RESUMO
In this paper, we have evaluated the diagnostic utility of three antigenic regions of the Mycoplasma pneumoniae P1, P30, and MPN456 gene products in order to replace the soluble, whole-cell bacterial extract in serological assays. Antigenic regions, being previously identified as B-cell epitopes, were used individually or assembled in a recombinant chimeric antigen by genetic engineering. Paired serum samples from 47 patients with M. pneumoniae infection and from 39 subjects with a clinical picture of atypical pneumonia but without a defined diagnosis of M. pneumoniae infection were included. Immunoglobulin G (IgG) antibodies against epitopes carried by recombinant antigens were measured by performing recombinant enzyme-linked immunosorbent assays (Rec-ELISAs). Rec-ELISA results were compared to those obtained by a commercial assay using the whole-cell Mycoplasma antigen. Our study demonstrates that all IgG Rec-ELISAs using recombinant antigens have better sensitivity with respect to the commercial assay. Furthermore, we show that the use of chimeric antigens improve the performance of the assays. The use of recombinant antigens is effective in distinguishing M. pneumoniae-infected patients from uninfected individuals and shows that immunoassays based on recombinant antigens could provide the basis for standardized commercial tests for the serodiagnosis of M. pneumoniae diseases.
Assuntos
Anticorpos Antibacterianos/sangue , Imunoglobulina G/sangue , Mycoplasma pneumoniae/imunologia , Pneumonia por Mycoplasma/diagnóstico , Testes Sorológicos , Adolescente , Adulto , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos de Linfócito B/imunologia , Humanos , Imunoglobulina G/imunologia , Lactente , Pneumonia por Mycoplasma/imunologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/imunologia , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Excreted secreted antigens of the protozoan parasite Toxoplasma gondii play a key role in stimulating the host immune system during acute and chronic infection. With the aim of identifying the immunodominant epitopes of T. gondii antigens involved in the human B-cell response against the parasite, we employed a novel immunological approach. A library of cDNA fragments from T. gondii tachyzoites was displayed as fusion proteins to the amino-terminus of lambda bacteriophage capsid protein D. The lambda D-tachyzoite library was then affinity-selected by using a panel of sera of pregnant women, all infected with the parasite. Some of the clones identified through this procedure matched the sequence of the dense granule GRA1 protein (p24), allowing us to identify its antigenic regions. In particular, the analysis of human antibody response against the recombinant GRA1 antigen fragments revealed the existence of an immunodominant epitope (epi-24 peptide).
Assuntos
Antígenos de Protozoários/imunologia , Epitopos de Linfócito B/imunologia , Epitopos Imunodominantes/imunologia , Toxoplasma/imunologia , Toxoplasmose/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Bacteriófago lambda/química , DNA de Protozoário/química , DNA de Protozoário/genética , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/genética , Feminino , Biblioteca Gênica , Glutationa Transferase/metabolismo , Humanos , Epitopos Imunodominantes/genética , Dados de Sequência Molecular , Biblioteca de Peptídeos , Reação em Cadeia da Polimerase , Gravidez , Homologia de Sequência de Aminoácidos , Toxoplasma/química , Toxoplasma/genéticaRESUMO
The mechanism of pathogenicity in Shigella and enteroinvasive Escherichia coli (EIEC) requires the co-ordinated expression of several genes located on both the virulence plasmid and the chromosome. We found that cells lacking a functional FIS protein (factor for inversion stimulation) are partially impaired in expressing the virulence genes and that full expression is totally restored when Shigella wild-type fis gene is offered in trans. We also identified virF, among the virulence genes, as a target of FIS-mediated activation and showed that FIS binds to four specific sites in the promoter region of virF. Previous studies have demonstrated that the expression of VirF, the first positive activator of a multistep regulatory cascade, is subject to temperature-dependent regulation by H-NS, one of the main nucleoid-associated proteins. We now demonstrate that two of the four FIS sites overlap one of the two H-NS sites responsible for thermoregulation (H-NS site I). FIS was found to exercise a direct positive transcriptional control at permissive temperature (37 degrees C), when H-NS fails to repress virF, as well as an indirect effect by partially counteracting H-NS inhibition at the transition temperature (32 degrees C). Our data indicate that FIS may be relevant for the rapid increase in virF expression after penetration of bacteria into the host.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/patogenicidade , Regulação Bacteriana da Expressão Gênica , Shigella/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/genética , Pegada de DNA , Proteínas de Ligação a DNA/genética , Fator Proteico para Inversão de Estimulação , Fatores Hospedeiros de Integração , Dados de Sequência Molecular , Plasmídeos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Elementos de Resposta/genética , Shigella/fisiologia , Temperatura , Fatores de Transcrição/genética , Transcrição Gênica , Virulência/genéticaRESUMO
We have investigated the major Escherichia coli histone-like proteins (H-NS, HU, FIS, and IHF) as putative factors involved in the maintenance of the overall DNA looped arrangement of the bacterial nucleoid. The long-range architecture of the chromosome has been studied by means of an assay based on in vivo genomic fragmentation mediated by endogenous DNA gyrase in the presence of oxolinic acid. The fragmentation products were analysed by CHEF electrophoresis. The results indicate that in vivo a large fraction of the bacterial chromatin constitutes an adequate substrate for the enzyme. DNA fragments released upon oxo-treatment span a size range from about 1000 kb to a limit-size of about 50 kb. The latter value is in excellent agreement with the average size reported for bacterial chromosomal domains. The DNA gyrase-mediated fragmentation does not appear to be significantly altered in strains depleted in histone-like proteins as compared to an E. coli wild type strain. This suggests that these proteins may not represent critical determinants for the maintenance of the supercoiled loop organisation of the E. coli chromosome.
