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J D Radolf

Publications and source records attributed to J D Radolf.

At least 91 records · Page 5Linked to original sources
PubMed1992

Virulent Treponema pallidum activates human vascular endothelial cells.

Perivascular lymphocytic infiltration, fibrin deposition, and endothelial cell abnormalities consistent with cellular activation are prominent histopathologic features of syphilis, a sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. Because activated endothelial cells play important roles in lymphocyte homing and hemostasis, the ability of virulent T. pallidum to activate cultured human umbilical vein endothelial cells (HUVEC) was investigated. T. pallidum induced the expression of intercellular adhesion molecule-1 (ICAM-1) and procoagulant activity on the surface of HUVEC. Electron microscopy of T. pallidum-stimulated HUVEC revealed extensive networks of fibrin strands not observed in cultures without treponemes. ICAM-1 expression in HUVEC also was promoted by a 47-kDa integral membrane lipoprotein purified from T. pallidum, implicating a role for spirochete membrane lipoproteins in endothelial cell activation. The combined findings are consistent with the pathology of syphilis and provide the first evidence that a pathogenic spirochetal bacterium such as T. pallidum or its constituent integral membrane lipoprotein(s) can activate directly host vascular endothelium.

Antigens, CD↗
PubMed1992

The outer membrane, not a coat of host proteins, limits antigenicity of virulent Treponema pallidum.

Virulent Treponema pallidum reacts poorly with the specific antibodies present in human and rabbit syphilitic sera, a phenomenon often attributed to an outer coat of host serum proteins. Here we present additional evidence that the limited antigenicity of virulent organisms actually is due to a paucity of proteins in the outer membrane. Initially, we used electron microscopy to demonstrate that the outer membrane is highly susceptible to damage from physical manipulation (i.e., centrifugation and resuspension) and nonionic detergents. Organisms with disrupted outer membranes were markedly more antigenic than intact treponemes as determined by immunoelectron microscopy (IEM) with rabbit syphilitic and antiendoflagellar antisera. Data obtained with a new radioimmunoassay, designated the T. pallidum surface-specific radioimmunoassay, corroborated these IEM findings by demonstrating that the major T. pallidum immunogens are not surface exposed; the assay also was unable to detect serum proteins, including fibronectin, on the surfaces of intact organisms. Furthermore, IEM of T. pallidum on ultrathin cryosections with monospecific anti-47-kDa-immunogen antiserum confirmed the intracellular location of the 47-kDa immunogen. On the basis of these and previous findings, we proposed a new model for T. pallidum ultrastructure in which the outer membrane contains a small number of transmembrane proteins and the major membrane immunogens are anchored by lipids to the periplasmic leaflet of the cytoplasmic membrane. This unique ultrastructure explains the remarkable ability of virulent organisms to evade the humoral immune response of the T. pallidum-infected host.

Animals↗
PubMed1992

Use of electroporation to construct isogenic mutants of Haemophilus ducreyi.

Little is known about the genetics of Haemophilus ducreyi, the etiologic agent of chancroid. To develop a method for constructing isogenic mutants of this organism that could be utilized in pathogenesis-related studies, electroporation techniques were evaluated as a means of introducing DNA into this organism. Electroporation of the plasmid shuttle vector pLS88 into H. ducreyi yielded approximately 10(6) antibiotic-resistant transformants per microgram of plasmid DNA. Studies of the feasibility of moving mutated genes into H. ducreyi were initiated by using NotI linker insertion and mini-Tn10kan mutagenesis techniques to introduce insertion mutations into cloned H. ducreyi genes encoding cell envelope antigens. In the former case, a gene encoding chloramphenicol acetyltransferase was then inserted into the NotI linker site created in the cloned H. ducreyi gene. The recombinant Escherichia coli strains containing these mutated plasmids no longer expressed the homologous H. ducreyi cell envelope antigens, as evidenced by their lack of reactivity with monoclonal antibody probes for these H. ducreyi proteins. Subsequent electroporation of both circular and linearized forms of plasmids carrying these mutated H. ducreyi genes into the homologous wild-type strain of H. ducreyi yielded antibiotic-resistant transformants which also lacked reactivity with the cell envelope antigen-specific monoclonal antibodies. Southern blot analysis confirmed that homologous recombination had occurred in these monoclonal antibody-unreactive transformants, resulting in the replacement of the wild-type allele with the mutated allele. Allelic exchange was most efficient when linear DNA molecules were used for electroporation. These results indicate that electroporation methods can be utilized to construct isogenic mutants of H. ducreyi.

Blotting, Southern↗
PubMed1991

Lipoproteins of Borrelia burgdorferi and Treponema pallidum activate cachectin/tumor necrosis factor synthesis. Analysis using a CAT reporter construct.

Lipoproteins from two pathogenic spirochetes (Borrelia burgdorferi and Treponema pallidum) induced the biosynthesis of TNF in murine macrophages and in permanently transformed macrophages of the cell line RAW 264.7. Induction was studied by measuring the secretion of biologically active TNF and by measuring the activity of the reporter enzyme chloramphenicol acetyltransferase (CAT) produced within macrophages transfected with an endotoxin-responsive CAT construct. Several lines of evidence indicated that the induction of TNF and CAT was attributable to the spirochete lipoproteins rather than to contaminating or endogenous LPS: 1) the dose response curves observed for the lipoproteins were markedly different from those obtained with LPS; 2) lipoprotein-mediated activation was unaffected by amounts of polymyxin B that completely neutralized the induction of TNF and CAT by LPS, 3) low concentrations of the lipoproteins induced TNF in macrophages from endotoxin-unresponsive C3H/HeJ mice as effectively as in macrophages from normal C3H/HeN mice, and 4) isolated spirochete lipoproteins, but not a non-lipoprotein immunogen, were potent inducers of CAT in the transformed macrophages. Moreover, LPS was not detected in the B. burgdorferi lipoprotein mixtures by Limulus amebocyte lysate assay. Proteolytic digestion of the intact bacterial protein preparations only modestly diminished their ability to activate the cells, suggesting that small lipopeptides comprise the biologically active portions of the molecules, as is the case with the murein lipoprotein of Escherichia coli. Through their ability to induce TNF production by macrophages, spirochete lipoproteins may play important roles in the development of the local inflammatory changes and the systemic manifestations that characterize syphilis and Lyme disease.

Animals↗
PubMed1991

A temperature-dependent rabbit model for production of dermal lesions by Haemophilus ducreyi.

The fact that optimal in vitro growth of Haemophilus ducreyi occurs at 33 degrees C prompted evaluation of the effect of temperature on the ability of this organism to produce skin lesions in rabbits after intradermal inoculation. Animals housed at a reduced ambient temperature (15-17 degrees C) consistently developed necrotic lesions when injected intradermally with 10(5) cfu of H. ducreyi; this inoculum did not produce necrotic lesions in animals housed at normal room temperature (23-25 degrees C). Lesion production in this new model was dependent on both viability of the H. ducreyi inoculum and replication of these organisms after intradermal injection. Histopathologic examination of the lesions revealed that H. ducreyi infection of the rabbit dermis evolves from an acute inflammatory reaction to abscess formation. Evaluation of three additional strains of H. ducreyi in this model confirmed that lesion formation was not bacterial strain-dependent. This new temperature-dependent rabbit model for productive H. ducreyi infection will facilitate investigation of the molecular pathogenesis of chancroid.

Animals↗
PubMed1991

Localization of outer surface proteins A and B in both the outer membrane and intracellular compartments of Borrelia burgdorferi.

Borrelia burgdorferi B31 with and without outer membranes contained nearly identical amounts of outer surface proteins A and B. The majority of each immunogen also was localized intracellularly by immunocryoultramicrotomy. These results are inconsistent with the widely held belief that outer surface proteins A and B are exclusively outer membrane proteins.

Bacterial Outer Membrane Proteins↗
PubMed1991

Sensitive detection of Treponema pallidum by using the polymerase chain reaction.

We have developed a sensitive assay for Treponema pallidum subsp. pallidum (T. pallidum), the agent of veneral syphilis, based upon the polymerase chain reaction (PCR). A 658-bp portion of the gene encoding the 47-kDa membrane immunogen was amplified, and the PCR products were probed by DNA-DNA hybridization with a 496-bp fragment internal to the amplitifed DNA. The assay detected approximately 0.01 pg of purified T. pallidum DNA, and positive results were obtained routinely from suspensions of treponemes calculated to contain 10 or more organism and from some suspensions calculated to contain a single organism. Specific PCR products were obtained for the closely related agent of yaws, Treponema pallidum subsp. pertenue, but not with human DNA or DNAs from other spirochetes (including Borrelia burgdoferi), skin microorganisms, sexually transmitted disease pathogens, and central nervous system pathogens. T. pallidum DNA was detected in serum, cerebrospinal fluids, and amniotic fluids from syphilis patients but not in in nonsyphilitic controls. T. pallidum DNA was also amplified from paraffin-embedded tissue. The diagnosis of syphillis by using PCR may become a significant addition to the diagnostic armamentarium and a valuable technique for the investigation of syphilis pathogenesis.

Animals↗
PubMed1991

Use of polymerase chain reaction and rabbit infectivity testing to detect Treponema pallidum in amniotic fluid, fetal and neonatal sera, and cerebrospinal fluid.

The diagnosis of congenital syphilis continues to pose a difficult clinical challenge. Because the serodiagnosis of congenital syphilis has significant limitations, the direct detection of Treponema pallidum in suspect neonatal tissues or body fluids represents a desirable alternate diagnostic strategy. We developed and applied the polymerase chain reaction (PCR) for the detection of T. pallidum in clinical material relevant to the diagnosis of congenital syphilis but which typically contain factors inhibitory for the PCR. Four methods of specimen processing were examined to circumvent PCR inhibition; clinical materials included amniotic fluids, neonatal sera, and neonatal cerebrospinal fluids. The PCR was 100% specific for T. T. pallidum compared with the sensitive rabbit infectivity test (RIT) for all clinical materials tested. For amniotic fluids, the PCR was 100% sensitive when correlated with the RIT but had a lesser sensitivity when applied to sera or cerebrospinal fluids, which typically contain few treponemes. The combined sensitivity of the PCR for all clinical samples was 78%. Positive PCR results also were obtained among some clinical specimens for which RIT was not performed; these results correlated well with either stigmata or risk factors for congenital syphilis. The combined results suggest that the PCR can be a useful adjunct to the diagnosis and clinical management of congenital syphilis and that it will provide a valuable tool for investigations of the pathogenesis of the disorder.

Amniotic Fluid↗
PubMed1990

Lipid modification of the 15 kiloDalton major membrane immunogen of Treponema pallidum.

The 15 kiloDalton major membrane immunogen was included among the Treponema pallidum polypeptides selectively labelled with [3H]-palmitate. The cloned gene for this immunogen, tpp15, encoded a signal peptide of 17 amino acids, a consensus signal peptidase II cleavage site, and a mature protein of 124 amino acids (13,967 Daltons). As predicted by the DNA sequence, the recombinant 15 kiloDalton immunogen labelled selectively with [3H]-palmitate, and globomycin inhibited processing of the precursor to the mature polypeptide. While the native and recombinant immunogens are amphiphilic, the 15 kiloDalton immunogen synthesized in a cell-free system was hydrophilic. The covalent attachment of fatty acids appears to be responsible for the amphiphilicity of the immunogen and its membrane attachment.

Acylation↗
PubMed1990

The 34-kilodalton membrane immunogen of Treponema pallidum is a lipoprotein.

Treponema pallidum subsp. pallidum and Escherichia coli incorporated exogenous [3H]palmitate into the 34-kilodalton (kDa) pathogen-specific antigen of T. pallidum. Radiolabeled fatty acid remained associated with the protein upon immunoprecipitation and after boiling in sodium dodecyl sulfate, acetone precipitation, and extensive extractions in organic solvents, suggesting that the fatty acid was covalently bound to the protein. Detection of [3H]palmitate after alkaline and acid hydrolyses confirmed the identity of the incorporated label. Globomycin inhibited maturation of the recombinant 34-kDa antigen, suggesting that E. coli uses the lipoprotein-specific signal peptidase II to process the treponemal antigen. Globomycin also inhibited processing of the 34-kDa antigen, as well as the 44.5- and 15-kDa antigens, in T. pallidum, implying that T. pallidum also possesses the lipoprotein export pathway common to both gram-negative and gram-positive bacteria. Ethanol inhibited processing of the 34-kDa antigen in minicells, suggesting that the 34-kDa antigen normally is translocated through the cytoplasmic membrane. Comparison of the Triton X-114 phase partitioning behavior of the 34-kDa antigen produced either by minicells or by a cell-free translation system indicated that the covalent attachment of fatty acid conferred hydrophobic biochemical properties to the 34-kDa antigen, consistent with the hypothesis that the attached lipid anchors the 34-kDa antigen into the membrane.

Acylation↗
PubMed1990

Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins.

The pathogenic spirochete Borrelia burgdorferi contains a set of integral membrane proteins which were selectively extracted into the detergent phase upon solubilization of intact B. burgdorferi with the nonionic detergent Triton X-114. Virtually all of these hydrophobic proteins were recognized by antibodies in pooled sera from patients with chronic Lyme arthritis, demonstrating that proteins partitioning into the detergent phase of Triton X-114 encompass the major B. burgdorferi immunogens. Furthermore, most of these immunogenic proteins, including the previously characterized OspA and OspB membrane antigens, could be biosynthetically labeled when B. burgdorferi was incubated in vitro with [3H]palmitate. The OspA and OspB antigens were radioimmunoprecipitated from [3H]palmitate-labeled detergent-phase proteins with monoclonal antibodies, and [3H]palmitate was recovered unaltered from these proteins after sequential alkaline and acid hydrolyses. The combined results provide formal confirmation that the major B. burgdorferi immunogens extracted by Triton X-114 are lipoproteins. The demonstration that B. burgdorferi integral membrane antigens are lipoproteins may explain the basis of their immunogenicity and may help to improve our understanding of the surface topology of B. burgdorferi.

Antigens, Bacterial↗
PubMed1990

Expression in Escherichia coli of the 37-kilodalton endoflagellar sheath protein of Treponema pallidum by use of the polymerase chain reaction and a T7 expression system.

We previously reported the complete primary structure of the 37-kilodalton endoflagellar sheath protein (FlaA) of Treponema pallidum. However, we were unable to determine the nucleotide sequence of flaA upstream of amino acid 10. The desired nucleotide sequence was obtained by use of a strategy based upon the polymerase chain reaction and was found to contain a consensus Escherichia coli promoter, a ribosomal binding site, and a 20-amino-acid signal peptide. Expression of FlaA in E. coli was achieved by cloning polymerase chain reaction-derived constructs lacking the native T. pallidum promoter into a temperature-inducible T7 expression system. Pulse-chase and ethanol inhibition analyses of protein processing in E. coli cells and minicells, respectively, indicated that processing of the FlaA precursor was incomplete. Native and recombinant FlaA were identical as assessed by antibody reactivity and sodium dodecyl sulfate- and two-dimensional polyacrylamide gel electrophoretic mobilities. Soluble FlaA was not detected in either the cytoplasmic or the periplasmic fractions of E. coli transformants. Fractionation of E. coli cell envelopes unexpectedly revealed that FlaA precursor and FlaA were associated with both the cytoplasmic and outer membranes. This is the first report of expression in E. coli of a T. pallidum protein which could not be cloned or expressed with its native promoter. Our data also indicate that information obtained in E. coli regarding the subcellular location of cloned treponemal proteins must be cautiously extrapolated to T. pallidum.

Amino Acid Sequence↗
PubMed1989

Outer membrane ultrastructure explains the limited antigenicity of virulent Treponema pallidum.

Freeze fracture and deep etching were used to investigate the ultrastructural basis for the observation that anti-treponemal antibodies bind poorly to the surface of virulent Treponema pallidum. Fractures of T. pallidum outer membranes contained scarce, uniformly sized intramembranous particles (IMPs). IMPs on the convex faces often appeared to form linear arrays that wound in spirals about the organism. In contrast to the outer membrane, IMPs of the cytoplasmic membrane were randomly distributed, numerous, and heterogeneous in size. In Escherichia coli and T. pallidum cofractures, IMPs of the E. coli outer membranes were densely packed within the concave fracture faces, while the T. pallidum fractures were identical to the experiments lacking the E. coli internal controls. Outer membranes of two representative nonpathogenic treponemes, Treponema phagedenis biotype Reiter and Treponema denticola, contained numerous IMPs, which segregated preferentially with the concave halves. Examination of apposed replicas and deep-etched specimens indicated that at least some of the IMPs extend through the T. pallidum outer membrane and are exposed on the surface of the organism. The outer membrane of intact T. pallidum appears to contain a paucity of integral membrane proteins that can serve as targets for specific antibodies. These findings appear to represent an unusual parasitic strategy for evasion of host humoral defenses.

Antigens, Bacterial↗
PubMed1989

Sequence analysis of the 47-kilodalton major integral membrane immunogen of Treponema pallidum.

The complete primary amino acid sequence for the 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum subsp. pallidum was obtained by using a combined strategy of DNA sequencing (of the cloned gene in Escherichia coli) and N-terminal amino acid sequencing of the native (T. pallidum subsp. pallidum-derived) antigen. An open reading frame believed to encode the 47-kDa antigen comprised 367 amino acid codons, which gave rise to a calculated molecular weight for the corresponding antigen of 40,701. Of the 367 amino acids, 113 (31%) were sequenced by N-terminal amino acid sequencing of trypsin and hydroxylamine cleavage fragments of the native molecule isolated from T. pallidum subsp. pallidum; amino acid sequence data had a 100% correlation with that of the amino acid sequence predicted from DNA sequencing of the cloned gene in E. coli. Although no consensus sequences for the initiation of transcription or translation were readily identifiable immediately 5' to the putative methionine start codon, a 63-base-pair PstI fragment located 159 nucleotides upstream was required for expression of the 47-kDa antigen in E. coli. The 47-kDa antigen sequence did not reveal a typical leader sequence. The overall G+C content for the DNA corresponding to the structural gene was 53%. Hydrophilicity analysis identified at least one major hydrophilic domain of the protein near the N terminus of the molecule which potentially represents an immunodominant epitope. No repetitive primary sequence epitopes were found. The combined data provide the molecular basis for further structural and functional studies regarding the role of the antigen in the immunopathogenesis of treponemal disease.

Amino Acid Sequence↗
PubMed1989

Structural and antigenic conservation of the P2 porin protein among strains of Haemophilus influenzae type b.

The P2 porin protein is the most abundant protein in the outer membrane of Haemophilus influenzae type b (Hib). Biochemical and immunochemical techniques were used to characterize the P2 proteins from a number of different Hib strains. P2 proteins from Hib outer membrane vesicles were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for in situ tryptic digestion. Solid-phase tryptic digests of P2 from eight Hib strains were resolved by high-pressure liquid chromatography and shown to be similar if not identical. Radioimmunoprecipitation analysis involving Hib cells (containing intrinsically radiolabeled proteins or lipooligosaccharide) and Western blot (immunoblot) analysis were used to identify two P2-specific murine monoclonal antibodies (MAbs). These MAbs were shown to be reactive with 120 Hib strains tested in a colony blot radioimmunoassay. One of these MAbs bound to a surface-exposed P2 epitope that was antibody accessible on all Hib strains tested; the other MAb was directed against a P2 epitope that either was not exposed on the cell surface or was otherwise inaccessible to antibody.

Amino Acid Sequence↗
PubMed1989

Molecular characterization of the pathogen-specific, 34-kilodalton membrane immunogen of Treponema pallidum.

The 34-kilodalton (kDa) antigen of Treponema pallidum subsp. pallidum (T. pallidum) is a pathogen-specific integral membrane protein. DNA sequence analysis of the cloned gene revealed an open reading frame encoding a primary product of 204 residues with a molecular mass of 22,087 daltons. Sequences that correspond to a consensus Escherichia coli promoter and a ribosome-binding site were found upstream from the AUG start codon that begins the open reading frame, suggesting that the cloned gene can use its own regulatory sequences for expression. Examination of the deduced amino acid sequence revealed the presence of a typical procaryotic leader peptide 19 amino acids long; processing results in a mature molecule with a molecular mass of 20,123 daltons. Pulse-chase experiments with E. coli minicells confirmed that the 34-kDa antigen is synthesized as a higher-molecular-weight precursor that is processed to a mature form with the electrophoretic mobility that is characteristic for this protein. The presence in the leader peptide of the sequence Phe-Ser-Ala-Cys suggested that the 34-kDa antigen is a proteolipid. Although hydropathy analysis of the deduced amino acid sequence of the mature 34-kDa antigen predicted that the molecule was primarily hydrophilic, both the native and recombinant 34-kDa molecules displayed hydrophobic biochemical behavior by fractionating into the detergent phase after extraction of intact organisms with Triton X-114. Cell fractionation experiments with E. coli showed that the 34-kDa molecule was localized in both the inner and outer membranes of the recombinant host. The combined data demonstrate that the 34-kDa antigen is an integral membrane protein that behaves in a biochemically consistent manner in both T. pallidum and E. coli.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum.

We have used a combination of nucleotide and N-terminal-amino-acid-sequence analyses to determine the primary structure of the 37-kilodalton (kDa) endoflagellar outer layer, or sheath, protein. Initially, a lambda gt11 clone (designated lambda A34) expressing a portion of the 37-kDa protein was selected from a Treponema pallidum genomic library with a murine monoclonal antibody (H9-2) directed against an epitope of the 37-kDa protein. The insert from lambda A34 provided a probe with which a chimeric plasmid (pR14) encoding all but the nine N-terminal amino acids of the entire protein was selected from a T. pallidum(pBR322) genomic library. The nine N-terminal amino acids determined by amino acid sequencing were combined with the DNA sequence encoded by pR14 to determine the primary structure of the entire 37-kDa protein; the combined sequence made up a polypeptide with a calculated molecular mass of 36,948 Da. Approximately one-third of the deduced sequence was confirmed by N-terminal amino acid analysis of tryptic peptides from the purified 37-kDa protein. Repeated attempts to clone upstream portions of the gene (flaA) by using a variety of strategies were unsuccessful, suggesting that unregulated expression of the intact sheath protein or of its most amino-terminal portions is toxic in Escherichia coli. These studies should provide the basis for further molecular investigations of the endoflagellar apparatus and of treponemal motility.

Amino Acid Sequence↗