RESUMEN
The pattern of meningococcal surface structure expression in different microenvironments following bloodstream invasion in vivo is not known. We used immunohistochemistry to determine the expression of capsule, type IV pili, and PorA by meningococci residing in the skin lesions of children with purpura fulminans. All the skin biopsy samples showed evidence of thrombosis and, frequently, a perivascular inflammatory cell infiltrate consisting of neutrophils (elastase positive) and monocytes/macrophages (CD68 positive). Modified Gram staining revealed 20 to over 100 gram-negative diplococci in each 4-microm-thick section, usually grouped into microcolonies. Immunoperoxidase staining demonstrated that the invading meningococci expressed PorA, capsule, and type IV pilin. Expression of these antigens was not restricted to any particular environment and was found in association with meningococci located in leukocytes, small blood vessels, and the dermal interstitium. Confocal laser scanning microscopy demonstrated coexpression of pilin and capsule by numerous microcolonies. However, there was some discordance in capsule and pilin expression within the microcolonies, suggesting phase variation. The strategy employed in this study will be helpful in investigating invasive bacterial diseases where antigenic and phase variation has a significant impact on virulence and on vaccine design.
Asunto(s)
Vasculitis por IgA/inmunología , Vasculitis por IgA/microbiología , Infecciones Meningocócicas/inmunología , Infecciones Meningocócicas/microbiología , Neisseria meningitidis/inmunología , Neisseria meningitidis/patogenicidad , Enfermedades Cutáneas Bacterianas/inmunología , Enfermedades Cutáneas Bacterianas/microbiología , Adolescente , Anticuerpos Antibacterianos , Variación Antigénica , Niño , Preescolar , Proteínas Fimbrias , Fimbrias Bacterianas/inmunología , Humanos , Vasculitis por IgA/patología , Inmunohistoquímica , Técnicas In Vitro , Lactante , Inflamación/patología , Proteínas de la Membrana/inmunología , Infecciones Meningocócicas/patología , Microscopía Confocal , Porinas/inmunología , Enfermedades Cutáneas Bacterianas/patología , Trombosis/patología , Virulencia/inmunologíaRESUMEN
The Mycobacterium tuberculosis complex is associated with a remarkably low level of structural gene polymorphism. As part of a search for alternative forms of genetic variation that may act as a source of biological diversity in M. tuberculosis, we have identified a region of the genome that is highly variable amongst a panel of unrelated clinical isolates. Fifteen of 24 isolates examined contained one or more copies of the M. tuberculosis-specific IS6110 insertion element within this 20 kb variable region. In nine of the isolates, including the laboratory-passaged strain H37Rv, genomic deletions were identified, resulting in loss of between two and 13 genes. In each case, deletions were associated with the presence of a copy of the IS6110 element. Absence of flanking tri- or tetra-nucleotide repeats identified homologous recombination between adjacent IS6110 elements as the most likely mechanism of the deletion events. IS6110 insertion into hot-spots within the genome of M. tuberculosis provides a mechanism for generation of genetic diversity involving a high frequency of insertions and deletions.
Asunto(s)
Elementos Transponibles de ADN , Eliminación de Gen , Variación Genética , Genoma Bacteriano , Mycobacterium tuberculosis/genética , Tuberculosis/microbiología , Hidrolasas de Éster Carboxílico/genética , Femenino , Humanos , Masculino , Repeticiones de Microsatélite , Mycobacterium tuberculosis/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Recombinación Genética , Repeticiones de Trinucleótidos , Fosfolipasas de Tipo C/genéticaRESUMEN
To identify Mycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure to M. leprae from exposure to Mycobacterium tuberculosis or to environmental mycobacteria or from immune responses following Mycobacterium bovis BCG vaccination, 15-mer synthetic peptides were synthesized based on data from the M. leprae genome, each peptide containing three or more predicted HLA-DR binding motifs. Eighty-one peptides from 33 genes were tested for their ability to induce T-cell responses, using peripheral blood mononuclear cells (PBMC) from tuberculoid leprosy patients (n = 59) and healthy leprosy contacts (n = 53) from Brazil, Ethiopia, Nepal, and Pakistan and 20 United Kingdom blood bank donors. Gamma interferon (IFN-gamma) secretion proved more sensitive for detection of PBMC responses to peptides than did lymphocyte proliferation. Many of the peptides giving the strongest responses in leprosy donors compared to subjects from the United Kingdom, where leprosy is not endemic, have identical, or almost identical, sequences in M. leprae and M. tuberculosis and would not be suitable as diagnostic tools. Most of the peptides recognized by United Kingdom donors showed promiscuous recognition by subjects expressing differing HLA-DR types. The majority of the novel T-cell epitopes identified came from proteins not previously recognized as immune targets, many of which are cytosolic enzymes. Fifteen of the tested peptides had > or =5 of 15 amino acid mismatches between the equivalent M. leprae and M. tuberculosis sequences; of these, eight gave specificities of > or =90% (percentage of United Kingdom donors who were nonresponders for IFN-gamma secretion), with sensitivities (percentage of responders) ranging from 19 to 47% for tuberculoid leprosy patients and 21 to 64% for healthy leprosy contacts. A pool of such peptides, formulated as a skin test reagent, could be used to monitor exposure to leprosy or as an aid to early diagnosis.
Asunto(s)
Antígenos Bacterianos/inmunología , Epítopos de Linfocito T/inmunología , Lepra Tuberculoide/inmunología , Mycobacterium leprae/inmunología , Péptidos/inmunología , Secuencia de Aminoácidos , Antígenos Bacterianos/genética , Epítopos de Linfocito T/química , Genoma Bacteriano , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Lepra Tuberculoide/diagnóstico , Lepra Tuberculoide/microbiología , Activación de Linfocitos , Datos de Secuencia Molecular , Mycobacterium leprae/química , Mycobacterium leprae/genética , Péptidos/síntesis química , Péptidos/química , Especificidad de la Especie , Linfocitos T/inmunologíaRESUMEN
Mannose-binding lectin (MBL) is an important component of the innate immune system. It binds to the arrays of sugars commonly presented by microorganisms and activates the complement system independently of antibody. Despite detailed knowledge of the stereochemical basis of MBL binding, relatively little is known about how bacterial surface structures influence binding of the lectin. Using flow cytometry, we have measured the binding of MBL to a range of mutants of Salmonella enterica serovar Typhimurium and Neisseria gonorrhoeae which differ in the structure of expressed lipopolysaccharide (LPS). For both organisms, the possession of core LPS structures led to avid binding of MBL, which was abrogated by the addition of O antigen (Salmonella serovar Typhimurium) or sialic acid (N. gonorrhoeae). Truncation of the LPS within the core led to lower levels of MBL binding. It was not possible to predict the magnitude of MBL binding from the identity of the LPS terminal sugar alone, indicating that the three-dimensional disposition of LPS molecules is probably also of importance in determining MBL attachment. These results further support the hypothesis that LPS structure is a major determinant of MBL binding.
Asunto(s)
Proteínas Portadoras/metabolismo , Lectinas/metabolismo , Lipopolisacáridos/metabolismo , Manosa/metabolismo , Neisseria gonorrhoeae/inmunología , Neisseria gonorrhoeae/metabolismo , Salmonella typhimurium/inmunología , Salmonella typhimurium/metabolismo , Secuencia de Carbohidratos , Colectinas , Humanos , Técnicas In Vitro , Lipopolisacáridos/química , Datos de Secuencia Molecular , Mutación , Neisseria gonorrhoeae/genética , Unión Proteica , Salmonella typhimurium/genéticaRESUMEN
To date, only a limited number of antigens have been described as specific for Mycobacterium leprae, and in many cases, homologues have subsequently been shown to exist in mycobacteria such as M. avium and M. intracellulare. A Leprosy Synthetic Peptide Skin Test Initiative was established by the Steering Committee on the Immunology of Mycobacteria of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, to investigate the potential of synthetic peptides that encode T-cell epitopes as diagnostic tools, which could be used to develop a skin-test reagent specific for leprosy. Such M. leprae-specific peptides should have unique amino acid sequences, or significant sequence-dissimilarity from those in other mycobacteria. Synthetic peptides, 15 amino acids long, were synthesised from 33 genes or open reading frames within the M. leprae genome. Tuberculoid leprosy patients from four leprosy-endemic countries, Brazil, Ethiopia, Nepal and Pakistan, were tested as subjects known to have been infected with M. leprae, and to make good T-cell responses to antigens of M. leprae; UK blood donors were used as non-exposed or non-infected subjects. Peptides inducing potentially specific responses in leprosy patients and not in UK controls, and those inducing cross-reaction responses, present in both leprosy patients and non-exposed, non-infected controls, were identified. A difference from the equivalent M. tuberculosis sequence of five or more amino acid residues did not, by itself, identify peptides that were M. leprae-specific, suggesting that many of these peptides may have homologues in environmental mycobacteria. To date, this approach has identified a number of peptides with greater than 90% specificity and 19-47% sensitivity, which are undergoing further specificity-testing. Such peptides would have great potential as T-cell reagents with which to monitor exposure to M. leprae within communities, formulated either as skin-test reagents, or as antigens for tests in vitro.
Asunto(s)
Epítopos de Linfocito T , Lepra/diagnóstico , Mycobacterium leprae/inmunología , Humanos , Lepra/inmunología , Sensibilidad y EspecificidadRESUMEN
penB is a chromosomal mutation that confers resistance to beta-lactams and tetracyclines and reduced susceptibility to quinolones in Neisseria gonorrhoeae. It is linked to the porin gene (por) and requires the increased expression of an efflux pump due to mtr. Transformation of a susceptible gonococcus (strain H1) with chromosomal DNA from strain FA140 (penA mtr penB; porin serovar IB1) and conjugal transfer of a beta-lactamase-expressing plasmid was used to produce isogenic strains for determination of equilibrium periplasmic penicillin concentrations by the method of Zimmermann and Rosselet (W. Zimmermann and A. Rosselet, Antimicrob. Agents Chemother. 12:368-372, 1977). In transformants with the Mtr and PenB phenotypes, equilibrium concentrations of penicillin were reduced. DNA sequence analysis of por from isogenic penB and penB+ transformants revealed 14 sequence differences; nine of these differences resulted in amino acid changes. Three amino acid changes were found in the putative gonococcal equivalent of the pore-constricting loop 3 of Escherichia coli OmpF. Two of these changes (Gly-101-Ala-102-->Asp-Asp) result in an increased negative charge at this position in por loop 3. PCR products comprising the complete por gene from strain FA140 were transformed into strain H1-2 (penA mtr; porin serovar IB-3), with the resulting transformants having the antibiotic susceptibility phenotype associated with penB. penB-like mutations were found in loop 3 of clinical isolates of gonococci with chromosomally mediated resistance to penicillin. We conclude that penB is a mutation in loop 3 of por that reduces porin permeability to hydrophilic antibiotics and plays an important role in the development of chromosomally mediated resistance to penicillin and tetracycline in gonococci.
Asunto(s)
Antibacterianos/farmacología , Neisseria gonorrhoeae/efectos de los fármacos , Porinas/genética , Tetraciclinas/farmacología , Mapeo Cromosómico , Farmacorresistencia Microbiana/genética , Mutación , Neisseria gonorrhoeae/genética , Porinas/química , Transformación Bacteriana , beta-LactamasAsunto(s)
Vacuna BCG , Mycobacterium tuberculosis/genética , Tuberculosis/tratamiento farmacológico , Tuberculosis/prevención & control , Adulto , Antituberculosos/uso terapéutico , Niño , Diseño de Fármacos , Humanos , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/fisiopatología , Vacunas de ADNRESUMEN
Low-level resistance of Neisseria gonorrhoeae to toxic hydrophobic agents (HAs), including some antibiotics, is chromosomally mediated via the multiple transferable resistance (mtr) efflux system. The gene encoding the 48:3 kDa outer-membrane protein MtrE, which is associated with the mtr phenotype, was identified and is homologous to export-associated outer-membrane proteins, including the OprM (formerly OprK) lipoprotein of Pseudomonas aeruginosa. Insertional inactivation of the mtrE gene in N. gonorrhoeae strain FA19 resulted in the loss o the outer-membrane protein, with concomitant hypersusceptibility of the mutant strain to a range of HAs. The properties of this mutant confirmed the role of MtrE in multidrug resistance mediated by an active efflux mechanism. Secondary structure predictions for MtrE indicated a largely hydrophilic protein with a single alpha-helical transmembrane region. A transposon-like element, similar to that found downstream of the region containing the promoters for mtrR and mtrC in Neisseria meningitidis, was identified 63 bp downstream of the mtrE gene.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros , Antibacterianos/toxicidad , Proteínas de la Membrana Bacteriana Externa/genética , Farmacorresistencia Microbiana/genética , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Neisseria gonorrhoeae/genética , Secuencia de Aminoácidos , Clonación Molecular , Datos de Secuencia Molecular , Mutación , Neisseria gonorrhoeae/efectos de los fármacos , Análisis de SecuenciaRESUMEN
Tissue factor (TF), a small membrane bound high affinity receptor for factor VII, has an important procoagulant role in the haemostatic dysfunction associated with severe sepsis. Using an in vitro model of human endothelial TF expression, defined strains of Neisseria meningitidis were found to upregulate endothelial cell procoagulant activity (PCA) in a dose dependent manner. This TF response was detected with as little as 10(4) cfu/ml and reached similar levels to those seen with high concentrations of purified endotoxin (> 1 ng/ml). Treatment of N. meningitidis with either adult donor immune serum, penicillin or gentamicin failed to enhance this PCA. Limulus amoebocyte lysate assay of lipopolysaccharide in bacterial culture filtrates together with polymyxin B inhibition experiments suggest that endotoxin is largely responsible for endothelial TF induction by N. meningitidis. Incubation of endothelial cells with N. meningitidis B1940 and B1940 siaD- (an eight-fold more adherent unencapsulated isogenic strain), revealed a significantly greater TF response to B1940 siaD- (P < 0.01). In conclusion, bacterial adhesion to the vessel wall and therefore local levels of endotoxin may be important determinants of the endothelial procoagulant response to N. meningitidis and the consequent coagulopathy commonly associated with the disease.
Asunto(s)
Endotelio Vascular/microbiología , Neisseria meningitidis/fisiología , Tromboplastina/biosíntesis , Adulto , Adhesión Bacteriana , Cápsulas Bacterianas , Coagulación Sanguínea , Células Cultivadas , Endotelio Vascular/citología , Endotoxinas/farmacología , Humanos , Polisacáridos Bacterianos/farmacología , Venas UmbilicalesRESUMEN
Transmission of multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) presents a serious problem for infection control in hospitals, particularly in the context of co-infection with the human immunodeficiency virus (HIV). We report on the use of molecular genetic tools to allow rapid assessment of samples from patients potentially infected with MDR-TB. Sputum and bronchoalveolar lavage samples were obtained from two HIV-positive patients with suspected tuberculosis, who had previous contact with a known MDR-TB index case. Polymerase chain reaction (PCR) was used directly on clinical samples to amplify genetic loci associated with rifampicin resistance (rpoB), and strain-specific polymorphisms (the direct repeat (DR) region). Drug resistance was determined using a commercially available kit for detection of point mutations in the rpoB gene (Inno-Lipa RifTB; Innogenetics, Belgium), and confirmed by nucleotide sequencing. Strain variation was determined using the spoligotyping method, based on the presence or absence of variable linker sequences within the DR region. In one patient, infection with a MDR strain identical to that of a known index case was demonstrated. A second patient, although positive for M. tuberculosis, was found to be infected with a rifampicin-sensitive strain. Results were obtained within 48 h, allowing appropriate treatment to be initiated and infection control measures to be implemented. PCR-based tests for strain-typing and for identification of rifampicin resistance provide important tools for identifying patients with MDR-TB and for rapid monitoring of potential nosocomial spread of MDR-TB. Prompt confirmation or exclusion of possible transmission allows early clinical intervention to prevent future outbreaks of multidrug-resistant M. tuberculosis.
Asunto(s)
Mycobacterium tuberculosis/genética , Reacción en Cadena de la Polimerasa/métodos , Tuberculosis/diagnóstico , Antibióticos Antituberculosos/farmacología , Líquido del Lavado Bronquioalveolar/microbiología , Infección Hospitalaria/transmisión , Farmacorresistencia Microbiana/genética , Resistencia a Múltiples Medicamentos/genética , Infecciones por VIH/complicaciones , Infecciones por VIH/microbiología , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Biología Molecular , Mycobacterium tuberculosis/aislamiento & purificación , Polimorfismo Genético , Secuencias Repetitivas de Ácidos Nucleicos , Rifampin/farmacología , Esputo/microbiología , Tuberculosis/tratamiento farmacológico , Tuberculosis/transmisiónRESUMEN
A homolog of the meningococcal cps locus region E has been identified in Neisseria gonorrhoeae immediately upstream of the gonococcal region D locus. Region E has no detectable function in capsule biosynthesis in Neisseria meningitidis or in lipopolysaccharide biosynthesis in either organism. The open reading frame is homologous to proteins of unknown function in Escherichia coli and Haemophilus influenzae. Further analysis of the N. meningitidis cps cluster has identified a second copy of region D encoding three additional open reading frames, including homologs of DNA methyltransferases. The organization of the region D and E genes in N. gonorrhoeae and N. meningitidis in relation to the cps genes provides some insight into the evolutionary origin of encapsulation in N. meningitidis.
Asunto(s)
Cápsulas Bacterianas/metabolismo , Genes Bacterianos , Neisseria gonorrhoeae/genética , Neisseria meningitidis/genética , Secuencia de Bases , Lipopolisacáridos/biosíntesis , Datos de Secuencia Molecular , Sistemas de Lectura AbiertaRESUMEN
A full-length mRNA encoding a secreted 26-kDa antigen of infective larvae of the ascarid nematode parasite Toxocara canis has been identified. This was characterized as a 1,082-base pair clone highly abundant (0.8-1.9%) in cDNA prepared from infective stage larvae but absent from cDNA from adult male worms. Sequence analysis revealed an open reading frame corresponding to a hydrophilic 263-amino acid residue polypeptide with a 20-residue N-terminal signal peptide, indicating that it is secreted. The 5' end of the cDNA was isolated by polymerase chain reaction using a primer containing the nematode-spliced leader sequence, SL1, showing that the mRNA is trans-spliced. The molecular mass of the putative protein with the signal peptide removed is 26.01 kDa, and antibody to the recombinant protein expressed in bacterial vectors reacts with a similarly sized protein in T. canis excretory/secretory (TES) products. An identical sequence was obtained from a genomic clone isolated by expression screening with mouse antibody to TES. The 72 amino acid residues adjacent to the signal peptide form two homologous 36-residue motifs containing 6 cysteine residues; this motif is found also in the T. canis-secreted glycoprotein TES-120 and in genes of Caenorhabditis elegans. Sequence data base searches revealed significant similarity to 7 other sequences in a newly recognized gene family of phosphatidylethanolamine-binding proteins that includes yeast, Drosophila, rat, bovine, simian, and human genes and a representative from the filarial nematode Onchocerca volvulus. Assays with the T. canis recombinant 26-kDa protein expressed as a fusion with maltose-binding protein have confirmed phosphatidylethanolamine-binding specificity for this novel product.
Asunto(s)
Antígenos Helmínticos/genética , Proteínas del Helminto/genética , Empalme del ARN , ARN Mensajero/genética , Toxocara canis/genética , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/inmunología , Antígenos Helmínticos/metabolismo , Secuencia de Bases , Proteínas Portadoras/genética , ADN Complementario/genética , Biblioteca Genómica , Proteínas del Helminto/inmunología , Proteínas del Helminto/metabolismo , Larva , Masculino , Datos de Secuencia Molecular , Fosfatidiletanolaminas/metabolismo , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Toxocara canis/crecimiento & desarrolloRESUMEN
The lipopolysaccharides of the pathogenic Neisseria species are subject to structural variation owing to a combination of intrinsic changes in lipopolysaccharide (LPS) biosynthesis and external modification of the LPS molecule with sialic acid. This variation appears to control bacterial behaviour by altering their ability to interact with human cells and to evade host immune defences. This interconversion of LPS phenotypes, which is also observed during the natural infection, is probably due to environmental regulation of LPS biosynthesis superimposed on spontaneous changes in the DNA of distinct LPS loci. LPS variation may be a common strategy of mucosal pathogens to colonize and persist within the human host.
Asunto(s)
Lipopolisacáridos/química , Neisseria/inmunología , Neisseria/patogenicidad , Conformación de Carbohidratos , Secuencia de Carbohidratos , Variación Genética , Humanos , Lipopolisacáridos/biosíntesis , Datos de Secuencia Molecular , Membrana Mucosa/microbiología , Neisseria/genética , Infecciones por Neisseriaceae/microbiología , Oligosacáridos/químicaRESUMEN
We have investigated the function of the Isi-1 gene of Neisseria gonorrhoeae previously implicated in lipopolysaccharide (LPS)-inner-core biosynthesis (Petricoin et al., 1991). Disruption of the gene in gonococcal strain MS11 resulted in the production of LPS that migrated faster than that from an isogenic galE mutant, typical for a mutation that influences the inner-core region. Complementation of a panel of Salmonella typhimurium mutants with defined defects in rfa loci demonstrated conclusively that the Isi-1 gene of MS11 is functionally homologous to the rfaF gene, which encodes heptosyltransferase II in both E. coli and S. typhimurium. Comparison of deduced amino acid sequences of the gonococcal and the Salmonella RfaF demonstrated 70% similarity, including 47% identical amino acid residues. Immunochemical analysis of the LPS using monoclonal antibodies directed against chemically defined inner-core glycoconjugates revealed that the gonococcal and Salmonella Rd2-chemotypes were antigenically similar, further extending the genetic and functional homology. Infection experiments in vitro demonstrated that the Isi-1 mutant could not invade human Chang epithelial cells despite expression of a genetically defined invasion-promoting gonococcal opacity protein. These data imply that the LPS phenotype is a critical factor for gonococcal invasiveness.
Asunto(s)
Lipopolisacáridos/biosíntesis , Neisseria gonorrhoeae/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Genes Bacterianos , Prueba de Complementación Genética , Glicosiltransferasas/genética , Glicosiltransferasas/fisiología , Lipopolisacáridos/clasificación , Lipopolisacáridos/inmunología , Datos de Secuencia Molecular , Neisseria gonorrhoeae/clasificación , Neisseria gonorrhoeae/inmunología , Neisseria gonorrhoeae/patogenicidad , Salmonella typhimurium/genética , Salmonella typhimurium/inmunología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Virulencia/genéticaRESUMEN
The variable incorporation of sialic acid into the LPS of Neisseria gonorrhoeae modulates the invasive behavior of this bacterium towards cultured human epithelial cells. Here we report that the inhibitory effect of LPS sialylation on the gonococcal entry into Chang epithelial cells and ME-180 endocervical cells (van Putten, EMBO J 12:4043-4051, 1993) is located early in the uptake pathway upstream of the invasion-associated recruitment of F-actin at the sites of bacterial entry, but beyond the level of bacterial adherence. Receptor equilibrium studies using purified radiolabelled opacity protein receptor demonstrated that LPS sialylation caused a 3-5 fold reduction in binding of the bacterial invasion-promoting opacity outer membrane protein to its receptor. In HEC-1B and PC-3 cells, LPS sialylation did only partially inhibit the bacterial entry process, suggesting the existence of a second uptake mechanism for gonococci in these cell lines. Experiments with non-sialylatable and truncated isogenic LPS variants, and with genetically defined LPS mutants demonstrated that the invasive phenotype of N. gonorrhoeae requires a minimum of an Rc (or Rd1) chemotype of LPS. Variation within the LPS outer core region did not influence the invasive properties of the bacteria as long as there was no attached sialic acid.
Asunto(s)
Lipopolisacáridos/toxicidad , Neisseria gonorrhoeae/patogenicidad , Actinas/metabolismo , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Línea Celular , Gonorrea/etiología , Humanos , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Membrana Mucosa/metabolismo , Membrana Mucosa/microbiología , Unión Proteica , Ácidos Siálicos/químicaRESUMEN
Neisseria gonorrhoeae synthesizes a rough lipopolysaccharide that does not contain any of the repetitive units characteristic of the smooth lipopolysaccharide of members of the family Enterobacteriaceae. Three gonococcal homologs of Salmonella serovar typhimurium genes involved in the synthesis of the rhamnose component of the repetitive subunits have been isolated. Gonococcal homologs for rfbB, rfbA, and rfbD were found downstream of the galE gene in a region of the chromosome which shows overall homology with the meningococcal capsule gene complex region D. Sequence alignment demonstrated that the gonococcal gene products have 69, 65, and 54% amino acid identity with the Salmonella proteins RfbB, RfbA, and RfbD. The gonococcal RfbB and RfbA amino acid sequences share even more identical residues (73 and 65%, respectively) with the amino acid sequences derived from Escherichia coli genes o355 and o292, respectively. These genes are clustered with the genes involved in the biosynthesis of enterobacterial common antigen, and o355 is listed in the GenBank and Swiss Protein data banks as rffE (encoding UDP-GlcNAc-2-epimerase). However, complementation studies demonstrated that o355 does not encode the enzyme UDP-GlcNAc-2-epimerase. Gonococcal strains constructed with null mutations in the rfbBAD genes were unchanged in lipopolysaccharide phenotype and in the synthesis of gonococcal carbohydrate-containing C antigen. We were unable to detect any changes in gonococcal phenotype with respect to lipopolysaccharide sialylation, monoclonal-antibody binding, serum sensitivity, or interaction with eukaryotic cells in vitro. We conclude that the absence of a homolog for rfbC precludes the existence of a functional dTDP-rhamnose biosynthesis pathway in the gonococcal strains examined and that these genes are only maintained in N. gonorrhoeae either because of the presence of the galE gene or because of another as yet unrecognized function.
Asunto(s)
Proteínas Bacterianas , Galactosiltransferasas/genética , Genes Bacterianos/genética , Lipopolisacáridos/biosíntesis , Manosa-6-Fosfato Isomerasa , Neisseria gonorrhoeae/genética , Ramnosa/biosíntesis , Antígenos Bacterianos/genética , Secuencia de Bases , Carbohidrato Epimerasas/genética , Clonación Molecular , Citotoxicidad Inmunológica , Prueba de Complementación Genética , Hidroliasas/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Neisseria gonorrhoeae/inmunología , Nucleotidiltransferasas/genética , Fenotipo , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Prueba Bactericida de SueroRESUMEN
Within the capsule gene complex (cps) of Neisseria meningitidis B a 5.5 kb DNA fragment encodes proteins with strong homologies to enzymes of the lipopolysaccharide biosynthetic pathway of Salmonella typhimurium and Escherichia coli, GalE, RfbB, RfbC and RfbD. A meningococcal galE mutant expressed a truncated lipooligosaccharide (LOS), which terminated at the glucose residue between inner and outer core, and a second galE gene present outside the cps cluster was found to be transcriptionally and functionally inactive and, thus, unable to complement this defect. Because of the defect in the outer core, the LOS of the galE-defective meningococcal mutant was not sialylated. In contrast, carbohydrate analysis of the LOS of an rfb-defective meningococcal mutant revealed no difference from the LOS of the wild-type strain, suggesting that the rfb genes are inactive. This was supported by Northern blot analysis, which showed that expression of the rfb gene products was transcriptionally regulated. The inability of the meningococcal galE mutant, which cannot sialylate the LOS, allowed us to investigate the significance of LOS sialylation in relation to the presence of the polysialic acid capsule. Sialylated LOS, but not the polysialic acid capsule, is necessary to confer complete serum resistance on the meningococcus by inhibition of the alternative complement pathway.
Asunto(s)
Proteínas Bacterianas/genética , Carbohidrato Epimerasas/genética , Genes Bacterianos , Hidroliasas/genética , Lipopolisacáridos/metabolismo , Neisseria meningitidis/genética , UDPglucosa 4-Epimerasa , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Carbohidrato Epimerasas/metabolismo , Secuencia de Carbohidratos , Vía Alternativa del Complemento , Proteínas del Sistema Complemento/farmacología , Farmacorresistencia Microbiana , Enterobacteriaceae/genética , Hidroliasas/metabolismo , Datos de Secuencia Molecular , Ácido N-Acetilneuramínico , Neisseria meningitidis/efectos de los fármacos , Neisseria meningitidis/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ácidos Siálicos/fisiología , Especificidad de la EspecieRESUMEN
AIMS: To evaluate an antigen capture enzyme linked immunosorbent assay (ELISA) which detects a carbohydrate epitope on the excretory-secretory (ES) antigens of Toxocara canis in clinical practice. METHODS: Serum specimens from healthy adults, patients with acute visceral larva migrans, ocular and inactive toxocariasis, and with other helminth infections were examined by two site antigen capture ELISA. RESULTS: Over half of the patients (19/28) with acute toxocariasis had a positive result in contrast to a small proportion of those with inactive disease (1/10) or ocular infection (2/7). False positive reactions, however, were found in 25% of the patients with serologically confirmed schistosomiasis and filariasis. CONCLUSIONS: This assay is useful in confirming the diagnosis of acute visceral larva migrans but could not be used alone in diagnosis because of false positive reactions in patients with other helminth infections.
Asunto(s)
Antígenos Helmínticos/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Larva Migrans Visceral/diagnóstico , Adulto , Animales , Niño , Estudios de Evaluación como Asunto , Femenino , Humanos , Larva Migrans Visceral/inmunología , Masculino , Sensibilidad y Especificidad , Toxocara canis/inmunologíaRESUMEN
Lipopolysaccharide is an essential component of the outer membrane of Gram-negative bacteria and an important virulence factor of many pathogens, such as Neisseria gonorrhoeae. We have cloned the gonococcal galE gene which was found to be located in the gonococcal homologue of the meningococcal capsule gene complex region D. Sequence alignment indicated extensive homology with the Escherichia coli and Salmonella GalE proteins. Mutants with insertions in the galE gene were used as a tool to characterize the structure and function of gonococcal lipopolysaccharide. They displayed deep rough phenotypes, and chemical analysis confirmed the loss of galactose from the mutant lipopolysaccharide. Functional analysis indicated that the terminal oligosaccharides contain galactose and that these are lost in galE mutants. The importance of these oligosaccharides in gonococcal biology is clear from the fact that they contain the epitopes that are the targets for killing by normal human serum, and the acceptor site for sialic acid, which acts to protect the gonococcus from this killing. Furthermore, infection experiments in vitro indicate that the galE mutants exhibit unaltered intergonococcal adhesion as well as adhesion to, and invasion of, epithelial cells.