Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen

Fehniger, Thomas E.; Walfield, Alan M.; Cunningham, Thomas M.; Radolf, Justin D.; Miller, James N.; Lovett, Michael

doi:10.1128/iai.46.2.598-607.1984

Cited by 55 publications

(34 citation statements)

References 18 publications

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“…Identification of exposed determinants on intact T. phagedenis endoflagella. Determinants exposed on intact T. phagedenis endoflagella were identified by a modification of immunoblotting that we have termed epitope bridging (10). By this technique, nitrocellulose transfers are probed with intact '25I-labeled T. phagedenis endoflagella rather than l25l-labeled staphylococcal protein A following reaction with the appropriate antiserum.…”

Section: Resultsmentioning

confidence: 99%

“…We utilized a modification of immunoblotting calbed epitope bridging (10) to determine which of the endoflagellar polypeptides are located on the surface of intact endoflagella. The technique relies on the ability of multivalent immunoglobulins to bind simultaneously to endoflagellar polypeptide determinants on Western blots and to surface-exposed on August 5, 2020 by guest http://iai.asm.org/ Downloaded from determinants of intact, radiolabeled endoflagella.…”

Section: Discussionmentioning

confidence: 99%

“…Nevertheless, investigators using immunoprecipitation of surface or intrinsically radiolabeled organisms (1,3,17,23,33,37) and Western blotting (2,11,12,25,37) have identified most, if not all, of the antigenic polypeptides of T. pallidum. More recently, recombinant DNA technology has made possible the expression of T. pallidum polypeptides in Escherichia coli (24,34,40,41) and their subsequent purification in quantities sufficient for biochemical characterization (10). However, cloning strategies used to date have resulted in the expression of relatively few of the total number of T. pallidum proteins.…”

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confidence: 99%

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Antigenic interrelationship between endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum (Nichols): molecular characterization of endoflagellar proteins

Radolf¹,

Blanco²,

Miller³

et al. 1986

Infect Immun

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Purified endoflagella from nonpathogenic Treponema phagedenis bioptye Reiter were characterized biochemically and compared antigenically with the endoflageilar proteins of Treponema pallidum. T. phagedenis biotype Reiter endoflagella were dissociated into constituent polypeptides by incubation under conditions which disrupt noncovalent bonds. Chymotrypsin peptide maps of T. phagedenis biotype Reiter endoflageliar proteins revealed that the 37and 33-kilodalton (kDa) major components shared significant homology with the 27and 30-kDa minor components, respectively. The peptide maps also suggested that the major components shared a lesser degree of structural similarity with each other. These relationships were confirmed by Western blots of T. phagedenis biotype Reiter endoflagellar proteins employing antibodies that were purified against the individual endoflagellar polypeptides. Western blots of T. pallidum with the purified antibodies also demonstrated strong cross-reactivity between the T. phagedenis biotype Reiter endoflageliar proteins and T. pallidum proteins of identical or similar molecular weights. A unique Western blotting technique that we called epitope bridging was used to determine that the 37-kDa subunit contains most of the external epitopes on T. phagedenis biotype Reiter endoflagella. Immunoelectron microscopy with human syphilitic serum and rabbit T. phagedenis biotype Reiter endoflagellar antiserum confirmed the presence of cross-reactive epitopes on the surface of intact T. phagedenis and T. paUidum endoflagella.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

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Antigenic interrelationship between endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum (Nichols): molecular characterization of endoflagellar proteins

Radolf¹,

Blanco²,

Miller³

et al. 1986

Infect Immun

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“…Solubilised promastigotes (one million cells), equivalent to 30/xg protein, were loaded per well. Following separation, the proteins were transfered onto nitrocellulose paper, pore size 0.45/^m (BIO RAD) using high concentration glycine buffer [15] in combination with a minitrans-blot cell system (Bio RAD).…”

Section: Methodsmentioning

confidence: 99%

Visceral Leishmaniasis in Ververt Monkeys: Immunological Responses During Asymptomatic Infections

Gicheru¹,

Olobo²,

Kariuki³

et al. 1995

Scand J Immunol

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Nine vervet monkeys (Cercopithecus aethiops) were infected intradermally with 8 x 10(7) virulent L. donovani promastigotes. Four animals developed clinical visceral leishmaniasis and died over a period of 18 months. The remaining five animals have remained asymptomatic for a period of 3 years now. Attempts to isolate parasites from spleen and liver through biopsies were fruitless. Immunological responses of these subclinically infected animals were examined. Enzyme-linked immunosorbent assay (ELISA) and western blot analyses demonstrated Leishmania specific antibodies in these animals, but the antibody titres were low. When proliferation of peripheral blood monocytes (PBMC) to Concanavalin A (Con A) of these animals was compared with control 'disease free animals' there were no significant differences in response. However, L. donovani antigen (fixed promastigotes) specific proliferation was demonstrated in the five subclinically infected animals. High and varying levels of interferon gamma (IFN-gamma) were secreted in PBMC cultures from the five vervet monkeys when stimulated with either Con A or L. donovani antigens. In control animals, IFN-gamma was only detected when PBMC were stimulated with Con A. Marked delayed-type hypersensitivity (DTH) responses were demonstrated in the five subclinically infected animals 48 h after injection with formalin fixed promastgotes. It was concluded that the visceral Leishmania disease spectrum due to L. donovani observed in humans could be induced in vervet monkeys and that L. donovani asymptomatic/cryptic infected animals have competent humoral and cellular responses to homologous parasites.

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“…Reports differ as to whether spirochetes contain lipopolysaccharide (LPS) in their outer membrane. Leptospira interrogans, Treponema phagedenis (biotype Reiter), and Serpula (Treponema) hyodysenteriae apparently do contain an LPSlike substance (3,21,23,28,35,36,41,63,66,76,77), while Treponema pallidum apparently does not (3,63). There is conflicting evidence regarding the presence of LPS in Borrelia spp.…”

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Purification and immunological characterization of a major low-molecular-weight lipoprotein from Borrelia burgdorferi

1992

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Borrelia burgdorferi resembles gram-negative bacteria in having both cellular and outer membranes. We previously showed that a lipopolysaccharide (LPS)-like material could be extracted from B. burgdorferi with phenol-chloroform-petroleum ether (PCP). The PCP extract of B. burgdorferi exhibited biological activity in several in vitro assays (e.g., mitogenicity, pyrogenicity, and cytokine release). These activities suggested the presence of endotoxin. The PCP extract of B. burgdorieri, however, also contained a small amount of protein.Preliminary studies showed that monoclonal antibody prepared against this protein inhibited the mitogenic activity of the PCP extract toward murine spleen cells. The current study was therefore undertaken to characterize this protein and to establish methods for its separation from the LPS. The PCP-extracted protein consisted of a single, low-molecular-weight lipoprotein (apparent M,, 10,000 by sodium dodecyl sulfatepolyacrylamide gel electrophoresis) (SDS-PAGE). By protein analysis, it accounted for 2% of the dry weight of defatted cells, thus making it a major constituent of the spirochete. It was purified from the LPS by initial extraction into 10%lY Triton X-100 followed by immunoaffinity chromatography in the presence of detergent. On removal of the LPS, the purified lipoprotein formed aggregates stable to SDS-PAGE which were detectable on Western blots (immunoblots) probed with either the monoclonal antibody or polyclonal antiserum. From a plot of the aggregate molecular weight versus aggregate size, a monomer molecular weight of 7,500 was obtained. Indirect immunofluorescence with the monoclonal antibody showed that the lipoprotein was exposed at the surface of the spirochete in only a small percentage of cells. The lipoprotein was present in several strains of B. burgdorferi but absent in other Borrelia spp., treponemes, and gram-negative human pathogens, indicating species specificity. 4995 on August 4, 2020 by guest http://iai.asm.org/ Downloaded from

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Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen

Cited by 55 publications

References 18 publications

Antigenic interrelationship between endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum (Nichols): molecular characterization of endoflagellar proteins

Antigenic interrelationship between endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum (Nichols): molecular characterization of endoflagellar proteins

Visceral Leishmaniasis in Ververt Monkeys: Immunological Responses During Asymptomatic Infections

Purification and immunological characterization of a major low-molecular-weight lipoprotein from Borrelia burgdorferi

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