RESUMEN
Recruitment of immune cells to infection sites is a critical component of the host response to pathogens. This process is facilitated partly through interactions of chemokines with cognate receptors. Here, we examine the importance of fractalkine (CX3CL1) receptor, CX3CR1, which regulates function and trafficking of macrophages and dendritic cells, in the host's ability to control respiratory infections with Mycobacterium tuberculosis or Francisella tularensis. Following low-dose aerosol challenge with M. tuberculosis, CX3CR1(-/-) mice were no more susceptible to infection than wild-type C57BL/6 mice as measured by organ burden and survival time. Similarly, following inhalation of F. tularensis, CX3CR1(-/-) mice displayed similar organ burdens to wild-type mice. CX3CR1(-/-) mice had increased recruitment of monocytes and neutrophils in the lung; however, this did not result in increased abundance of infected monocytes or neutrophils. We conclude that CX3CR1-deficiency affects immune-cell recruitment; however, loss of CX3CR1 alone does not render the host more susceptible to M. tuberculosis or F. tularensis.
Asunto(s)
Francisella tularensis , Pulmón/inmunología , Receptores de Quimiocina/deficiencia , Tuberculosis Pulmonar/metabolismo , Tularemia/metabolismo , Animales , Receptor 1 de Quimiocinas CX3C , Células Dendríticas/inmunología , Susceptibilidad a Enfermedades , Femenino , Citometría de Flujo , Inmunofenotipificación , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Mycobacterium tuberculosis , Neutrófilos/inmunología , Receptores de Quimiocina/genética , Tularemia/inmunologíaRESUMEN
Haemophilus ducreyi causes chancroid, a sexually transmitted cutaneous genital ulcer disease associated with increased heterosexual transmission of human immunodeficiency virus. H. ducreyi expresses a periplasmic copper-zinc superoxide dismutase (Cu, Zn SOD) that protects the bacterium from killing by exogenous superoxide in vitro. We hypothesized that the Cu,Zn SOD would protect H. ducreyi from immune cell killing, enhance survival, and affect ulcer development in vivo. In order to test this hypothesis and study the role of the Cu,Zn SOD in H. ducreyi pathogenesis, we compared a Cu,Zn SOD-deficient H. ducreyi strain to its isogenic wild-type parent with respect to survival and ulcer development in immunocompetent and immunosuppressed pigs. The Cu,Zn SOD-deficient strain was recovered from significantly fewer inoculated sites and in significantly lower numbers than the wild-type parent strain or a merodiploid (sodC+ sodC) strain after infection of immunocompetent pigs. In contrast, survival of the wild-type and Cu,Zn SOD-deficient strains was not significantly different in pigs that were rendered neutropenic by treatment with cyclophosphamide. Ulcer severity in pigs was not significantly different between sites inoculated with wild type and sites inoculated with Cu,Zn SOD-deficient H. ducreyi. Our data suggest that the periplasmic Cu,Zn SOD is an important virulence determinant in H. ducreyi, protecting the bacterium from host immune cell killing and contributing to survival and persistence in the host.
Asunto(s)
Chancroide/inmunología , Haemophilus ducreyi/enzimología , Neutropenia/inmunología , Superóxido Dismutasa/fisiología , Animales , Actividad Bactericida de la Sangre , Modelos Animales de Enfermedad , Femenino , Haemophilus ducreyi/inmunología , Haemophilus ducreyi/patogenicidad , Neutrófilos/inmunología , Piel/patología , Superóxido Dismutasa/deficiencia , Porcinos , VirulenciaRESUMEN
Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease. Keratinocytes are likely the first cell type encountered by H. ducreyi upon infection of human skin; thus, the interaction between H. ducreyi and keratinocytes is probably important for the ability of H. ducreyi to establish infection. We have used the HaCaT keratinocyte cell line grown in monolayers and in cocultures with HS27 fibroblasts to investigate H. ducreyi interactions with keratinocytes and the host-cell response to H. ducreyi infection. Using quantitative adherence and gentamicin protection assays, we determined that approximately 13% of H. ducreyi adhered to HaCaT cell monolayers, while only a small proportion (0.0052%) was intracellular. By transmission electron microscopy, we observed numerous H. ducreyi organisms adherent to but rarely within HaCaT cells cocultured with fibroblasts. Both live H. ducreyi and purified H. ducreyi lipooligosaccharide (LOS) induced significant interleukin 8 (IL-8) expression from HaCaT cell-HS27 cell cocultures. However, the level of IL-8 expression in response to LOS alone was not as pronounced. H. ducreyi LOS was a more potent inducer of IL-8 from cocultures than Escherichia coli lipopolysaccharide (LPS) at the same concentration, suggesting a unique effect of H. ducreyi LOS on cocultures. Neither live H. ducreyi nor purified H. ducreyi LOS or E. coli LPS induced tumor necrosis factor alpha expression from cocultures. H. ducreyi induced drastically different cytokine profiles from cocultures than from HS27 or HaCaT cells cultured separately. IL-8 expression by skin cells in response to H. ducreyi infection in vivo may be responsible for the massive influx of polymorphonuclear leukocytes and other inflammatory cells to the site of infection. This influx of inflammatory cells may be partly responsible for the tissue destruction characteristic of chancroid.
Asunto(s)
Fibroblastos/microbiología , Haemophilus ducreyi/patogenicidad , Queratinocitos/microbiología , Adhesión Bacteriana , Células Cultivadas , Chancroide/inmunología , Técnicas de Cocultivo , Gentamicinas/farmacología , Haemophilus ducreyi/efectos de los fármacos , Humanos , Interleucina-8/biosíntesis , Lipopolisacáridos/toxicidad , Microscopía Electrónica , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Cutaneous lesions of the human sexually transmitted genital ulcer disease chancroid are characterized by the presence of intraepidermal pustules, keratinocyte cytopathology, and epidermal and dermal erosion. These lesions are replete with neutrophils, macrophages, and CD4(+) T cells and contain very low numbers of cells of Haemophilus ducreyi, the bacterial agent of chancroid. We examined lesion formation by H. ducreyi in a pig model by using cyclophosphamide (CPA)-induced immune cell deficiency to distinguish between host and bacterial contributions to chancroid ulcer formation. Histologic presentation of H. ducreyi-induced lesions in CPA-treated pigs differed from ulcers that developed in immune-competent animals in that pustules did not form and surface epithelia remained intact. However, these lesions had significant suprabasal keratinocyte cytotoxicity. These results demonstrate that the host immune response was required for chancroid ulceration, while bacterial products were at least partially responsible for the keratinocyte cytopathology associated with chancroid lesions in the pig. The low numbers of H. ducreyi present in lesions in humans and immune-competent pigs have prevented localization of these organisms within skin. However, H. ducreyi organisms were readily visualized in lesion biopsies from infected CPA-treated pigs by immunoelectron microscopy. These bacteria were extracellular and associated with necrotic host cells in the epidermis and dermis. The relative abundance of H. ducreyi in inoculated CPA-treated pig skin suggests control of bacterial replication by host immune cells during natural human infection.
Asunto(s)
Chancroide/inmunología , Chancroide/patología , Úlcera Cutánea/inmunología , Animales , Chancroide/microbiología , Ciclofosfamida/farmacología , Modelos Animales de Enfermedad , Haemophilus ducreyi/inmunología , Inmunosupresores/farmacología , Leucocitos/citología , Leucocitos/inmunología , Piel/microbiología , Piel/patología , Úlcera Cutánea/microbiología , Úlcera Cutánea/patología , PorcinosRESUMEN
H-NS is a major Escherichia coli nucleoid-associated protein involved in bacterial DNA condensation and global modulation of gene expression. This protein exists in cells as at least two different isoforms separable by isoelectric focusing. Among other phenotypes, mutations in hns result in constitutive expression of the proU and fimB genes, increased fimA promoter inversion rates, and repression of the flhCD master operon required for flagellum biosynthesis. To understand the relationship between H-NS structure and function, we transformed a cloned hns gene into a mutator strain and collected a series of mutant alleles that failed to repress proU expression. Each of these isolated hns mutant alleles also failed to repress fimB expression, suggesting that H-NS-specific repression of proU and fimB occurs by similar mechanisms. Conversely, alleles encoding single amino acid substitutions in the C-terminal DNA-binding domain of H-NS resulted in significantly reduced affinity for DNA yet conferred a wild-type fimA promoter inversion frequency, indicating that the mechanism of H-NS activity in modulating promoter inversion is independent of DNA binding. Furthermore, two specific H-NS amino acid substitutions resulted in hypermotile bacteria, while C-terminal H-NS truncations exhibited reduced motility. We also analyzed H-NS isoform composition expressed by various hns mutations and found that the N-terminal 67 amino acids were sufficient to support posttranslational modification and that substitutions at positions 18 and 26 resulted in the expression of a single H-NS isoform. These results are discussed in terms of H-NS domain organization and implications for biological activity.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/fisiología , Proteínas Fimbrias , Regiones Promotoras Genéticas , Proteínas Bacterianas/metabolismo , Inversión Cromosómica , Codón de Terminación , Escherichia coli/genética , Mutación del Sistema de Lectura , Regulación Bacteriana de la Expresión Génica , Movimiento , Mutagénesis Sitio-Dirigida , Operón , Fenotipo , Pili Sexual/genética , Pili Sexual/fisiología , Mutación Puntual , Proteínas Recombinantes/metabolismoRESUMEN
H-NS is an Escherichia coli nucleoid protein known only to function as a modulator of gene expression. In this study, we found that specific single amino acid substitutions in H-NS caused an approximately 50% increase in flagellum rotational speed. In fluorescence anisotropy and chemical cross-linking assays, H-NS interacted with the flagellar torque-generating rotor protein FliG to form a complex with a Kd of 2.15 microM. Furthermore, one of the altered H-NS proteins that exhibited high speed flagellum rotation bound FliG 50% tighter than wild-type H-NS. These results demonstrate the first non-regulatory role for H-NS and provide a direct correlation between H-NS-FliG binding affinities, flagellar rotation, and motor torque generation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/fisiología , Flagelos/fisiología , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Reactivos de Enlaces Cruzados , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/ultraestructura , Flagelos/ultraestructura , Polarización de Fluorescencia , Cinética , Microscopía Electrónica de Rastreo , Modelos Estructurales , Movimiento , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. Predominantly a cutaneous pathogen, H. ducreyi is present in chancroid ulcers that are characterized by extensive neutrophil accumulation in intraepidermal lesions accompanied by a mononuclear infiltrate in the dermis. We used an in vitro human skin model composed of foreskin fibroblasts and keratinocytes to examine host skin cell interactions with H. ducreyi 35000. Bacteria replicated and persisted in artificial skin for at least 14 days. We observed H. ducreyi inside suprabasal keratinocytes using transmission electron microscopy. Although no bacteria were seen in the basal keratinocyte region, these cells were disrupted in infected cocultures. H. ducreyi infection stimulated increased secretion of interleukin-6 (IL-6) and IL-8 by skin cells. Conversely, tumor necrosis factor alpha and IL-1alpha levels were not elevated. IL-8 produced in response to H. ducreyi infection may be involved in recruiting polymorphonuclear leukocytes and other inflammatory cells, thereby contributing to the tissue necrosis and ulcer formation characteristic of chancroid.
Asunto(s)
Citocinas/metabolismo , Haemophilus ducreyi/inmunología , Queratinocitos/patología , Piel/inmunología , Células Cultivadas , Chancroide/etiología , Haemophilus ducreyi/crecimiento & desarrollo , Humanos , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Masculino , Piel/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Haemophilus ducreyi is the etiologic agent of the sexually transmitted disease chancroid, an ulcerative condition implicated in increased HIV transmission. There is increasing evidence for the roles of oxidative stress proteins including superoxide dismutase enzymes in the survival and persistence of pathogenic organisms within the host. The sodA gene of Haemophilus ducreyi was isolated from a genomic plasmid library on the basis of its ability to rescue the hydrogen peroxide hypersensitivity of an Escherichia coli sodA sodB strain. The H. ducreyi SodA protein also complemented the aerobic growth defect of the E. coli sodA sodB strain in minimal medium. The deduced amino-acid sequence of the H. ducreyi sodA gene product is 74 and 70% identical to the Mn-SODs of Haemophilus influenzae and E. coli, respectively. However, unlike Mn-SODs, the H ducreyi SodA protein was inhibited by hydrogen peroxide in native gels stained for SOD activity.
Asunto(s)
Proteínas Bacterianas/genética , Haemophilus ducreyi/enzimología , Superóxido Dismutasa/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Secuencia de Bases , ADN Bacteriano , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos , Prueba de Complementación Genética , Biblioteca Genómica , Haemophilus ducreyi/genética , Peróxido de Hidrógeno/metabolismo , Datos de Secuencia Molecular , Estrés Oxidativo , Plásmidos , Superóxido Dismutasa/antagonistas & inhibidoresRESUMEN
Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcer disease implicated in increased heterosexual transmission of HIV. As part of an effort to identify H. ducreyi gene products involved in virulence and pathogenesis, we created random TnphoA insertion mutations in an H. ducreyi 35000 library cloned in Escherichia coli. Inserts encoding exported or secreted PhoA fusion proteins were characterized by DNA sequencing. One such clone encoded a Cu-Zn superoxide dismutase (SOD) enzyme. The Cu-Zn SOD was periplasmic in H. ducreyi and accounted for most of the detectable SOD activity in whole-cell lysates of H. ducreyi grown in vitro. To investigate the function of the Cu-Zn SOD, we created a Cu-Zn SOD-deficient H. ducreyi strain by inserting a cat cassette into the sodC gene. The wild-type and Cu-Zn SOD null mutant strains were equally resistant to excess cytoplasmic superoxide induced by paraquat, demonstrating that the Cu-Zn SOD did not function in the detoxification of cytoplasmic superoxide. However, the Cu-Zn SOD null strain was significantly more susceptible to killing by extracellular superoxide than the wild type. This result suggests that the H. ducreyi Cu-Zn SOD may play a role in bacterial defence against oxidative killing by host immune cells during infection.
Asunto(s)
Cobre , Haemophilus ducreyi/enzimología , Superóxido Dismutasa/metabolismo , Superóxidos/farmacología , Zinc , Fosfatasa Alcalina/genética , Clonación Molecular , Citoplasma/metabolismo , Elementos Transponibles de ADN , Farmacorresistencia Microbiana , Haemophilus ducreyi/efectos de los fármacos , Mutagénesis , Sistemas de Lectura Abierta , Paraquat/farmacología , Superóxido Dismutasa/genética , Superóxidos/metabolismoRESUMEN
The H-NS protein is a major component of the Escherichia coli nucleoid. Mutations in hns, the gene encoding H-NS, have pleiotropic effects on the cell altering both the expression of a variety of unlinked genes and the inversion rate of the DNA element containing the fimA promoter. We investigated the interaction between H-NS and fimB, the gene encoding the bidirectional recombinase that catalyzes fimA promoter flipping. In beta-galactosidase assays, we found that fimB expression increased approximately fivefold in an hns2-tetR insertion mutant. In gel mobility shift assays with purified H-NS, we have also shown that H-NS bound directly and cooperatively to the fimB promoter region with greater affinity than for any other known H-NS-regulated gene. Furthermore, this high-affinity interaction resulted in a promoter-specific inhibition of fimB transcription. The addition of purified H-NS to an in vitro transcription system yielded a fivefold or greater reduction in fimB-specific mRNA production. However, the marked increase in cellular FimB levels in the absence of H-NS was not the primary cause of the mutant rapid inversion phenotype. These results are discussed in regard to both H-NS as a transcriptional repressor of fimB expression and its role in regulating type 1 pilus promoter inversion.
Asunto(s)
Proteínas Bacterianas/biosíntesis , ADN Nucleotidiltransferasas/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas Fimbrias , Regulación Bacteriana de la Expresión Génica , Integrasas , Regiones Promotoras Genéticas , Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Mutación , Unión Proteica , ARN Bacteriano/análisis , ARN Mensajero/análisis , Transcripción GenéticaRESUMEN
A physical and rudimentary genetic map of the Haemophilus ducreyi strain 35000 genome was constructed. Pulsed-field gel electrophoresis was used to separate restriction fragments of H. ducreyi DNA digested with Sfil, I-Ceul, or Sfil plus I-Ceul. The sizes of the fragments were determined, and the circular chromosome was estimated to be 1757 kbp. The six I-Ceul fragments and four Sfil fragments were ordered into macrorestriction maps using Southern blot hybridization with random H. ducreyi clones as probes. It was shown that both H. ducreyi and the distantly related Haemophilus influenzae have six rrn operons marked by the locations of the I-Ceul sites. However, the two species displayed distinct I-Ceul restriction patterns. A second H. ducreyi strain, CIP542, displayed an identical I-Ceul pattern to that of H. ducreyi 35000, but Sfil digests of teh two strains were distinct. The orientation of the six rrn operons was determined and thirteen identified H. ducreyi genes positioned on the map of strain 35000.
Asunto(s)
Mapeo Cromosómico , Haemophilus ducreyi/genética , Southern Blotting , Clonación Molecular , Electroforesis en Gel de Campo Pulsado , Genoma , Haemophilus influenzae/genética , Operón , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
An inbred strain of the southern platyfish, Xiphophorus maculatus, was used as a host for Aeromonas hydrophila and Yersinia ruckeri infections. The infections were initiated by holding the platyfish in inoculation baths containing dilutions of virulent A. hydrophila or Y. ruckeri strains. Inoculating the platyfish in this manner resulted in a dose-dependent mortality over a range of bacterial input from 10(5) to 10(8) A. hydrophila and 10(6) to 10(8) Y. ruckeri/ml. Clinical manifestations of A. hydrophila infections were noted in infected platyfish that eventually died, but not in platyfish that survived. In this model, the Y. ruckeri infected fish died before obvious signs of infection were detected. The A. hydrophila strain used to establish the infections was recovered from the kidney and intestine of infected fish that died, but not from survivors receiving the same inoculation dose. Both infective bacteria were tested for the ability to invade a number of different fish and human cultured cells. A hydrophila strain TF7 did not invade of the cells tested, whereas the Y. ruckeri strain invaded fish derived cultured cells, but not human derived Hep-2 cells.
Asunto(s)
Aeromonas hydrophila/patogenicidad , Ciprinodontiformes/microbiología , Infecciones por Bacterias Gramnegativas , Yersiniosis , Yersinia/patogenicidad , Animales , Antibacterianos/uso terapéutico , Southern Blotting , Células Cultivadas , Cyprinidae/microbiología , Gentamicinas/uso terapéutico , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/mortalidad , Humanos , Microscopía Electrónica , Modelos Biológicos , Oncorhynchus mykiss/microbiología , Yersiniosis/tratamiento farmacológico , Yersiniosis/microbiología , Yersiniosis/mortalidadRESUMEN
Hsc66 is the second identified Hsp70 protein in Escherichia coli. Mutations in hscA, the gene encoding Hsc66, compensate for some phenotypic effects of a mutation in hns, a gene encoding the cold-inducible, nucleoid-associated protein H-NS. Expression of hscA was not induced upon heat shock but was induced approximately 11-fold 3 h after a shift from 37 to 10 degrees C. Furthermore, hscA was induced upon chloramphenicol addition, which induces the synthesis of other cold-inducible genes. Mapping of the transcription initiation site showed that hscA was cotranscribed with an upstream dnaJ-like gene, hscB; thus, hscB was also cold inducible. The hscBA promoter did not contain a Y-box element found in some cold-inducible promoters. Using two-dimensional electrophoresis, we identified Hsc66 under static 37 degrees C growth conditions and showed that Hsc66 was induced, as well as hscA, 3 h after a cold shock. Growth of an hscA mutant following cold shock was monitored relative to that of an isogenic wild-type strain. While cold shock adaptation as a function of growth rate was not significantly impaired in an hscA mutant, the expression of at least five other proteins was altered in this mutant following cold shock. On the basis of the homology to Hsp70 proteins and the induction following cold shock, we speculate that Hsc66 functions as a cold shock molecular chaperone.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Chaperonas Moleculares , Proteínas Bacterianas/biosíntesis , Secuencia de Bases , Cloranfenicol/farmacología , Frío , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/genética , Calor , Datos de Secuencia Molecular , Mutagénesis Insercional , Regiones Promotoras Genéticas/genética , Transcripción GenéticaRESUMEN
Haemophilus ducreyi is a strict human pathogen that causes sexually transmitted genital ulcer disease. We infected domestic swine with H. ducreyi 35000, resulting in the development of cutaneous ulcers histologically resembling human chancroid lesions. Intraepidermal lesions progressed from pustules to ulcers containing polymorphonuclear leukocytes and were accompanied by a dermal inflammatory infiltrate containing T cells and macrophages. H. ducreyi was recovered from lesions up to 17 days after inoculation, and pigs did not develop immunity to reinfection with the challenge strain. Features of the model include inoculation through abrasions in the epidermis, ambient housing temperatures for infected pigs, the ability to deliver multiple different inocula to a single host, and the availability of monoclonal antibodies against porcine immune cells permitting immunohistochemical characterization of the host immune response to H. ducreyi infection.
Asunto(s)
Chancroide/fisiopatología , Haemophilus ducreyi/patogenicidad , Animales , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/inmunología , Chancroide/microbiología , Chancroide/patología , Modelos Animales de Enfermedad , Oído , Femenino , Haemophilus ducreyi/inmunología , Masculino , PorcinosRESUMEN
Mutations in hns, the gene encoding the nucleoid-associated protein H-NS, affect both the expression of many specific unlinked genes and the inversion rate of the DNA segment containing the pilA promoter in Escherichia coli. A second-site mutation, termed hscA1, compensated for the effect of an hns-1 mutant allele on the pilA promoter inversion rate and on activation of the bgl operon. The proU operon, induced in an hns-1 background, remained derepressed in an hns-1 hscA1 strain and was induced at an intermediate level in an hns hscA1 strain. An insertion mutant allele, hscA2-cat, conferred the same partial hns-1 compensatory phenotype as the hscA1 allele. The hscA gene encoded a 66-kDa protein product that is a member of the Hsp70 protein class. The gene encoding this product is part of a bicistronic operon that is preceded by a possible sigma 32 promoter and also encodes a 21-kDa protein with significant homology to the DnaJ protein family. The mutation defining the hscA1 allele resulted in a phenylalanine substituting a conserved serine residue located in the ATP-binding region of other Hsp70 proteins.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Proteínas HSP70 de Choque Térmico , Proteínas de Choque Térmico/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Proteínas de Unión al ADN/genética , Genes Supresores , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Mapeo RestrictivoRESUMEN
Escherichia coli pilG mutants are thought to have a dramatically higher DNA inversion rate as measured by the site-specific DNA inversion of the type 1 pili pilA promoter. DNA sequence of the pilG gene confirmed its identity to the gene encoding the bacterial histonelike protein H-NS. Unlike other histonelike protein complexes that enhance site-specific DNA recombination, the H-NS protein inhibited this process. This inhibition was indicated by the increased inversion rate of the pilA promoter region effected by two different mutant pilG alleles. One of these alleles, pilG1, conferred a mutant phenotype only at low temperature attributable to a T-to-G transversion in the -35 sequence of the pilG promoter. The other allele, pilG2-tetR, was an insertion mutation in the pilG coding region that conferred the mutant phenotype independent of temperature. We measured an approximately 100-fold-increased pilA promoter inversion rate in the mutant by exploiting the temperature-dependent expression of pilG1 and using a novel rapid-population-sampling method. Contrary to one current view on how the H-NS protein might act to increase DNA inversion rate, we found no evidence to support the hypothesis that DNA supercoiling affected pilA promoter inversion.
Asunto(s)
Proteínas Bacterianas/genética , Inversión Cromosómica , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Mutagénesis Sitio-Dirigida , Alelos , Frío , Elementos Transponibles de ADN , ADN Bacteriano/química , ADN Superhelicoidal/química , Regiones Promotoras Genéticas , Recombinación GenéticaRESUMEN
The pathogenic Neisseria have multiple genes encoding proteins that bind monoclonal antibody (MAb) H.8. We previously reported the cloning and sequencing of a meningococcal gene (laz) encoding an H.8 MAb-binding protein with a consensus lipoprotein processing site, an N-terminal domain containing the epitope for H.8 MAb binding, and a C-terminal domain with extensive similarity to the sequences of azurins from other organisms. In the current study, we showed that the product of the cloned gene could be labelled with palmitic acid, that it was subject to globomycin-sensitive processing, and that it was immunologically cross-reactive with azurin from Pseudomonas aeruginosa. All neisserial species tested, both pathogens and commensals, produced a protein recognized by anti-azurin serum. Southern blots with oligonucleotide probes specific for the azurin domain of the gene showed that it was present in a single copy in the chromosome; it was highly conserved in gonococci and meningococci, and less conserved in commensal Neisseria species.
Asunto(s)
Azurina/genética , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/genética , Epítopos/genética , Genes Bacterianos , Lipoproteínas/metabolismo , Neisseria/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Secuencia de Bases , Clonación Molecular , Reacciones Cruzadas , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Datos de Secuencia Molecular , Neisseria/genética , Ácido Palmítico , Ácidos Palmíticos , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/inmunología , Mapeo Restrictivo , Especificidad de la EspecieRESUMEN
Expression of Neisseria gonorrhoeae Protein II (P.II) is subject to phase variation and antigenic variation. The P.II proteins made by one strain possess both unique and conserved antigenic determinants. To study the mechanism of antigenic variation, we cloned several P.II genes, using as probes a panel of monoclonal antibodies (MAbs) specific for unique determinants. The DNA sequences of three P.II genes showed that they shared a conserved framework, with two short hypervariable (HV) regions being responsible for most of the differences among them. We demonstrated that unique epitopes recognized by the MAbs were at least partially encoded by one of the HV regions. Moreover, we found that reassortment of the two HV regions among P.II genes occurs, generating increased structural and antigenic variability in the P.II protein family.
Asunto(s)
Antígenos Bacterianos/genética , Genes Bacterianos , Genes , Neisseria gonorrhoeae/genética , Recombinación Genética , Secuencia de Bases , Clonación Molecular , Conjugación Genética , Epítopos/análisis , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Neisseria meningitidis class 5 proteins are major outer membrane proteins that share many properties with the proteins II (P.II) of Neisseria gonorrhoeae. We generated two bactericidal monoclonal antibodies, each of which bound specifically to one of the two identified class 5 proteins produced by N. meningitidis FAM18. The monoclonal antibodies also bound to class 5 proteins of a limited number of other meningococcal strains. Using the bactericidal activity of the monoclonal antibodies, we demonstrated that expression of both class 5 proteins was subject to reversible phase variation in vitro. The N-terminal amino acid sequence of a purified class 5 protein revealed striking similarity to the N-terminal amino acid sequence of gonococcal P.II proteins. Using a cloned class 5 gene, we identified three potential class 5 gene loci in N. meningitidis FAM18. These class 5 sequences also had homology with gonococcal P.II gene sequences and contained the CTCTT repeat sequence believed to be important in the regulation of gonococcal P.II expression.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Neisseria gonorrhoeae/inmunología , Neisseria meningitidis/inmunología , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales , Variación Antigénica , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia de Bases , ADN Bacteriano/genética , Regulación de la Expresión Génica , Genes Bacterianos , Datos de Secuencia Molecular , Neisseria gonorrhoeae/genética , Neisseria meningitidis/genéticaRESUMEN
The pathogenic neisseriae, Neisseria gonorrhoeae and Neisseria meningitidis, possess an outer membrane protein, H.8, which contains a conserved monoclonal antibody (MAb)-binding epitope in all strains tested. We have cloned and sequenced a meningococcal H.8 gene, and determined the characteristics of the predicted protein. The predicted signal peptide has features characteristic of a prokaryotic lipoprotein. The region at the N-terminal end of the mature protein (39 amino acids) is primarily composed of alanine, glutamate and proline residues arranged in imperfect repeats with the consensus sequence AAEAP. The epitope for H.8 MAb-binding was localized to a 20-amino-acid sequence within this region. The remainder of the predicted amino acid sequence shows extensive homology to azurins, which are small blue copper-binding proteins found in a limited number of species of pathogenic bacteria.