RESUMO
Borrelia miyamotoi is a relapsing fever Borrelia group spirochete that is transmitted by the same hard-bodied (ixodid) tick species that transmit the agents of Lyme disease. It was discovered in 1994 in Ixodes persulcatus ticks in Japan. B. miyamotoi species phylogenetically cluster with the relapsing fever group spirochetes, which usually are transmitted by soft-bodied (argasid) ticks or lice. B. miyamotoi infects at least six Ixodes tick species in North America and Eurasia that transmit Lyme disease group spirochetes and may use small rodents and birds as reservoirs. Human cases of B. miyamotoi infection were first reported in 2011 in Russia and subsequently in the United States, Europe and Japan. These reports document the public health importance of B. miyamotoi, as human B. miyamotoi infection appears to be comparable in frequency to babesiosis or human granulocytic anaplasmosis in some areas and may cause severe disease, including meningoencephalitis. The most common clinical manifestations of B. miyamotoi infection are fever, fatigue, headache, chills, myalgia, arthralgia, and nausea. Symptoms of B. miyamotoi infection generally resolve within a week of the start of antibiotic therapy. B. miyamotoi infection should be considered in patients with acute febrile illness who have been exposed to Ixodes ticks in a region where Lyme disease occurs. Because clinical manifestations are nonspecific, etiologic diagnosis requires confirmation by blood smear examination, PCR, antibody assay, in vitro cultivation, and/or isolation by animal inoculation. Antibiotics that have been used effectively include doxycycline for uncomplicated B. miyamotoi infection in adults and ceftriaxone or penicillin G for meningoencephalitis.
Assuntos
Borrelia/isolamento & purificação , Febre Recorrente/epidemiologia , Febre Recorrente/patologia , Doenças Transmitidas por Carrapatos/epidemiologia , Doenças Transmitidas por Carrapatos/patologia , Animais , Antibacterianos/uso terapêutico , Vetores Artrópodes , Aves , Técnicas de Laboratório Clínico , Reservatórios de Doenças , Europa (Continente)/epidemiologia , Humanos , Ixodes/microbiologia , Japão/epidemiologia , Febre Recorrente/tratamento farmacológico , Febre Recorrente/transmissão , Roedores , Doenças Transmitidas por Carrapatos/tratamento farmacológico , Doenças Transmitidas por Carrapatos/transmissão , Estados Unidos/epidemiologiaRESUMO
Some copies of the large subunit rRNA genes (LSU rDNA) of most arthropods studied to date are inactivated by R-element retrotransposons at a specific target region that is highly conserved in sequence across all kingdoms of organisms. Here we report finding R2 elements in low copy numbers in the LSU rDNA of hard and soft ticks. Although the elements were inserted at the same LSU rDNA location as in insects, there were substitutions in the consensus R2 endonuclease cleavage site in the ticks and some other parasitiform mites. The substituted region comprises a critical contact point with small subunit rRNA, but in vitro structure probing analysis revealed novel, presumably stabilizing base-pairing.
Assuntos
DNA Ribossômico/genética , Ácaros/genética , Retroelementos/genética , Carrapatos/genética , Animais , Sequência de Bases , DNA Ribossômico/química , Filogenia , Homologia de Sequência do Ácido NucleicoRESUMO
The relapsing fever spirochete, Borrelia hermsii, escapes immune selection by alternating expression of surface lipoprotein alleles. The switch results from a duplicative transposition of one of several surface lipoprotein-encoding nucleotide sequences into the singular expression site. These nucleotide sequences constitute a large gene family whose diversity originated, in some cases, before the major divergences of Borrelia species. We have examined the B. hermsii vsp subfamily of alleles, which are carried on linear plasmids within each cell and maintained in several diverse copies as an antigenic archive. Each encodes a distinct serotype-specific protein. We sequenced more than 90% of the alleles within a single strain-B. hermsii strain HS1. A preponderance of allelic mosaicism suggests that intragenic recombination, coupled with selection imposed by host immune response, has driven diversification of the archived ensemble of vsp alleles. The recombinational diversification of vsp alleles generates change in the associated serotypes of the magnitude (30-40% amino acid differentiation) necessary for overcoming cross-reactivity of neutralizing antibodies. We conclude that evolution of vsp has occurred by punctuated occurrence of allelic differentiation, rather than by gradual selection of incremental point mutations that do not meet the threshold for antigenic diversity.
Assuntos
Antígenos de Bactérias/genética , Borrelia/imunologia , Polimorfismo Genético , Sequência de Bases , DNA Bacteriano , Sequências Repetitivas de Ácido NucleicoRESUMO
Studying tick feeding and digestion, we discovered in a cDNA library from partially fed Amblyomma americanum ticks the first known arthropod homologue of a human cytokine, the pro-inflammatory Macrophage Migration Inhibitory Factor (MIF). The tick origin of the MIF cDNA clone was confirmed by sequencing a genomic fragment that contained the full-length tick MIF gene with two introns. Antiserum to a tick MIF-specific peptide as well as antiserum to complete tick MIF revealed the expression of tick MIF in the salivary gland and midgut tissues of A. americanum ticks. In an in vitro functional assay, recombinant tick MIF inhibited the migration of human macrophages to the same extent that recombinant human MIF did.
Assuntos
Citocinas/genética , Fatores Inibidores da Migração de Macrófagos/genética , Carrapatos/genética , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Proteínas de Artrópodes , Sequência de Bases , Citocinas/classificação , Citocinas/isolamento & purificação , Citocinas/fisiologia , DNA Complementar , Feminino , Expressão Gênica , Humanos , Fatores Inibidores da Migração de Macrófagos/classificação , Fatores Inibidores da Migração de Macrófagos/isolamento & purificação , Fatores Inibidores da Migração de Macrófagos/fisiologia , Macrófagos/fisiologia , Dados de Sequência Molecular , Coelhos , Homologia de Sequência de Aminoácidos , Carrapatos/imunologia , Distribuição TecidualRESUMO
Mice with severe combined immunodeficiency (scid mice) and infected with the relapsing fever agent Borrelia turicatae develop manifestations that resemble those of disseminated Lyme disease. We have characterized two isogenic serotypes, A and B, which differ in their variable small proteins (Vsps) and disease manifestations. Serotype A but not serotype B was cultured from the brain during early infection, and serotype B caused more severe arthritis, myocarditis, and vestibular dysfunction than serotype A. Here we compared the localization and number of spirochetes and the severity of inflammation in scid mice, using immunostained and hematoxylin-and-eosin-stained coronal sections of decalcified heads. Spirochetes in the brain localized predominantly to the leptomeninges, and those in peripheral tissues localized mainly to the extracellular matrix. There were significantly more serotype A than B spirochetes in the leptomeninges and more serotype B than A spirochetes in the skin. The first tissue where spirochetes were observed outside the vasculature was the dura mater. Inflammation was more severe in the skin than in the brain. VspA, VspB, and the periplasmic flagellin protein were expressed in all tissues examined. These findings indicate that isogenic but antigenically distinct Borrelia serotypes can have marked differences in their localization in tissues.
Assuntos
Infecções por Borrelia/microbiologia , Borrelia/isolamento & purificação , Encéfalo/microbiologia , Pele/microbiologia , Animais , Proteínas de Bactérias/análise , Borrelia/classificação , Feminino , Inflamação/etiologia , Camundongos , Camundongos SCID , Sorotipagem , Medula Espinal/microbiologiaRESUMO
Genetic transformation of Borrelia spp. is limited in development and has found application in only one species. For a non-genetic approach for manipulating the phenotype of these spirochaetes, we determined whether exogenous recombinant lipoproteins would incorporate in the cell's outer membrane. Using unlabelled or 125I-labelled Osp proteins, Osp-specific monoclonal antibodies, proteinase K and formaldehyde as reagents, we found that decoration of spirochaetes had the following characteristics. (i) Purified recombinant OspA or OspD lipoproteins associated with Borrelia burgdorferi and B. hermsii cells that lacked abundant lipoproteins of their own. (ii) This decoration of the cells with exogenous OspA did not affect cell's viability. (iii) The decoration was concentration and temperature dependent and stable for at least 24 h. (iv) Like native OspA, the recombinant OspA decorating the cells was accessible to antibodies and proteases and could be cross-linked to the integral outer membrane protein, P66. (v) Decoration of viable B. burgdorferi and B. hermsii with OspA rendered the cells susceptible to killing by OspA-specific antiserum. Such non-genetic alteration of the surface of a bacterium may be used to study functions and properties of lipoproteins in situ.
Assuntos
Antígenos de Superfície/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Borrelia/classificação , Borrelia/metabolismo , Membrana Celular/imunologia , Membrana Celular/metabolismo , Lipoproteínas , Vacinas contra Doença de Lyme/metabolismo , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Superfície/genética , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas , Borrelia/crescimento & desenvolvimento , Borrelia/imunologia , Grupo Borrelia Burgdorferi/classificação , Grupo Borrelia Burgdorferi/crescimento & desenvolvimento , Grupo Borrelia Burgdorferi/imunologia , Grupo Borrelia Burgdorferi/metabolismo , Reagentes de Ligações Cruzadas , Endopeptidase K/metabolismo , Formaldeído , Vacinas contra Doença de Lyme/genética , Fenótipo , Propriedades de SuperfícieRESUMO
The antigenic variation of the relapsing fever agent Borrelia hermsii is associated with changes in the expression of the Vlp and Vsp outer membrane lipoproteins. To investigate whether these serotype-defining proteins are the target of a neutralizing and protective antibody response, monoclonal antibodies were produced from spleens of infected mice just after clearance of serotype 7 cells from the blood. Two immunoglobulin M monoclonal antibodies, H7-7 and H7-12, were studied in detail. Both antibodies specifically agglutinated serotype 7 cells and inhibited their growth in vitro. Administered to mice before or after infection, both antibodies provided protection against infection or substantially reduced the number of spirochetes in the blood of mice after infection. Whereas antibody H7-12 bound to Vlp7 in Western blotting, enzyme-linked immunosorbent assay, and immunoprecipitation assays, as well as to whole cells in other immunoassays, antibody H7-7 only bound to wet, intact cells of serotype 7. Antibody H7-7 selected against cells expressing Vlp7 in vitro and in vivo, an indication that Vlp7 was a conformation-sensitive antigen for the antibody. Vaccination of mice with recombinant Vlp7 with adjuvant elicited antibodies that bound to fixed whole cells of serotype 7 and to Vlp7 in Western blots, but these antibodies did not inhibit the growth of serotype 7 in vitro and did not provide protection against an infectious challenge with serotype 7. The study established that a Vlp protein was the target of a neutralizing antibody response, and it also indicated that the conformation and/or the native topology of Vlp were important for eliciting that immunity.
Assuntos
Anticorpos Antibacterianos/imunologia , Borrelia/imunologia , Imunoglobulina M/imunologia , Febre Recorrente/microbiologia , Testes de Aglutinação , Animais , Anticorpos Monoclonais/imunologia , Western Blotting , Feminino , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Testes de Neutralização , Testes de Precipitina , Sorotipagem , Linfócitos T/imunologiaRESUMO
Vsp surface lipoproteins are serotype-defining antigens of relapsing fever spirochetes that undergo multiphasic antigenic variation to avoid the immune response. One of these proteins, VspA of Borrelia turicatae, is also associated with neurotropism in infected mice. Vsp proteins are highly polymorphic in sequence, which may relate to their specific antibody reactivities and host cell interactions. To determine whether sequence variations affect protein structure, we compared B. turicatae VspA with three related proteins: VspB of B. turicatae, Vsp26 of the relapsing fever agent Borrelia hermsii, and OspC of the Lyme disease spirochete Borrelia burgdorferi. Recombinant non-lipidated proteins were purified by affinity or ion exchange chromatography. Circular dichroism spectra revealed similar, highly alpha-helical secondary structures for all four proteins. In vitro assays demonstrated protease-resistant, thermostable Vsp cores starting at a conserved serine at position 34 (Ser(34)). All proteins aggregate as dimers in solution. In situ trypsin treatment and surface protein cross-linking showed that the native lipoproteins also form protease-resistant dimers. These findings indicate that Vsp proteins have a common compact fold and that their established functions are based on localized polymorphisms. Two forms of VspA crystals suitable for structure determination by x-ray diffraction methods have been obtained.
Assuntos
Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/química , Borrelia/química , Lipoproteínas/química , Sequência de Aminoácidos , Antígenos de Bactérias/isolamento & purificação , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Western Blotting , Borrelia/classificação , Borrelia/imunologia , Cromatografia em Gel , Dicroísmo Circular , Cristalização , Cristalografia por Raios X , Dimerização , Endopeptidases/metabolismo , Evolução Molecular , Lipoproteínas/isolamento & purificação , Lipoproteínas/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , SoluçõesRESUMO
Recombinant outer surface protein A (OspA) vaccination of wild animal reservoirs has potential application for reducing Borrelia burgdorferi transmission in nature and subsequent risk of human infection. As a major reservoir host, the white-footed mouse (Peromyscus leucopus) is a candidate for a vaccination program designed to reduce infection prevalence in vector ticks. In this study we characterized the effect of various levels of immunization with recombinant OspA-glutathione transferase fusion protein on transmission dynamics from infected P. leucopus to larval ticks. Control mice were vaccinated with glutathione transferase alone. All mice were experimentally infected with B. burgdorferi before vaccination. The immune responses of the immunized mice were assessed by enzyme-linked immunosorbent assay for antibodies to OspA. Transmission of B. burgdorferi from infected mice was determined by xenodiagnosis with uninfected larval ticks. Spirochetes in ticks were counted by direct immunofluorescence assay. The concentration of antibody to OspA increased with each OspA vaccination but most markedly after the first and second vaccinations. In comparison with control mice, there was reduced transmission by OspA-vaccinated mice to uninfected ticks. One, two, or three doses of OspA reduced infection prevalence in xenodiagnostic ticks by 48%, 92%, or 99% and the numbers of spirochetes per tick by 84%, 98%, or 99%, respectively. This study suggests that vaccination of P. leucopus with OspA could reduce transmission to the tick vector in nature despite prior infection of the reservoir host.
Assuntos
Antígenos de Superfície/imunologia , Vetores Aracnídeos/microbiologia , Proteínas da Membrana Bacteriana Externa/imunologia , Borrelia burgdorferi/imunologia , Ixodes/microbiologia , Lipoproteínas , Doença de Lyme/prevenção & controle , Peromyscus/microbiologia , Animais , Animais Selvagens , Anticorpos Antibacterianos/imunologia , Antígenos de Superfície/administração & dosagem , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Vacinas Bacterianas , Reservatórios de Doenças/veterinária , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática/veterinária , Técnica Direta de Fluorescência para Anticorpo , Interações Hospedeiro-Parasita , Larva/microbiologia , Doença de Lyme/transmissãoRESUMO
Borrelia hermsii, an agent of relapsing fever, undergoes antigenic variation of serotype-specifying membrane proteins during mammalian infections. When B. hermsii is cultivated in broth medium, one serotype, 33, eventually predominates in the population. Serotype 33 has also been found to be dominant in ticks but not in mammalian hosts. We investigated the biology and genetics of two independently derived clonal populations of serotype 33 of B. hermsii. Both isolates infected immunodeficient mice, but serotype 33 cells were limited in number and were only transiently present in the blood. Probes for vsp33, which encodes the serotype-specifying Vsp33 outer membrane protein, revealed that the gene was located on a 53-kb linear plasmid and that there was only one locus for the gene in serotype 33. The vsp33 probe and probes for other variable membrane protein genes showed that expression of Vsp33 was determined at the level of transcription and that when the vsp33 expression site was active, an expression site for other variable proteins was silent. The study confirmed that serotype 33 is distinct from other serotypes of B. hermsii in its biology and demonstrated that B. hermsii can change its major surface protein through switching between two expression sites.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Borrelia/genética , Febre Recorrente/microbiologia , Animais , Escherichia coli/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Regiões Promotoras Genéticas , Telômero , Transcrição GênicaRESUMO
Several pathogens of humans and domestic animals depend on hematophagous arthropods to transmit them from one vertebrate reservoir host to another and maintain them in an environment. These pathogens use antigenic variation to prolong their circulation in the blood and thus increase the likelihood of transmission. By convergent evolution, bacterial and protozoal vector-borne pathogens have acquired similar genetic mechanisms for successful antigenic variation. Borrelia spp. and Anaplasma marginale (among bacteria) and African trypanosomes, Plasmodium falciparum, and Babesia bovis (among parasites) are examples of pathogens using these mechanisms. Antigenic variation poses a challenge in the development of vaccines against vector-borne pathogens.
Assuntos
Anaplasma , Variação Antigênica , Babesia bovis , Infecções Bacterianas/transmissão , Borrelia , Vetores de Doenças , Doenças Parasitárias/transmissão , Plasmodium falciparum , Anaplasma/genética , Anaplasma/patogenicidade , Animais , Variação Antigênica/genética , Variação Antigênica/imunologia , Babesia bovis/genética , Babesia bovis/patogenicidade , Infecções Bacterianas/imunologia , Borrelia/genética , Borrelia/patogenicidade , Humanos , Doenças Parasitárias/imunologia , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidadeRESUMO
Tick-borne relapsing fever, caused by pathogenic Borrelia such as B. hermsii and B. turicatae, features recurrent episodes of bacteraemia, each of which is caused by a population of spirochaetes that expresses a different variable major protein. Relapsing fever is also associated with the infection of a variety of tissues, such as the central nervous system. In this study, we show that glycosaminoglycans (GAGs) mediate the attachment of relapsing fever spirochaetes to mammalian cells. B. hermsii strain DAH bound to immobilized heparin, and heparin and dermatan sulphate blocked bacterial binding to host cells. Bacterial binding was diminished by inhibition of host cell GAG synthesis or sulphation, or by the enzymatic removal of GAGs. GAGs mediated the attachment of relapsing fever spirochaetes to potentially relevant target cells, such as endothelial and glial cells. B. hermsii was able to attach to GAGs independently of variable major proteins, because strains expressing the variable major proteins Vsp33, Vlp7 or no variable major protein at all each recognized GAGs. Nevertheless, we found that a variable major protein of B. turicatae directly promoted GAG binding by this relapsing fever spirochaete. B. turicatae strain Oz1 serotype B, which expresses the variable major protein VspB, bound to GAGs more efficiently than did B. turicatae Oz1 serotype A, which expresses VspA. Recombinant VspB, but not VspA, bound to heparin and dermatan sulphate. Previous studies have shown that strain Oz1 serotype B grows to higher concentrations in the blood than does Oz1 serotype A. Thus, relapsing fever spirochaetes have the potential to express Vsp-dependent and Vsp-independent GAG-binding activities and, for one pair of highly related B. turicatae strains, differences in GAG binding correlate with differences in tissue tropism.
Assuntos
Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/metabolismo , Borrelia/metabolismo , Glicosaminoglicanos/metabolismo , Animais , Antígenos de Bactérias , Borrelia/fisiologia , Células CHO , Linhagem Celular , Sulfatos de Condroitina/metabolismo , Cricetinae , Heparina/metabolismo , Humanos , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Febre Recorrente/microbiologia , Células Tumorais CultivadasRESUMO
The free-living spirochete Spirochaeta aurantia was nearly as susceptible to diacetyl chloramphenicol, the product of chloramphenicol acetyltransferase, as it was to chloramphenicol itself. This unexpected susceptibility to diacetyl chloramphenicol was wholly or partly the consequence of intrinsic carboxylesterase activity, as indicated by high-performance liquid chromatography, thin-layer chromatography, and microbiological assays. The esterase converted the diacetate to chloramphenicol, thus inhibiting spirochete growth. The esterase activity was cell associated, reduced by proteinase K, eliminated by boiling, and independent of the presence of either chloramphenicol or diacetyl chloramphenicol. S. aurantia extracts also hydrolyzed other esterase substrates, and two of these, alpha-napthyl acetate and 4-methylumbelliferyl acetate, identified an esterase of approximately 75 kDa in a nondenaturing gel. Carboxylesterases occur in Streptomyces species, but in this study their activity was weaker than that of S. aurantia. The S. aurantia esterase could reduce the effectiveness of cat as either a selectable marker or a reporter gene in this species.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Cloranfenicol/análogos & derivados , Spirochaeta/enzimologia , Hidrolases de Éster Carboxílico/química , Cloranfenicol/metabolismo , Cloranfenicol/farmacologia , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Endopeptidase K/metabolismo , Escherichia coli/citologia , Escherichia coli/enzimologia , Temperatura Alta , Hidrólise , Cinética , Testes de Sensibilidade Microbiana , Peso Molecular , Spirochaeta/citologia , Spirochaeta/efeitos dos fármacos , Spirochaeta/crescimento & desenvolvimento , Streptomyces/citologia , Streptomyces/enzimologia , Especificidade por SubstratoRESUMO
Serial serum samples from a 2-year human trial of outer surface protein (Osp) A vaccine were analyzed by Borrelia burgdorferi growth-inhibition assay (GIA) and anti-OspA ELISA to assess the antibody responses of vaccine recipients and subjects with Lyme disease. Although 74% of OspA recipients had a reciprocal GIA titer >/=64 after 3 vaccinations, none of the placebo recipients, even those with Lyme disease, had a GIA titer this high. The correlation between GIA and ELISA titers after 3 doses of vaccine was.84; however, more vaccine recipients had an elevated ELISA titer paired with low GIA titer than had a low ELISA titer with a high GIA titer. OspA-vaccine recipients who acquired Lyme disease had significantly lower serum GIA and ELISA titers after 3 immunizations than did age- and sex-matched OspA recipients without Lyme disease. Thus, vaccinated subjects had antibodies to native antigen on viable cells, and antibody assays with this specificity may predict protection of vaccinees against infection.
Assuntos
Anticorpos Antibacterianos/biossíntese , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Grupo Borrelia Burgdorferi/imunologia , Doença de Lyme/imunologia , Vacinas Sintéticas/imunologia , Idoso , Grupo Borrelia Burgdorferi/crescimento & desenvolvimento , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , VacinaçãoRESUMO
In the two years after publication of the genome sequence of Borrelia burgdorferi and reports on human field trials of a vaccine against Lyme borreliosis, there has been further progress in understanding of host-parasite interactions during Lyme borreliosis and relapsing fever. Some mechanisms that Borrelia spirochetes use to avoid elimination and to persist in the host are novel. In addition, the recent discovery of antigenic variation in the Lyme disease agent B. burgdorferi adds to the complexity of the possible virulence properties of this human pathogen.
Assuntos
Infecções por Borrelia/microbiologia , Borrelia/patogenicidade , Animais , Vacinas Bacterianas , Borrelia/genética , Borrelia/imunologia , Infecções por Borrelia/imunologia , Infecções por Borrelia/patologia , Genoma Bacteriano , Humanos , Camundongos , VirulênciaRESUMO
The Lyme disease spirochete Borrelia burgdorferi expresses diverse subsurface yet antigenically cross-reactive Bdr protein paralogs from distinct circular- and linear-plasmid loci. We assessed the possible effects of in vitro and in vivo growth on bdr locus structure, searching for recombinational events leading to either deletions or insertions of central repeat units or novel amino- and carboxy-terminus combinations. Our data indicate that, apart from plasmid loss during in vitro cultivation, the bdr paralog loci of strain B31 are stable. This suggests that recombinatorial variation of bdr genes is not essential for persistent mammalian infection.
Assuntos
Proteínas de Bactérias/genética , Grupo Borrelia Burgdorferi/genética , Mapeamento Cromossômico , Animais , Grupo Borrelia Burgdorferi/classificação , Grupo Borrelia Burgdorferi/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C3H , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Sequências de Repetição em TandemRESUMO
The passage of immunoglobulins in the blood meal into hemolymph prompts development of vaccines against internal antigens of ticks, but little is known about kinetics and specificity of the immunoglobulin uptake. We used capillary feeding of adult Amblyomma americanum hard ticks to introduce compounds into the midgut and then examined the hemolymph after various times for their presence and concentration. Immunoglobulins of different sources, albumin, choramphenicol acetyltransferase, inulin, and mannitol were labeled with (125)I, (14)C, or biotin. With the exception of the carbohydrate inulin, all the compounds entered the hemolymph of tick during capillary feeding. The small molecule mannitol had the highest rate of entry at 9% after 6 h. Among proteins, the entry of immunoglobulin G (IgG) of different species into the hemolymph was greater at 6% after 6 h than for the smaller proteins albumin or choramphenicol acetyltransferase at 1 and 3%, respectively. The entry of denatured IgG was equal to that of nondenatured protein. There was no evidence of degradation of the IgG or of its binding to cells once it entered the hemolymph. A monoclonal IgG antibody labeled with biotin entered the hemolymph and retained its ability to bind to its specific antigen in an immunoassay. Although different proteins entered the hemolymph after capillary feeding, there was evidence of a specific mechanism for immunoglobulin uptake.
Assuntos
Vetores Aracnídeos/imunologia , Hemolinfa/metabolismo , Imunoglobulinas/metabolismo , Carrapatos/imunologia , Animais , Ingestão de Alimentos/fisiologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Hemolinfa/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Inulina/metabolismo , Manitol/metabolismoRESUMO
The relapsing fever agent Borrelia turicatae has two antigenically distinct serotypes, A and B, which differ in their variable small proteins (Vsps) and in their degree of virulence and neurotropism in mice. Each Vsp gene (vspA or vspB) had an expression-linked copy that was unique to the serotype expressing it. This was located on one linear plasmid, which was defined by the upstream sequence. The archived copies of vspA and vspB were each located on different linear plasmids that were the same in both serotypes. In this feature, the mechanism of antigenic variation is similar to that of another relapsing fever agent, B. hermsii. However, in other features, the mechanisms of the two organisms differ. The expressed and archived loci for vspA and vspB of B. turicatae were near the centre of linear plasmids instead of near the telomeres. The vspA and vspB expression loci were duplicate copies of their respective silent loci: from the vsp itself to at least 13-14 kb downstream. Despite the extensive interplasmidic duplications and the internal position of the expression locus, the only detectable difference between serotypes A and B was in whether they expressed VspA or VspB.
Assuntos
Borrelia/classificação , Borrelia/patogenicidade , Genes Duplicados , Lipoproteínas/genética , Proteínas de Membrana/genética , Proteínas de Plantas , Plasmídeos/genética , Febre Recorrente/microbiologia , Animais , Antígenos de Bactérias , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Southern Blotting , Borrelia/genética , Clonagem Molecular , Eletroforese em Gel Bidimensional , Genes Bacterianos , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fenótipo , Mapeamento Físico do Cromossomo , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Sorotipagem , Transcrição Gênica , VirulênciaRESUMO
Serotypes A and B of the relapsing fever spirochete Borrelia turicatae produce different disease manifestations in infected mice. Whereas serotype B causes more severe arthritis and reaches higher densities in the blood of mice than serotype A, serotype A invades the central nervous system earlier than serotype B during infection. These differences between serotypes A and B in mice are associated with the expression of different surface proteins, VspA and VspB, respectively, in the culture medium. To determine whether these proteins, in particular, VspB, are also expressed in vivo, scid mice infected with B. turicatae were studied. The expression of VspB by spirochetes in the blood was demonstrated in Coomassie blue-stained polyacrylamide gels and Western blots with a specific monoclonal antibody. Indirect immunofluorescence and immunoperoxidase studies confirmed the expression of VspB in the blood and also demonstrated VspB expression in the joints and heart. The gene for VspB was next identified and cloned by using partial amino acid sequencing, reverse transcriptase PCR, and a specific monoclonal antibody. The vspB gene encodes a protein of 216 amino acids that is 68% identical to VspA of B. turicatae and 44 to 56% identical to representative Vsp and OspC lipoproteins of other Borrelia spp. The processed VspB protein was distinguished from 26 other Vsp and OspC proteins by a high predicted isoelectric point at 9.39. The promoter region for vspB was similar to the promoter region for the vsp33 gene of Borrelia hermsii and for the ospC gene of Borrelia burgdorferi, two genes known to be environmentally regulated. These studies established that the virulence-associated VspB protein is expressed by spirochetes in the mouse and that VspB is a novel member of the Vsp-OspC family of proteins.
Assuntos
Antígenos de Bactérias , Proteínas da Membrana Bacteriana Externa/biossíntese , Infecções por Borrelia/metabolismo , Borrelia/metabolismo , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/sangue , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Western Blotting , Infecções por Borrelia/sangue , Infecções por Borrelia/patologia , Enzimas de Restrição do DNA , DNA Bacteriano , Eletroforese em Gel de Poliacrilamida , Feminino , Imunofluorescência , Técnicas Imunoenzimáticas , Masculino , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido NucleicoRESUMO
The in vitro activity of the everninomicin antibiotic SCH 27899 against 17 isolates of Borrelia spp. was investigated. MICs ranged from 0.06 to 0.5 microg/ml. Time-kill studies with the B31 strain of B. burgdorferi demonstrated >/=3-log10-unit killing after 72 h with concentrations representing four times the MIC. The in vitro activity of four other newer antimicrobial agents, meropenem, cefepime, quinupristin-dalfopristin, and linezolid, was also tested against the B31 strain. Meropenem was the most potent of the latter agents, with an MIC of 0.125 microg/ml.