possessed a low titer of treponemicidal antibody (≤1:1 in killing ≥50% of a treponemal suspension) and showed a correspondingly low level of TROMP aggregation (16.5% of the total number of outer membrane particles counted) similar to normal serum controls (13.4%); the number of particles within these aggregates never exceeded three. Sera from partially immune rabbits, which were susceptible to local reinfection but had no evidence of dissemination, showed an increase in the titer of treponemicidal antibody (1:16) compared to the completely susceptible group (≤1:1). Although no significant increase was observed in the total number of TROMP particles aggregated (18.9%) compared to the number in controls (13.4%), approximately 15% of these aggregates did exhibit a significant increase in the number of particles per aggregate (4 to 5 particles) compared to controls (≤3 particles), indicating a measurable increase in anti-TROMP antibody. Finally, sera from rabbits completely immune to both local and disseminated reinfection possessed both high titers of treponemicidal antibody (1:128) and significant aggregation of TROMP (88.6%); approximately 50% of these aggregates contained four to six particles. The results indicate that complete immunity in experimental rabbit syphilis correlates with antibody that kills T. pallidumand aggregates TROMPs, suggesting that TROMPs are molecules which contribute to the development of acquired immunity.

scholarly journals Sequence variation of rare outer membrane protein β-barrel domains in clinical strains provides insights into the evolution ofTreponema pallidumsubsp.pallidum, the syphilis spirochete

2018 ◽  
Author(s):  
Sanjiv Kumar ◽  
Melissa J. Caimano ◽  
Arvind Anand ◽  
Abhishek Dey ◽  
Kelly L. Hawley ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Evolutionary Biology ◽  
Sequence Variation ◽  
Vaccine Development ◽  
Outer Membrane Proteins ◽  
Treponema Pallidum ◽  
Clinical Strains ◽  
Extracellular Loops ◽  
Reference Strains ◽  
Reference Genomes

ABSTRACTIn recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) ofTreponema pallidumsubspeciespallidum(TPA), the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions oftprC,tprD, andbamA, in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we usedtp0548genotyping andtp0558sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants inTPAreference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. Based upon structural models, non-conservative amino acid substitutions in predicted transmembrane β-strands of TprC and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures driveTPAOMP diversity and that genetic exchange contributes to the evolutionary biology ofTPA. They also set the stage for topology-based analysis of antibody responses against OMPs and help frame strategies for syphilis vaccine development.IMPORTANCEDespite recent progress characterizing outer membrane proteins (OMPs) ofTreponema pallidum(TPA), little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci,tprC,tprD, andbamA,inTPAfrom a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurred predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function also were noted. Our findings suggest that selection pressures exerted by human populations driveTPAOMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance ofTPAand frame strategies for vaccine development based upon conserved OMP extracellular loops.

scholarly journals Sequence Variation of Rare Outer Membrane Protein β-Barrel Domains in Clinical Strains Provides Insights into the Evolution ofTreponema pallidumsubsp.pallidum, the Syphilis Spirochete

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Sanjiv Kumar ◽  
Melissa J. Caimano ◽  
Arvind Anand ◽  
Abhishek Dey ◽  
Kelly L. Hawley ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Evolutionary Biology ◽  
Vaccine Development ◽  
Outer Membrane Proteins ◽  
Treponema Pallidum ◽  
Content Type ◽  
Clinical Strains ◽  
Extracellular Loops ◽  
Reference Strains ◽  
Reference Genomes

ABSTRACTIn recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) ofTreponema pallidumsubspeciespallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions oftprC,tprD, andbamAin 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we usedT. pallidum0548 (tp0548) genotyping andtp0558sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants inT. pallidumsubsp.pallidumreference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands ofT. pallidumrepeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures driveT. pallidumsubsp.pallidumOMP diversity and that genetic exchange contributes to the evolutionary biology ofT. pallidumsubsp.pallidum. They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development.IMPORTANCEDespite recent progress characterizing outer membrane proteins (OMPs) ofTreponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci,tprC,tprD, andbamA, inT. pallidumsubsp.pallidumisolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations driveT. pallidumsubsp.pallidumOMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance ofT. pallidumsubsp.pallidumand frame strategies for vaccine development based upon conserved OMP extracellular loops.

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