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 SalivaryAntibodies Directed against Outer Membrane Proteins ofMoraxella catarrhalis in HealthyAdults

2003 ◽  
Vol 71 (12) ◽  
pp. 6793-6798 ◽  
Author(s):  
Patricia Stutzmann Meier ◽  
Nadja Heiniger ◽  
Rolf Troller ◽  
Christoph Aebi
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Immunoglobulin A ◽  
Immunoblot Analysis ◽  
Human Saliva ◽  
Mucosal Immune Response ◽  
Binding Activity ◽  
Human Respiratory Tract ◽  
Iga Response

ABSTRACT Moraxella catarrhalis is a major mucosal pathogen of the human respiratory tract, but the mucosal immune response directed against surface components of this organism has not been characterized in detail. The aim of this study was to investigate the salivary immunoglobulin A (IgA) response toward outer membrane proteins (OMP) of M. catarrhalis in healthy adults, the group of individuals least likely to be colonized and thus most likely to display mucosal immunity. Unstimulated saliva samples collected from 14 healthy adult volunteers were subjected to IgA immunoblot analysis with OMP preparations of M. catarrhalis strain O35E. Immunoblot analysis revealed a consistent pattern of IgA reactivity, with the appearance of five major bands located at >250, 200, 120, 80, and 60 kDa. Eleven (79%) of 14 saliva samples elicited reactivity to all five bands. Immunoblot analysis with a set of isogenic knockout mutants lacking the expression of individual OMP was used to determine the identities of OMP giving rise to IgA bands. Human saliva was shown consistently to exhibit IgA-binding activity for oligomeric UspA2 (>250 kDa), hemagglutinin (200 kDa), monomeric UspA1 (120 kDa), transferrin-binding protein B (TbpB), monomeric UspA2, CopB, and presumably OMP CD. TbpB, oligomeric UspA2, and CopB formed a cluster of bands at about 80 kDa. These data indicate that the human salivary IgA response is directed consistently against a small number of major OMP, some of which are presently considered vaccine candidates. The functional properties of these mucosal antibodies remain to be elucidated.

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 Assembly of TolC, a Structurally Unique and Multifunctional Outer Membrane Protein of Escherichia coli K-12

2003 ◽  
Vol 185 (22) ◽  
pp. 6540-6547 ◽  
Author(s):  
John Werner ◽  
Anne Marie Augustus ◽  
Rajeev Misra
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Outer Membrane Proteins ◽  
The Other ◽  
Proteinase K ◽  
Folded Structure ◽  
K 12

ABSTRACT TolC is a multifunctional outer membrane protein of Escherichia coli that folds into a novel α-β-barrel conformation absent in the other model outer membrane proteins used in assembly studies. The data presented in this work show that the unique folded structure of TolC reflects a unique assembly pathway. During its assembly, the newly translocated nascent TolC monomers are released in the periplasm. Maturation of these nascent monomers, and possibly their oligomerization, in the periplasm precedes their insertion in the outer membrane. The completion of the assembly process is signaled by the development of a characteristic proteinase K-resistant fragment generated by cleavage at a single, periplasmically exposed, protease-sensitive site of the membrane-anchored trimer. None of the assembly steps of TolC is affected by known folding factors, such as SurA, Skp, and lipopolysaccharide, which have profound effects on the assembly of other model trimeric outer membrane proteins. Two assembly-defective TolC mutants were isolated and characterized. One of the mutants (TolCI106N) was defective in the folding of nascent monomers, while the other (TolCS350F) was impaired in steps involving trimerization and membrane insertion of folded monomers.

scholarly journals Expression and Comparative Analysis of Genes Encoding Outer Membrane Proteins LipL21, LipL32 and OmpL1 in Epidemic Leptospires

2005 ◽  
Vol 37 (10) ◽  
pp. 649-656 ◽  
Author(s):  
Xiang-Yan Zhang ◽  
Yang Yu ◽  
Ping He ◽  
Yi-Xuan Zhang ◽  
Bao-Yu Hu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Immunoblot Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Leptospira Interrogans ◽  
Genes Encoding ◽  
Leptospira Borgpetersenii ◽  
High Degree

AbstractLeptospiral outer membrane proteins (OMPs) are highly conserved in different species, and play an essential role in the development of new immunoprotection and serodiagnosis strategies. The genes encoding LipL21, LipL32 and OmpL1 were cloned from the complete genome sequence of Leptospira interrogans serovar lai strain Lai and expressed in vitro. Sequence comparison analysis revealed that the three genes were highly conserved among distinct epidemic leptospires, including three major epidemic species Leptospira interrogans, Leptospira borgpetersenii and Leptospira weilii, in China. Immunoblot analysis was further performed to scrutinize 15 epidemic Leptospira reference strains using the antisera of the recombinant OMPs. Both immunoblot assay and reverse transcription-polymerase chain reaction demonstrated that these three OMPs were conservatively expressed in pathogenic L. interrogans strains and other pathogenic leptospires. Additionally, the use of these recombinant OMPs as antigens in enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of leptospirosis was evaluated. The recombinant LipL32 and OmpL1 proteins showed a high degree of ELISA reactivity with sera from patients infected with L. interrogans strain Lai and other pathogenic leptospires. These results may contribute to the identification of candidates for broad-range vaccines and immunodiagnostic antigens in further research.

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