scholarly journals Temporal Analysis of the Antigenic Composition of Borrelia burgdorferi during Infection in Rabbit Skin

2004 ◽  
Vol 72 (9) ◽  
pp. 5063-5072 ◽  
Author(s):  
Timothy R. Crother ◽  
Cheryl I. Champion ◽  
Julian P. Whitelegge ◽  
Rodrigo Aguilera ◽  
Xiao-Yang Wu ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Antigenic Variation ◽  
Outer Membrane Proteins ◽  
Temporal Analysis ◽  
Immunoblot Analysis ◽  
Rabbit Skin ◽  
Antigenic Composition ◽  
Marked Diminution ◽  
Time Point

ABSTRACT The numbers of host-adapted Borrelia burgdorferi (HAB) organisms in rabbit skin were assessed by real-time PCR over the first 3 weeks of infection. Maximal numbers were found at day 11, while spirochete numbers decreased by more than 30-fold by day 21. The antigenic composition of HAB in skin biopsy samples was determined by use of a procedure termed hydrophobic antigen tissue Triton extraction. Immune sera from rabbits, sera from chronically infected mice, and monospecific antiserum to the antigenic variation protein, VlsE, were used to probe parallel two-dimensional immunoblots representing each time point. Individual proteins were identified using either specific antisera or by matching protein spots to mass spectrometry-identified protein spots from in vitro-cultivated Borrelia. There were significant changes in the relative expression of a variety of known and previously unrecognized HAB antigens during the 21-day period. OspC and the outer membrane proteins OspA and OspB were prominent at the earliest time point, day 5, when the antigenic variation protein VlsE was barely detected. OspA and OspB were not detected after day 5. OspC was not detected after day 9. VlsE was the most prominent antigen from day 7 through day 21. BmpA, ErpN, ErpP, LA7, OppA-2, DbpA, and an unidentified 15-kDa protein were also detected from day 7 through day 21. Immunoblot analysis using monospecific anti-VlsE revealed the presence of prominent distinct VlsE lower forms in HAB at days 9, 11, and 14; however, these lower forms were no longer detected at day 21. This marked diminution in VlsE lower forms paralleled the clearance of the spirochete from skin.

scholarly journals Antigenic Composition of Borrelia burgdorferi during Infection of SCID Mice

2003 ◽  
Vol 71 (6) ◽  
pp. 3419-3428 ◽  
Author(s):  
Timothy R. Crother ◽  
Cheryl I. Champion ◽  
Xiao-Yang Wu ◽  
David R. Blanco ◽  
James N. Miller ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Antigenic Variation ◽  
Protein Composition ◽  
Surface Protein ◽  
Immunoblot Analysis ◽  
Scid Mice ◽  
Antigenic Composition ◽  
Mouse Tissues ◽  
Molecular Masses

ABSTRACT The general concept that during infection of mice the Borrelia burgdorferi surface protein composition differs profoundly from that of tick-borne or in vitro-cultivated spirochetes is well established. Specific knowledge concerning the differences is limited because the small numbers of spirochetes present in tissue have not been amenable to direct compositional analysis. In this report we describe novel means for studying the antigenic composition of host-adapted Borrelia (HAB). The detergent Triton X-114 was used to extract the detergent-phase HAB proteins from mouse ears, ankles, knees, and hearts. Immunoblot analysis revealed a profile distinct from that of in vitro-cultivated Borrelia (IVCB). OspA and OspB were not found in the tissues of SCID mice 17 days after infection. The amounts of antigenic variation protein VlsE and the relative amounts of its transcripts were markedly increased in ear, ankle, and knee tissues but not in heart tissue. VlsE existed as isoforms having both different unit sizes and discrete lower molecular masses. The hydrophobic smaller forms of VlsE were also found in IVCB. The amounts of the surface protein (OspC) and the decorin binding protein (DbpA) were increased in ear, ankle, knee, and heart tissues, as were the relative amounts of their transcripts. Along with these findings regarding VlsE, OspC, and DbpA, two-dimensional immunoblot analysis with immune sera also revealed additional details of the antigenic composition of HAB extracted from ear, heart, and joint tissues. A variety of novel antigens, including antigens with molecular masses of 65 and 30 kDa, were found to be upregulated in mouse tissues. Extraction of hydrophobic B. burgdorferi antigens from tissue provides a powerful tool for determining the antigenic composition of HAB.

scholarly journals Effects of vlsE Complementation on the Infectivity of Borrelia burgdorferi Lacking the Linear Plasmid lp28-1

2004 ◽  
Vol 72 (11) ◽  
pp. 6577-6585 ◽  
Author(s):  
Matthew B. Lawrenz ◽  
R. Mark Wooten ◽  
Steven J. Norris
Keyword(s):  
Borrelia Burgdorferi ◽  
Gene Conversion ◽  
Sequence Variation ◽  
Antigenic Variation ◽  
Shuttle Vector ◽  
Immunoblot Analysis ◽  
Linear Plasmid ◽  
Shuttle Plasmid ◽  
Immunocompetent Mice

ABSTRACT The loss of linear plasmid lp28-1, which contains the vls antigenic variation locus, is associated with reduced infectivity of Borrelia burgdorferi in immunocompetent mice. The recombinant shuttle vector pBBE22, which includes the virulence determinant BBE22 from lp25 and restores infectivity to readily transformable B. burgdorferi lacking lp25 and lp56, was used to determine the effect of trans expression of vlsE on virulence. Spirochetes lacking lp28-1 were complemented with the plasmid pBBE22:vlsE, containing both BBE22 and vlsE. VlsE protein produced by this construct was expressed and surface accessible in in vitro-cultured B. burgdorferi, as determined by surface proteolysis and immunoblot analysis. Clones lacking lp25 but containing lp28-1 and either pBBE22 or pBBE22:vlsE were reisolated consistently from immunocompetent mice 8 weeks after infection. In contrast, a clone lacking both lp25 and lp28-1 and complemented with pBBE22:vlsE was isolated from only a single tissue of one of six C3H/HeN mice 8 weeks postinfection. These results indicate that either an intact vls antigenic variation locus or another determinant on lp28-1 is required to restore complete infectivity. In addition, an isogenic clone that retained lp28-1 was complemented with the vlsE shuttle plasmid and was examined for vlsE sequence variation and infectivity. Sequence variation was not observed for the shuttle plasmid, indicating that the cis arrangement of vlsE and the vls silent cassettes in lp28-1 facilitate vlsE gene conversion. Lack of vlsE sequence variation on the shuttle plasmid thus did not result in clearance of the trans-complemented strain in immunocompetent mice under the conditions tested.

scholarly journals Role of the BBA64 Locus of Borrelia burgdorferi in Early Stages of Infectivity in a Murine Model of Lyme Disease

2007 ◽  
Vol 76 (1) ◽  
pp. 391-402 ◽  
Author(s):  
Mahulena Maruskova ◽  
M. Dolores Esteve-Gassent ◽  
Valerie L. Sexton ◽  
J. Seshu
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Immunoblot Analysis ◽  
Open Reading Frames ◽  
Mammalian Host ◽  
Environmental Signals ◽  
Significant Difference ◽  
Linear Plasmid 54

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, undergoes rapid adaptive gene expression in response to environmental signals encountered during different stages of its life cycle in the arthropod vector or the mammalian host. Among all the plasmid-encoded genes of B. burgdorferi, several linear plasmid 54 (lp54)-encoded open reading frames (ORFs) exhibit the greatest differential expression in response to mammalian host-specific temperature, pH, and other uncharacterized signals. These ORFs include members of the paralogous gene family 54 (pgf 54), such as BBA64, BBA65, and BBA66, present on lp54. In an attempt to correlate transcriptional up-regulation of these pgf 54 members to their role in infectivity, we inactivated BBA64 and characterized the phenotype of this mutant both in vitro and in vivo. There were no major differences in the protein profiles between the BBA64 mutant and the control strains, while immunoblot analysis indicated that inactivation of BBA64 resulted in increased levels of BBA65. Moreover, there was no significant difference in the ability of the BBA64 mutant to infect C3H/HeN mice compared to that of its parental or complemented control strains as determined by culturing of viable spirochetes from infected tissues. However, enumeration of spirochetes using quantitative real-time PCR revealed tissue-specific differences, suggesting a minimal role for BBA64 in the survival of B. burgdorferi in select tissues. Infectivity analysis of the BBA64 mutant suggests that B. burgdorferi may utilize multiple determinants to establish infection in mammalian hosts.

scholarly journals Multiple gonococcal opacity proteins are expressed during experimental urethral infection in the male.

1994 ◽  
Vol 179 (3) ◽  
pp. 911-920 ◽  
Author(s):  
A E Jerse ◽  
M S Cohen ◽  
P M Drown ◽  
L G Whicker ◽  
S F Isbey ◽  
...  
Keyword(s):  
Antigenic Variation ◽  
Outer Membrane Proteins ◽  
Predominant Type ◽  
Detectable Difference ◽  
Single Protein ◽  
Selection For ◽  
Opacity Proteins ◽  
Strain Fa1090

The opacity (Opa) proteins of Neisseria gonorrhoeae are a family of outer membrane proteins demonstrating phase and antigenic variation. N. gonorrhoeae strain FA0190 has 11 opa loci that encode at least 8 antigenically distinct Opa proteins. To determine if expression of one Opa protein or a subset of them is favored during gonococcal infection, we inoculated Opa-negative variants of strain FA1090 intraurethrally into male volunteers. The Opa phenotype of gonococci isolated from urine and urethral swab cultures from nine infected subjects was determined. Opa proteins were expressed in a large proportion of the reisolates from the infected subjects. Gonococci cultured from urine or urethral swab samples from six of the subjects were uniformly Opa positive, with the predominant Opa variants differing among subjects. Three different Opa proteins were represented as the predominant type in at least one subject each. In three subjects, there was more heterogeneity in Opa phenotype of the reisolates, including the presence of Opa-negative variants. An increase in the proportion of isolates expressing multiple Opa proteins occurred over time in most subjects. Passage of the inoculum in vitro did not result in similar changes in Opa expression. There was no detectable difference in infectivity of an Opa-negative variant and one expressing an Opa protein (OpaF) that was highly represented in reisolates from the original nine subjects. Reisolates from three infected volunteers inoculated with the OpaF variant showed continued expression of OpaF alone or in conjunction with other Opa proteins. These results demonstrate that there is strong selection for expression of one or more Opa proteins by strain FA1090 in vivo, but that no single protein is preferentially expressed during early infection in the male urethra.

scholarly journals Antibody-resistant mutants of Borrelia burgdorferi: in vitro selection and characterization.

1992 ◽  
Vol 176 (3) ◽  
pp. 799-809 ◽  
Author(s):  
A Sădziene ◽  
P A Rosa ◽  
P A Thompson ◽  
D M Hogan ◽  
A G Barbour
Keyword(s):  
Borrelia Burgdorferi ◽  
In Vitro Selection ◽  
Outer Membrane Proteins ◽  
Class I ◽  
Missense Mutations ◽  
Class Iii ◽  
Western Blots ◽  
Disease Agent ◽  
Resistant Mutants

We used polyclonal antisera and monoclonal antibodies (mAbs) to inhibit the growth of clonal populations of two strains of Borrelia burgdorferi, the Lyme disease agent, and thereby select for antibody-resistant mutants. mAbs were directed at the outer membrane proteins, OspA or OspB. Mutants resistant to the growth-inhibiting properties of the antibodies were present in the populations at frequencies ranging from 10(-5) to 10(-2). The several escape variants that were examined were of four classes. Class I mutants were resistant to all mAbs; they lacked OspA and OspB and the linear plasmid that encodes them. Two other proteins were expressed in larger amounts in class I mutants; mAbs to these proteins inhibited the mutant but not the wild-type cells. Class II mutants were resistant to some but not all mAbs; they had truncated OspA and/or OspB proteins. Class III mutants were resistant only to the selecting mAb; they had full-length Osp proteins that were not bound by the selecting antibody in Western blots. In two class III mutants resistant to different anti-OspA mAbs, missense mutations were demonstrated in the ospA genes. Class IV mutants were likewise resistant only to selecting antibody, but in this case the selecting antibody still bound in Western blots.

scholarly journals C-Terminal Invariable Domain of VlsE May Not Serve as Target for Protective Immune Response against Borrelia burgdorferi

2001 ◽  
Vol 69 (3) ◽  
pp. 1337-1343 ◽  
Author(s):  
Fang Ting Liang ◽  
Mary B. Jacobs ◽  
Mario T. Philipp
Keyword(s):  
Immune Response ◽  
Borrelia Burgdorferi ◽  
Antigenic Variation ◽  
Keyhole Limpet Hemocyanin ◽  
Protective Immune Response ◽  
Variable Surface ◽  
Entire Sequence ◽  
Mouse Antibody

ABSTRACT VlsE, the variable surface antigen of the Lyme disease spirochete,Borrelia burgdorferi, contains two invariable domains, at the amino and carboxyl termini, respectively, which collectively account for approximately one-half of the entire molecule's length and remain unchanged during antigenic variation. It is not known if these two invariable domains are exposed at the surface of either the antigen or the spirochete. If they are exposed at the spirochete's surface, they may elicit a protective immune response against B. burgdorferi and serve as vaccine candidates. In this study, a 51-mer synthetic peptide that reproduced the entire sequence of the C-terminal invariable domain of VlsE was conjugated to the carrier keyhole limpet hemocyanin and used to immunize mice. Generated mouse antibody was able to immunoprecipitate native VlsE extracted from cultured B. burgdorferi B31 spirochetes, indicating that the C-terminal invariable domain was exposed at the antigen's surface. However, this domain was inaccessible to antibody binding at the surface of cultured intact spirochetes, as demonstrated by both an immunofluorescence experiment and an in vitro killing assay. Mouse antibody to the C-terminal invariable domain was not able to confer protection against B. burgdorferi infection, indicating that this domain was unlikely exposed at the spirochete's surface in vivo. We concluded that the C-terminal invariable domain was exposed at the antigen's surface but not at the surface of either cultured or in vivo spirochetes and thus cannot elicit protection against B. burgdorferi infection.

scholarly journals Cryptic and Exposed Invariable Regions of VlsE, the Variable Surface Antigen of Borrelia burgdorferisl

2000 ◽  
Vol 182 (12) ◽  
pp. 3597-3601 ◽  
Author(s):  
Fang Ting Liang ◽  
Jena M. Nowling ◽  
Mario T. Philipp
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Synthetic Peptides ◽  
Antigenic Variation ◽  
Surface Protein ◽  
Variable Surface ◽  
Antibody Titers ◽  
Negative Surface ◽  
Antipeptide Antibody

ABSTRACT Borrelia burgdorferi, the Lyme disease spirochete, possesses a surface protein, VlsE, which undergoes antigenic variation. VlsE contains two invariable domains and a variable one that includes six variable and six invariable regions (IRs). Five of the IRs are conserved among strains and genospecies of B. burgdorferisensu lato. IR6 is conserved, immunodominant, and exposed at the VlsE surface but not at the spirochete surface, as assessed in vitro. In the present study, the remaining conserved IRs (IR2 to IR5) were investigated. Antisera to synthetic peptides based on each of the IR2 to IR5 sequences were produced in rabbits. Antipeptide antibody titers were similarly high in all antisera. Native VlsE was immunoprecipitable with antibodies to IR2, IR4, and IR5 but not to IR3, indicating that the first three sequences were exposed at the VlsE surface. However, negative surface immunofluorescence and in vitro antibody-mediated killing results indicated that none of the IRs were accessible to antibody at the spirochetal surface in vitro.

scholarly journals Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin

2004 ◽  
Vol 384 (3) ◽  
pp. 609-617 ◽  
Author(s):  
Jaruwan SIRITAPETAWEE ◽  
Heino PRINZ ◽  
Chartchai KRITTANAI ◽  
Wipa SUGINTA
Keyword(s):  
Burkholderia Pseudomallei ◽  
Polyclonal Antibodies ◽  
Outer Membrane Proteins ◽  
Immunoblot Analysis ◽  
Cd Spectroscopy ◽  
Cloning And Expression ◽  
Buffer System ◽  
Burkholderia Thailandensis ◽  
Peptide Mass

In the present paper, we describe cloning and expression of two outer membrane proteins, BpsOmp38 (from Burkholderia pseudomallei) and BthOmp38 (from Burkholderia thailandensis) lacking signal peptide sequences, using the pET23d(+) expression vector and Escherichia coli host strain Origami(DE3). The 38 kDa proteins, expressed as insoluble inclusion bodies, were purified, solubilized in 8 M urea, and then subjected to refolding experiments. As seen on SDS/PAGE, the 38 kDa band completely migrated to ∼110 kDa when the purified monomeric proteins were refolded in a buffer system containing 10% (w/v) Zwittergent® 3-14, together with a subsequent heating to 95 °C for 5 min. CD spectroscopy revealed that the 110 kDa proteins contained a predominant β-sheet structure, which corresponded completely to the structure of the Omp38 proteins isolated from B. pseudomallei and B. thailandensis. Immunoblot analysis using anti-BpsOmp38 polyclonal antibodies and peptide mass analysis by MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS confirmed that the expressed proteins were BpsOmp38 and BthOmp38. The anti-BpsOmp38 antibodies considerably exhibited the inhibitory effects on the permeation of small sugars through the Omp38-reconstituted liposomes. A linear relation between relative permeability rates and Mr of neutral sugars and charged antibiotics suggested strongly that the in vitro re-assembled Omp38 functioned fully as a diffusion porin.

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.

scholarly journals Kinetics and In Vivo Induction of Genetic Variation of vlsE in Borrelia burgdorferi

1998 ◽  
Vol 66 (8) ◽  
pp. 3689-3697 ◽  
Author(s):  
Jing-Ren Zhang ◽  
Steven J. Norris
Keyword(s):  
Borrelia Burgdorferi ◽  
Sequence Variation ◽  
Antigenic Variation ◽  
Pcr Amplification ◽  
Scid Mice ◽  
Mammalian Host ◽  
Expression Site ◽  
Disease Agent

ABSTRACT The Lyme disease agent, Borrelia burgdorferi, is able to persistently infect humans and animals for months or years in the presence of an active immune response. It is not known how the organisms survive immune attack in the mammalian host.vlsE, a gene localized near one end of linear plasmid lp28-1 and encoding a surface-exposed lipoprotein in B. burgdorferi B31, was shown recently to undergo extensive genetic and antigenic variation within 28 days of initial infection in C3H/HeN mice. In this study, we examined the kinetics of vlsEsequence variation in C3H/HeN mice at 4, 7, 14, 21, and 28 days and at 7 and 12 months postinfection. Sequence changes were detected by PCR amplification and sequence analysis as early as 4 days postinfection and accumulated progressively in both C3H/HeN and CB-17 severe combined immunodeficient (SCID) mice throughout the course of infection. The sequence changes were consistent with sequential recombination of segments from multiple silent vls cassette sites into thevlsE expression site. No vlsE sequence changes were detected in organisms cultured in vitro for up to 84 days. These results indicate that vlsE recombination is induced by a factor(s) present in the mammalian host, independent of adaptive immune responses. The possible inducing conditions appear to be present in various tissue sites because isolates from multiple tissues showed similar degrees of sequence variation. The rate of accumulation of predicted amino acid changes was higher in the immunologically intact C3H/HeN mice than in SCID mice, a finding consistent with immune selection of VlsE variants.

Sign in / Sign up

Export Citation Format

Share Document