RESUMO
The finding that Treponema pallidum, the syphilis spirochete, contains 12 orthologs of the Treponema denticola outer membrane major sheath protein has engendered speculation that members of this T. pallidum repeat (Tpr) family may be similarly surface exposed. In this regard, the TprK protein was reported to be a target of opsonic antibody and protective immunity and subject to immunologically driven sequence variation. Despite these findings, results from our previous analyses of treponemal outer membranes in concert with computer-based predictions for TprK prompted us to examine the cellular location of this protein. TprK-alkaline phosphatase fusions expressed in Escherichia coli demonstrate that TprK contains a signal peptide. However, opsonophagocytosis assays failed to indicate surface exposure of TprK. Moreover, results from three independent methodologies, i.e., (a) indirect immunofluorescence analysis of agarose-encapsulated organisms, (b) proteinase K treatment of intact spirochetes, and (c) Triton X-114 phase partitioning of T. pallidum conclusively demonstrated that native TprK is entirely periplasmic. Consistent with this location, immunization with the recombinant protein failed to induce either protective immunity or select for TprK variants in the rabbit model of experimental syphilis. These findings challenge the notion that TprK will be a component of an efficacious syphilis vaccine.
Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Periplasma/metabolismo , Treponema pallidum/imunologia , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , Membrana Celular/metabolismo , Códon de Iniciação , DNA Bacteriano , Endopeptidase K/metabolismo , Genes Bacterianos , Variação Genética , Dados de Sequência Molecular , Biossíntese de Proteínas , Coelhos , Homologia de Sequência de Aminoácidos , Sífilis/prevenção & controle , Transcrição Gênica , Treponema pallidum/genética , VacinaçãoRESUMO
Apoptosis is implicated in the generation and resolution of inflammation in response to bacterial pathogens. All bacterial pathogens produce lipoproteins (BLPs), which trigger the innate immune response. BLPs were found to induce apoptosis in THP-1 monocytic cells through human Toll-like receptor-2 (hTLR2). BLPs also initiated apoptosis in an epithelial cell line transfected with hTLR2. In addition, BLPs stimulated nuclear factor-kappaB, a transcriptional activator of multiple host defense genes, and activated the respiratory burst through hTLR2. Thus, hTLR2 is a molecular link between microbial products, apoptosis, and host defense mechanisms.
Assuntos
Apoptose , Proteínas de Bactérias/farmacologia , Proteínas de Drosophila , Lipoproteínas/farmacologia , Glicoproteínas de Membrana/metabolismo , Monócitos/citologia , Receptores de Superfície Celular/metabolismo , Anticorpos Monoclonais , Proteínas de Bactérias/metabolismo , Linhagem Celular/metabolismo , Cicloeximida/farmacologia , Citotoxicidade Imunológica , Genes Reporter , Humanos , Receptores de Lipopolissacarídeos/análise , Lipopolissacarídeos/imunologia , Lipoproteínas/metabolismo , Glicoproteínas de Membrana/imunologia , Monócitos/imunologia , Monócitos/metabolismo , NF-kappa B/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/imunologia , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Receptor 2 Toll-Like , Receptores Toll-Like , Transfecção , Células Tumorais CultivadasRESUMO
There is now substantial evidence that Borrelia burgdorferi, the Lyme disease spirochete, undergoes major alterations in antigenic composition as it cycles between its arthropod and mammalian hosts. In this report, we cultivated B. burgdorferi 297 within dialysis membrane chambers implanted into the peritoneal cavities of rats to induce antigenic changes similar to those which occur during mammalian infection. Chamber-grown spirochetes, which remained fully virulent, did not express either outer surface protein A or Lp6.6, lipoproteins known to be downregulated after mammalian infection. However, they did, express p21, a well characterized outer surface protein E homologue, which is selectively expressed during infection. SDS-PAGE, two-dimensional gel electrophoresis, and immunoblot analysis revealed that chamber-grown borreliae also expressed uncharacterized proteins not expressed by in vitro-cultivated spirochetes; reactivity with sera from mice chronically infected with B. burgdorferi 297 confirmed that many of these novel proteins are selectively expressed during experimental murine infection. Finally, we used differential display RT-PCR to identify transcripts of other differentially expressed B. burgdorferi genes. One gene (2.9-7lpB) identified with this technique belongs to a family of genes located on homologous 32- and 18-kb circular plasmids. The lipoprotein encoded by 2.9-7lpB was shown to be selectively expressed by chamber-grown spirochetes and by spirochetes during experimental infection. Cultivation of B. burgdorferi in rat peritoneal implants represents a novel system for studying Lyme disease spirochetes in a mammalian host-adapted state.
Assuntos
Grupo Borrelia Burgdorferi/imunologia , Doença de Lyme/fisiopatologia , Animais , Antígenos de Bactérias/análise , Antígenos de Bactérias/imunologia , Antígenos de Superfície/imunologia , Proteínas de Bactérias/análise , Grupo Borrelia Burgdorferi/patogenicidade , Diálise/métodos , Modelos Animais de Doenças , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos/genética , Lipoproteínas/metabolismo , Microscopia de Fluorescência , Cavidade Peritoneal/microbiologia , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Spirochaetales/crescimento & desenvolvimentoRESUMO
Combined ultrastructural and molecular studies have revealed that the syphilis and Lyme-disease spirochetes, Treponema pallidum and Borrelia burgdorferi, have distinctive molecular architectures. Both organisms persist in their hosts and have strategies for immune evasion that include the use of rare, poorly immunogenic surface-exposed proteins as potential virulence determinants.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Grupo Borrelia Burgdorferi/imunologia , Doença de Lyme/imunologia , Sífilis/imunologia , Treponema pallidum/imunologia , Animais , Grupo Borrelia Burgdorferi/patogenicidade , Humanos , Tolerância Imunológica , Doença de Lyme/microbiologia , Camundongos , Microscopia Eletrônica , Sífilis/microbiologia , Treponema pallidum/patogenicidadeAssuntos
Grupo Borrelia Burgdorferi , Doença de Lyme/tratamento farmacológico , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/líquido cefalorraquidiano , Grupo Borrelia Burgdorferi/imunologia , Grupo Borrelia Burgdorferi/isolamento & purificação , Doença Crônica , Ensaios Clínicos Controlados como Assunto , Diagnóstico Diferencial , Humanos , Doença de Lyme/complicações , Seleção de PacientesRESUMO
The sodC gene of Haemophilus ducreyi was cloned and sequenced. The deduced amino acid sequence of this protein exhibited 71.6% identity and 81.8% similarity to the H. influenzae and H. parainfluenzae copper (Cu), zinc (Zn)-superoxide dismutase (SOD) enzymes. This gene was localized to a 2.2-kb H. ducreyi chromosomal DNA insert in plasmid pHdSOD. SOD activity was expressed in cell-free extracts of Escherichia coli containing the recombinant plasmid pHdSOD and was localized to the periplasmic space. The Cu,Zn-SOD produced by the H. ducreyi sodC gene did not complement the aerobic growth defect of an E. coli SOD-deficient mutant.
Assuntos
Genes Bacterianos/genética , Haemophilus ducreyi/genética , Superóxido Dismutase/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Teste de Complementação Genética , Haemophilus ducreyi/enzimologia , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Superóxido Dismutase/metabolismoRESUMO
A 38-kDa lipoprotein of Treponema pallidum subsp. pallidum (T. pallidum), the syphilis spirochete, previously was identified as a putative homolog of E. coli MglB [Becker et al. (1994) Infect. Immun. 62, 1381-1391]. In the present study, genome walking in regions adjacent to the T. pallidum 38-kDa lipoprotein gene has identified three contiguous genes (tp-mglB [formerly tpp38], tp-mglA, and tp-mglC) which appear to comprise a mgl-like operon in T. pallidum. A prominent transcript corresponding to tp-mglB, the first gene of the operon which encodes the carbohydrate receptor, is synthesized by T. pallidum along with lesser abundant transcript(s) corresponding to the entire T. pallidum mgl operon. An active promoter 135 bp upstream of tp-mglB is believed to direct mRNA synthesis for the operon. This is the first membrane protein-encoding operon of T. pallidum for which a putative function (glucose import) has been assigned. Furthermore, by analogy with E. coli MglB which interacts with the sensory transducer Trg to induce a chemotactic response, it is possible that T. pallidum also contains a homolog of E. coli Trg or other methyl-accepting chemotaxis proteins. The existence of a mgl operon in T. pallidum thus may have important implications with respect to T. pallidum survival, tissue dissemination, and sensory transduction during virulence expression.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação ao Cálcio , Proteínas de Membrana/genética , Proteínas de Transporte de Monossacarídeos , Óperon , Proteínas Periplásmicas de Ligação , Treponema pallidum/genética , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/genética , DNA Bacteriano , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Família Multigênica , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
We have characterized a 5.2-kilobase (kb) putative transport related operon (tro) locus of Treponema pallidum subsp. pallidum (Nichols strain) (Tp) encoding six proteins: TroA, TroB, TroC, TroD, TroR and Phosphoglycerate mutase (Pgm). Four of these gene products (TroA-TroD) are homologous to members of the ATP-Binding Cassette (ABC) superfamily of bacterial transport proteins. TroA (previously identified as Tromp1) has significant sequence similarity to a family of Gram-negative periplasmic substrate-binding proteins and to a family of streptococcal proteins that may have dual roles as substrate binding proteins and adhesins. TroB is homologous to the ATP-binding protein component, whereas TroC and TroD are related to the hydrophobic membrane protein components of ABC transport systems. TroR is similar to Gram-positive iron-activated repressor proteins (DesR, DtxR, IdeR, and SirR). The last open reading frame (ORF) of the tro operon encodes a protein that is highly homologous to the glycolytic pathway enzyme, Pgm. Primer extension results demonstrated that the tro operon is transcribed from a sigma 70-type promoter element. Northern analysis and reverse transcriptase-polymerase chain reactions provided evidence for the presence of a primary 1-kb troA transcript and a secondary, less abundant, troA-pgm transcript. The tro operon is flanked by a Holliday structure DNA helicase homolog (upstream) and two ORFs representing a purine nucleoside phosphorylase homolog and tpp15, a previously characterized gene encoding a membrane lipoprotein (downstream). The presence of a complex operon containing a putative ABC transport system and a DtxR homolog indicates a possible linkage between transport and gene regulation in Tp.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Óperon/genética , Proteínas Periplásmicas de Ligação , Fosfoglicerato Mutase/genética , Proteínas Repressoras/genética , Treponema pallidum/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Genes Bacterianos/genética , Ferro/farmacologia , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , RNA Bacteriano/análise , RNA Mensageiro/análise , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
A murine monoclonal antibody (MAb) directed against a surface-exposed epitope of the lipooligosaccharide (LOS) of Haemophilus ducreyi strain 35000 was shown to be reactive with all 37 strains of this pathogen tested in a colony blot-radioimmunoassay. The LOS epitope bound by this MAb appeared to be stably expressed by H. ducreyi growing in vitro. The use of this MAb in the immunolimulus system revealed that it could detect purified H. ducreyi LOS at a level of 25 pg/ml. Similarly, this immunolimulus system could detect as few as 1000 colony forming units of in vitro-grown H. ducreyi cells per ml of buffer. When this MAb was utilized in the immunolimulus system together with lesion material from rabbits infected with two different H. ducreyi strains, a positive reaction was obtained with every sample tested, even when no viable organisms were present in the lesion material. In contrast, this MAb yielded consistently negative results when used in the immunolimulus system with lesion material from animals infected with Staphylococcus aureus.
Assuntos
Anticorpos Monoclonais , Antígenos de Bactérias/análise , Cancroide/diagnóstico , Haemophilus ducreyi/imunologia , Lipopolissacarídeos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Feminino , Testes Imunológicos , Teste do Limulus , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , Especificidade da EspécieRESUMO
Brucellosis is a highly pleomorphic zoonotic infection caused by one of the following four species of gram-negative facultative intracellular coccobacilli: Brucella melitensis, B. abortus, B. suis, or B. canis. The disease is a worldwide public health problem and a significant cause of economic losses in domestic live-stock. Although largely eradicated in most industrialized countries, in the United States there has been an upsurge of B. melitensis cases associated with the ingestion of unpasteurized goat's milk or goat's milk cheese from Mexico. Brucellosis can be either insidious or abrupt in onset and can affect virtually every organ system; skeletal involvement (spondylitis, arthritis) is the most frequent metastatic complication. Cases are diagnosed either by isolation of the bacterium (usually from blood) or by serologic testing. Treatment of brucellosis requires the administration of two antimicrobial agents. Doxycycline plus streptomycin or rifampin or trimethoprim-sulfamethoxazole plus rifampin appear to be the most effective regimens.
Assuntos
Brucella melitensis , Brucelose/terapia , Animais , Brucelose/complicações , Brucelose/microbiologia , HumanosRESUMO
A 36-kDa immunosuppressant protein (Da-p36) was isolated from salivary glands of feeding female ixodid ticks Dermacentor andersoni, using its affinity for UltraLink Biosupport Medium (Pierce, Rockford, Illinois)/protein complexes. Using a nested set of forward degenerate oligonucleotide primers corresponding to Da-p36 N-terminal amino acids, a cDNA encoding the immunosuppressant protein was isolated by 3' rapid amplification of cDNA ends. The resulting 772-base pair cDNA encodes a novel protein with predicted molecular weight of 24.9 kDa. Sequence analysis revealed the presence of 5 potential glycosylation sites and 1 myristylation site. Immunoblot analyses showed native Da-p36 is present in salivary glands and saliva from both male and female D. andersoni but not in salivary glands or saliva from Amblyomma americanum or Ixodes scapularis. Reverse transcription polymerase chain reaction and immunoblot analyses showed that Da-p36 expression is temporally regulated in salivary glands with maximum mRNA levels preceding maximum Da-p36 accumulation that occurred at day 6 of feeding. The levels of Da-p36 mRNA and protein were greatly reduced in salivary glands from near-replete females removed from sheep after 8 days of feeding. These data are consistent with a role of Da-p36 in immunosuppression during feeding.
Assuntos
Dermacentor/química , Imunossupressores/isolamento & purificação , Proteínas e Peptídeos Salivares/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA Complementar/química , DNA Complementar/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Feminino , Immunoblotting , Imunoglobulina G/imunologia , Imunossupressores/química , Imunossupressores/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , RNA Mensageiro/metabolismo , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândulas Salivares/química , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/imunologia , OvinosAssuntos
Doença de Lyme , Ensaios Clínicos Controlados Aleatórios como Assunto , Amoxicilina/administração & dosagem , Antibacterianos/administração & dosagem , Fatores de Confusão Epidemiológicos , Método Duplo-Cego , Humanos , Doença de Lyme/diagnóstico , Doença de Lyme/tratamento farmacológico , Seleção de Pacientes , Projetos de Pesquisa/normasAssuntos
Proteínas de Bactérias/isolamento & purificação , Detergentes , Proteínas de Membrana/isolamento & purificação , Polietilenoglicóis , Antígenos de Bactérias/análise , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/análise , Cromatografia/métodos , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Immunoblotting , Indicadores e Reagentes , Proteínas de Membrana/análise , Octoxinol , Treponema pallidum/químicaAssuntos
Aneurisma Aórtico , Sífilis Cardiovascular , Idoso , Idoso de 80 Anos ou mais , Aneurisma Infectado , Aorta Torácica , Feminino , HumanosRESUMO
Treponema pallidum, the syphilis spirochaete, has a remarkable ability to evade the humoral and cellular responses it elicits in infected hosts. Although formerly attributed to the presence of an outer coat comprised of serum proteins and/or mucopolysaccharides, current evidence indicates that the immuno-evasiveness of this bacterium is largely the result of its unusual molecular architecture. Based upon a combination of molecular, biochemical, and ultrastructural data, it is now believed that the T. pallidum outer membrane (OM) contains a paucity of poorly immunogenic transmembrane proteins ('rare outer membrane proteins') and that its highly immunogenic proteins are lipoproteins anchored predominantly to the periplasmic leaflet of the cytoplasmic membrane. The presence in the T. pallidum OM of a limited number of transmembrane proteins has profound implications for understanding syphilis pathogenesis as well as treponemal physiology. Two major strategies for molecular characterization of rare outer membrane proteins have evolved. The first involves the identification of candidate OM proteins as fusions with Escherichia coli alkaline phosphatase. The second involves the characterization of candidate OM proteins identified in outer membranes isolated from virulent T. pallidum. Criteria to define candidate OM proteins and for definitive identification of rare OM proteins are proposed as a guide for future studies.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Treponema pallidum/química , Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Antígenos de Superfície/química , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Humanos , Proteínas Recombinantes de Fusão , Sífilis/imunologia , Sífilis/microbiologia , Treponema pallidum/imunologia , Treponema pallidum/patogenicidade , Treponema pallidum/ultraestrutura , VirulênciaRESUMO
The antigenically conserved proteins of Treponema pallidum subsp. pallidum and four nonpathogenic cultivatable treponemes were investigated by phase partitioning with the nonionic detergent Triton X-114 and immunoblot analysis. None of the T. pallidum integral membrane proteins identified by phase partitioning (detergent-phase proteins) appeared to be antigenically related to proteins of the nonpathogens. Protease-resistant material similar to lipopolysaccharide was identified in the detergent phase from T. phagedenis biotype Reiter but was not detected in T. pallidum.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Detergentes , Proteínas de Membrana/isolamento & purificação , Polietilenoglicóis , Tensoativos , Treponema pallidum/patogenicidade , Animais , Centrifugação , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoensaio , Masculino , Proteínas de Membrana/imunologia , Octoxinol , Coelhos , Treponema pallidum/análise , Treponema pallidum/imunologiaRESUMO
The case presented here involves a 32-year-old homosexual man with human immunodeficiency virus (HIV) seropositivity and unusual manifestations of secondary syphilis. The patient presented with syphilitic keratoderma and chorioretinitis, and his appearance superficially resembled that of a patient with Reiter's syndrome. Although nontreponemal and treponemal tests for syphilis showed reactivity, the patient's humoral immune response to individual polypeptides of Treponema pallidum, measured by Western blot analysis, was markedly abnormal. The possible relationship between asymptomatic HIV infection and an abnormal humoral immune response to a second pathogen, in this case T. pallidum, is discussed. Our case is one of several recent cases of active syphilis reported in individuals with HIV seropositivity.
Assuntos
Soropositividade para HIV/complicações , Sífilis/complicações , Adulto , Anticorpos Antibacterianos/biossíntese , Artrite Reativa/patologia , Coriorretinite/tratamento farmacológico , Coriorretinite/patologia , Quimioterapia Combinada , Soropositividade para HIV/imunologia , Humanos , Ceratodermia Palmar e Plantar/tratamento farmacológico , Ceratodermia Palmar e Plantar/patologia , Masculino , Penicilina G/uso terapêutico , Prednisona/uso terapêutico , Sífilis/diagnóstico , Sífilis/tratamento farmacológico , Treponema pallidum/imunologiaRESUMO
Numerous case reports of atypical and/or severe forms of syphilis in individuals coinfected with human immunodeficiency virus (HIV) have led many authorities to conclude that HIV exacerbates early syphilitic infection. Herein we report prominent osseous and unusual dermatologic manifestations of early syphilis in two individuals whose serostatuses for HIV infection were discordant. Our cases emphasize the need for caution before conclusions are drawn from anecdotal data about the interactions between HIV infection and syphilis.
Assuntos
Soronegatividade para HIV , Soropositividade para HIV/complicações , Osteíte/microbiologia , Sífilis/fisiopatologia , Adulto , Feminino , Humanos , Osteíte/diagnóstico , Sífilis/diagnóstico , Sífilis/virologia , Sífilis Cutânea/fisiopatologia , Sífilis Cutânea/virologiaRESUMO
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.