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 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 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 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.

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.

Cubilin and megalin expression and their interaction in the rat intestine: effect of thyroidectomy

2001 ◽  
Vol 281 (5) ◽  
pp. E900-E907 ◽  
Author(s):  
Raghunatha R. Yammani ◽  
Shakuntla Seetharam ◽  
Bellur Seetharam
Keyword(s):  
Brush Border ◽  
Intrinsic Factor ◽  
Immunoblot Analysis ◽  
Rat Intestine ◽  
Complement Components ◽  
Protein Levels ◽  
Intestinal Brush Border ◽  
Molecular Masses ◽  
Epidermal Growth

Cubilin is a 460-kDa multipurpose, multidomain receptor that contains an NH2-terminal 110-residue segment followed by 8 epidermal growth factor (EGF)-like repeats and a contiguous stretch (representing nearly 88% of its mass) of 27 CUB (initially found in complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domains. Cubilin binds to intrinsic factor (IF)-cobalamin (cbl, vitamin B12) complex and promotes the ileal transport of cbl. The 460-kDa form of cubilin is the predominant form present in the apical brush-border membranes of rat intestine, kidney, and yolk sac, but a 230-kDa form of cubilin is also noted in the intestinal membranes. In thyroidectomized (TDX) rats, levels of intestinal brush-border IF-[57Co]-labeled cbl binding, 460-kDa cubilin protein levels and tissue (kidney) accumulation of cbl were reduced by ∼70%. Immunoblot analysis using cubilin antiserum of intestinal total membranes from TDX rats revealed cubilin fragments with molecular masses of 200 and 300 kDa. Both of these bands, along with the 230-kDa band detected in the total membranes of control rats and unlike the 460-kDa form, failed to react with antiserum to EGF. Mucosal membrane cubilin associated with megalin was reduced from ∼12% in control to ∼4% in TDX rats, and this decreased association was not due to altered megalin levels. Thyroxine treatment of TDX rats resulted in reversal of all of these effects, including an increase to nearly 24% of cubilin associated with megalin. In vitro, megalin binding to cubilin occurred with the NH2-terminal region that contained the EGF-like repeats and CUB domains 1 and 2 but not with a downstream region that contained CUB domains 2–10. These studies indicate that thyroxine deficiency in rats results in decreased uptake and tissue accumulation of cbl caused mainly by destabilization and deficit of cubilin in the intestinal brush border.

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats

1991 ◽  
Vol 69 (8) ◽  
pp. 499-508 ◽  
Author(s):  
Andrea G. Bodnar ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Immunoblot Analysis ◽  
In Vitro Translation ◽  
Peroxisome Proliferator ◽  
Peroxisomal Membrane ◽  
Molecular Masses ◽  
Fold Induction ◽  
Membrane Bound ◽  
Liver Peroxisomes

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.Key words: peroxisomes, integral membrane polypeptides, clofibrate, free polysomes, membrane-bound polysomes.

scholarly journals The Ubiquitin-Proteasome System Does Not Regulate the Degradation of Porcine β-Microseminoprotein during Sperm Capacitation

2020 ◽  
Vol 21 (11) ◽  
pp. 4151
Author(s):  
Lucie Tumova ◽  
Michal Zigo ◽  
Peter Sutovsky ◽  
Marketa Sedmikova ◽  
Pavla Postlerova
Keyword(s):  
Seminal Plasma ◽  
Plasma Proteins ◽  
Protein Composition ◽  
Surface Protein ◽  
Ubiquitin Proteasome System ◽  
Sperm Capacitation ◽  
Sperm Surface ◽  
Ubiquitin Proteasome ◽  
Seminal Plasma Proteins

Sperm capacitation, one of the key events during successful fertilization, is associated with extensive structural and functional sperm remodeling, beginning with the modification of protein composition within the sperm plasma membrane. The ubiquitin-proteasome system (UPS), a multiprotein complex responsible for protein degradation and turnover, participates in capacitation events. Previous studies showed that capacitation-induced shedding of the seminal plasma proteins such as SPINK2, AQN1, and DQH from the sperm surface is regulated by UPS. Alterations in the sperm surface protein composition also relate to the porcine β-microseminoprotein (MSMB/PSP94), seminal plasma protein known as immunoglobulin-binding factor, and motility inhibitor. MSMB was detected in the acrosomal region as well as the flagellum of ejaculated boar spermatozoa, while the signal disappeared from the acrosomal region after in vitro capacitation (IVC). The involvement of UPS in the MSMB degradation during sperm IVC was studied using proteasomal interference and ubiquitin-activating enzyme (E1) inhibiting conditions by image-based flow cytometry and Western blot detection. Our results showed no accumulation of porcine MSMB either under proteasomal inhibition or under E1 inhibiting conditions. In addition, the immunoprecipitation study did not detect any ubiquitination of sperm MSMB nor was MSMB detected in the affinity-purified fraction containing ubiquitinated sperm proteins. Based on our results, we conclude that UPS does not appear to be the regulatory mechanism in the case of MSMB and opening new questions for further studies. Thus, the capacitation-induced processing of seminal plasma proteins on the sperm surface may be more complex than previously thought, employing multiple proteolytic systems in a non-redundant manner.

scholarly journals An Immune Evasion Mechanism for Spirochetal Persistence in Lyme Borreliosis

2002 ◽  
Vol 195 (4) ◽  
pp. 415-422 ◽  
Author(s):  
Fang Ting Liang ◽  
Mary B. Jacobs ◽  
Lisa C. Bowers ◽  
Mario T. Philipp
Keyword(s):  
Immune Evasion ◽  
Protein C ◽  
Passive Immunization ◽  
Surface Protein ◽  
Scid Mice ◽  
Immune Evasion Mechanism ◽  
Immunocompetent Mice ◽  
Humoral Responses ◽  
Selection Of

Borrelia burgdorferi, the Lyme disease spirochete, persistently infects mammalian hosts despite the development of strong humoral responses directed against the pathogen. Here we describe a novel mechanism of immune evasion by B. burgdorferi. In immunocompetent mice, spirochetes that did not express ospC (the outer-surface protein C gene) were selected within 17 d after inoculation, concomitantly with the emergence of anti-OspC antibody. Spirochetes with no detectable OspC transcript that were isolated from immunocompetent mice reexpressed ospC after they were either cultured in vitro or transplanted to naive immunocompetent mice, but not in OspC-immunized mice. B. burgdorferi persistently expressed ospC in severe combined immune-deficient (SCID) mice. Passive immunization of B. burgdorferi–infected SCID mice with an anti-OspC monoclonal antibody selectively eliminated ospC-expressing spirochetes but did not clear the infection. OspC-expressing spirochetes reappeared in SCID mice after the anti-OspC antibody was eliminated. We submit that selection of surface-antigen nonexpressers is an immune evasion mechanism that contributes to spirochetal persistence.

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 Evaluation of RevA, a Fibronectin-Binding Protein of Borrelia burgdorferi, as a Potential Vaccine Candidate for Lyme Disease

2013 ◽  
Vol 20 (6) ◽  
pp. 892-899 ◽  
Author(s):  
Angela M. Floden ◽  
Tammy Gonzalez ◽  
Robert A. Gaultney ◽  
Catherine A. Brissette
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Passive Immunization ◽  
Surface Protein ◽  
Surface Expression ◽  
Content Type ◽  
Good Target ◽  
Potential Vaccine ◽  
Mammalian Infection

ABSTRACTPrevious studies indicated that the Lyme disease spirocheteBorrelia burgdorferiexpresses the RevA outer surface protein during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA appears to be a good target for preventive therapies. RevA proteins are highly conserved across all Lyme borreliae, and antibodies against RevA protein are cross-reactive among RevA proteins from diverse strains. Mice infected withB. burgdorferimounted a rapid IgM response to RevA, followed by a strong IgG response that generally remained elevated for more than 12 months, suggesting continued exposure of RevA protein to the immune system. RevA antibodies were bactericidalin vitro. To evaluate the RevA antigen as a potential vaccine, mice were vaccinated with recombinant RevA and challenged withB. burgdorferiby inoculation with a needle or by a tick bite. Cultured tissues from all treatment groups were positive forB. burgdorferi. Vaccinated animals also appeared to have similar levels ofB. burgdorferiDNA compared to nonvaccinated controls. Despite its antigenicity, surface expression, and the production of bactericidal antibodies against it, RevA does not protect againstBorrelia burgdorferiinfection in a mouse model. However, passive immunization with anti-RevA antibodies did prevent infection, suggesting the possible utility of RevA-based immunotherapeutics or vaccine.

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