RESUMEN

Chemokine receptors direct T lymphocytes to the site of an infection by following coordinated chemokine gradients, which allow their recruitment to specific tissues. Although identification of receptors needed for homing to some mucosal sites, such as skin and gut, have been elucidated, the receptors that direct lymphocytes to the genital mucosa remain relatively uncharacterized. In this study we identify that the chemokine receptors CXCR3 (chemokine (C-X-C motif) receptor 3) and CCR5 (chemokine (C-C motif) receptor 5) are pivotal for T-lymphocyte access to the genital tract during Chlamydia trachomatis infection. Chlamydia-specific CD4(+) transgenic T cells that lack CXCR3 or CCR5 do not accumulate in the genital mucosa following infection. Loss of either CXCR3 or CCR5 impairs the protective capacity of Chlamydia-specific T cells, whereas T cells lacking both receptors are completely nonprotective. These results show that CXCR3 and CCR5 are the predominant chemokine receptors that act cooperatively to promote homing to the genital mucosa during Chlamydia infection.

Asunto(s)

Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Membrana Mucosa/inmunología , Membrana Mucosa/microbiología , Receptores CCR5/inmunología , Receptores CXCR3/inmunología , Linfocitos T/inmunología , Animales , Movimiento Celular/inmunología , Femenino , Enfermedades de los Genitales Femeninos/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores CCR5/genética , Receptores CXCR3/genética , Receptores de Quimiocina/inmunología

RESUMEN

Chlamydia trachomatis is a bacterial pathogen that is a major cause of blindness and infertility in diverse populations across the world. In an effort to model genetic complexities that are observed in human populations and to identify novel genes involved in susceptibility to C. trachomatis, we have adapted a murine model of systemic infection for use in genetic analysis. In this model, chlamydial colonization and replication is measured in the spleens of mice shortly after intravenous delivery of C. trachomatis L2. Here, we show that C57BL/6J and C3H/HeJ inbred mice are differentially susceptible to this systemic infection. Additionally, fibroblasts cultured from C57BL/6J and C3H/HeJ embryos are differentially permissive for chlamydial replication. We have taken advantage of this natural variation to map quantitative trait loci on Chromosomes 2, 3, and 11 that segregate with the bacterial load in F2 cross progeny during the acute phase of C. trachomatis infection in vivo. To validate our mapping results, we also generated mice that are congenic for a portion of Chromosome 11 from the susceptible parent. This congenic interval confers increased susceptibility to C. trachomatis, both in vivo and in vitro, suggesting that our screen identified at least one gene that is involved in cellular resistance to C. trachomatis replication.

Asunto(s)

Infecciones por Chlamydia/genética , Chlamydia trachomatis/patogenicidad , Animales , Células Cultivadas , Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/clasificación , Chlamydia trachomatis/fisiología , Mapeo Cromosómico , Cromosomas , ADN Bacteriano/análisis , Susceptibilidad a Enfermedades , Femenino , Fibroblastos/citología , Fibroblastos/inmunología , Fibroblastos/microbiología , Ligamiento Genético , Marcadores Genéticos , Cinética , Ratones , Ratones Congénicos , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Embarazo , Sitios de Carácter Cuantitativo , Serotipificación , Especificidad de la Especie , Bazo/microbiología

RESUMEN

CD8(+) T cells are required for protective immunity against intracellular pathogens such as Listeria monocytogenes. In this study, we used class Ia MHC-deficient mice, which have a severe reduction in circulating CD8(+) T cells, to determine the protective capacity of class Ib MHC-restricted T cells during L. monocytogenes infection. The K(b-/-)D(b-/-) mutation was backcrossed onto a C.B10 (BALB/c congenic at H-2 locus with C57BL/10) background, because BALB/c mice are more susceptible to Listeria infection than other commonly studied mouse strains such as C57BL/6. C.B10 K(b-/-)D(b-/-) mice immunized with a sublethal dose of L. monocytogenes were fully protected against a subsequent lethal infection. Adoptive transfer of Listeria-immune splenocyte subsets into naive K(b-/-)D(b-/-) mice indicated that CD8(+) T cells were the major component of this protective immune response. A CD8(+) T cell line isolated from the spleen of a Listeria-infected class Ia MHC-deficient mouse was shown to specifically recognize Listeria-infected cells in vitro, as determined by IFN-gamma secretion and cytotoxicity assays. Adoptive transfer of this T cell line alone resulted in significant protection against L. monocytogenes challenge. These results suggest that even a limited number of class Ib MHC-restricted T cells are sufficient to generate the rapid recall response required for protection against secondary infection with L. monocytogenes.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica/genética , Antígenos H-2/genética , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Listeriosis/prevención & control , Activación de Linfocitos/genética , Traslado Adoptivo , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/trasplante , División Celular/genética , División Celular/inmunología , Línea Celular , Separación Celular , Cruzamientos Genéticos , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Antígenos H-2/inmunología , Antígeno de Histocompatibilidad H-2D , Inmunidad Activa/genética , Listeria monocytogenes/crecimiento & desarrollo , Listeriosis/genética , Listeriosis/microbiología , Ratones , Ratones Congénicos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Bazo/inmunología , Bazo/patología , Células Tumorales Cultivadas

RESUMEN

We have used a novel quantitative trait locus model to study the genetics of survival of F2 progeny of susceptible BALB/cByJ and resistant C57BL/6ByJ mice that have been infected with Listeria monocytogenes. This allowed us to map modifiers of L. monocytogenes susceptibility to chromosomes 5 and 13.

Asunto(s)

Listeriosis/genética , Listeriosis/inmunología , Animales , Mapeo Cromosómico , Cruzamientos Genéticos , Femenino , Listeriosis/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Carácter Cuantitativo Heredable

RESUMEN

During infection with Chlamydia trachomatis, CD8(+) T cells are primed, even though the bacteria remain confined to a host cell vacuole throughout their developmental cycle. Because CD8(+) T cells recognize antigens processed from cytosolic proteins, the Chlamydia antigens recognized by these CD8(+) T cells very likely have access to the host cell cytoplasm during infection. The identity of these C. trachomatis proteins has remained elusive, even though their localization suggests they may play important roles in the biology of the organism. Here we use a retroviral expression system to identify Cap1, a 31-kDa protein from C. trachomatis recognized by protective CD8(+) T cells. Cap1 contains no strong homology to any known protein. Immunofluorescence microscopy by using Cap1-specific antibody demonstrates that this protein is localized to the vacuolar membrane. Cap1 is virtually identical among the human C. trachomatis serovars, suggesting that a vaccine incorporating Cap1 might enable the vaccine to protect against all C. trachomatis serovars. The identification of proteins such as Cap1 that associate with the inclusion membrane will be required to fully understand the interaction of C. trachomatis with its host cell.

Asunto(s)

Proteínas Bacterianas/inmunología , Linfocitos T CD8-positivos/inmunología , Chlamydia trachomatis/inmunología , Proteínas de la Membrana/inmunología , Animales , Proteínas Bacterianas/genética , Secuencia de Bases , Línea Celular , Células Cultivadas , Chlamydia trachomatis/genética , Chlamydia trachomatis/patogenicidad , Femenino , Biblioteca de Genes , Inmunohistoquímica , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , Vacuolas/microbiología

Asunto(s)

Antígenos Bacterianos , Bacillus anthracis/inmunología , Toxinas Bacterianas/inmunología , Vacunas Bacterianas , Linfocitos T Citotóxicos/inmunología , Vacunas Sintéticas , Animales , Fusión Artificial Génica , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Secuencia de Bases , Citotoxicidad Inmunológica , Cartilla de ADN , Epítopos/genética , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes de Fusión/inmunología

RESUMEN

We have investigated the use of the protective antigen (PA) and lethal factor (LF) components of anthrax toxin as a system for in vivo delivery of cytotoxic T-lymphocyte (CTL) epitopes. During intoxication, PA directs the translocation of LF into the cytoplasm of mammalian cells. Here we demonstrate that antiviral immunity can be induced in BALB/c mice immunized with PA plus a fusion protein containing the N-terminal 255 amino acids of LF (LFn) and an epitope from the nucleoprotein (NP) of lymphocytic choriomeningitis virus. We also demonstrate that BALB/c mice immunized with a single LFn fusion protein containing NP and listeriolysin O protein epitopes in tandem mount a CTL response against both pathogens. Furthermore, we show that NP-specific CTL are primed in both BALB/c and C57BL/6 mice when the mice are immunized with a single fusion containing two epitopes, one presented by Ld and one presented by Db. The data presented here demonstrate the versatility of the anthrax toxin delivery system and indicate that this system may be used as a general approach to vaccinate outbred populations against a variety of pathogens.

Asunto(s)

Antígenos Bacterianos , Antígenos Virales/inmunología , Toxinas Bacterianas/inmunología , Citotoxicidad Inmunológica , Virus de la Coriomeningitis Linfocítica/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Presentación de Antígeno , Antígenos Virales/genética , Antivirales/inmunología , Toxinas Bacterianas/genética , Epítopos/genética , Epítopos/inmunología , Inmunoterapia/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/inmunología , Virosis/inmunología

RESUMEN

In this study, we used mice in which the gene for gamma interferon (IFN-gamma) has been disrupted (IFN-gamma-/- mice) to study the role of this cytokine in the resolution of Chlamydia trachomatis infection. We show that IFN-gamma-/- mice are impaired in the ability to clear infection with C. trachomatis compared to IFN-gamma+/+ control mice. Activated CD8(+) cytotoxic T lymphocytes (CTL) secrete IFN-gamma in response to intracellular infection, and we have shown previously that a Chlamydia-specific CTL line can reduce C. trachomatis infection when adoptively transferred into infected mice. In the present study, we found that when these IFN-gamma+/+ CTL lines are transferred into Chlamydia-infected IFN-gamma-/- mice, the transferred CTL cannot overcome the immune defect seen in the IFN-gamma-/- mice. We also show that Chlamydia-specific CTL can be cultured from IFN-gamma-deficient mice infected with C. trachomatis; however, the adoptive transfer of IFN-gamma-/- CTL into infected IFN-gamma+/+ mice does not reduce the level of infection. These results suggest that IFN-gamma production by CTL is not sufficient to overcome the defect that IFN-gamma-/- mice have in the resolution of Chlamydia infection, yet IFN-gamma production by CTL is required for the protective effect seen upon adoptive transfer of CTL into IFN-gamma+/+ mice.

Asunto(s)

Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Interferón gamma/biosíntesis , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Células 3T3 , Traslado Adoptivo , Animales , Línea Celular , Células Cultivadas , Infecciones por Chlamydia/genética , Infecciones por Chlamydia/prevención & control , Citotoxicidad Inmunológica/genética , Femenino , Inmunidad Innata/genética , Interferón gamma/deficiencia , Interferón gamma/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Citotóxicos/trasplante

RESUMEN

We have previously demonstrated that anthrax toxin-derived proteins, protective antigen (PA) and the amino-terminal portion of lethal factor (LFn), can be used in combination to deliver heterologous molecules to the cytosol of mammalian cells. In this study we examined the ability of an LFn-peptide disulfide-linked heterodimer to prime cytotoxic T lymphocytes (CTL) in the presence of PA. A mutant of LFn that contains a carboxy-terminal reactive cysteine was generated. This form of LFn could be oxidized with a synthetic cysteine containing peptide to form a heterodimer of the protein and peptide. Mice injected with the heterodimer plus PA mounted a peptide-specific CTL response, indicating that this molecule functioned similarly to the genetically fused forms used previously. We also report the results of an analysis of two aspects of this system important for the development of experimental vaccines. First, CD4 knockout mice were unable to generate a CTL response when treated with PA plus an LFn-epitope fusion protein, suggesting that CD4(+) helper responses are essential for stimulating specific CTL with the PA-LFn system. Second, we now show that primary injection with this system does not generate any detectable antibody response to the vaccine components and that prior immunization has no effect on priming a CTL response to an unrelated epitope upon subsequent injection.

Asunto(s)

Antígenos Bacterianos , Toxinas Bacterianas/inmunología , Epítopos/inmunología , Inmunoconjugados/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Toxinas Bacterianas/química , Antígenos CD4/genética , Linfocitos T CD4-Positivos , Cisteína , Disulfuros , Antígenos H-2/genética , Ratones , Ratones Noqueados , Ovalbúmina/inmunología

RESUMEN

We reported earlier that a nontoxic form of anthrax toxin was capable of delivering a cytotoxic T-lymphocyte (CTL) epitope in vivo, such that a specific CTL response was primed against the epitope. The epitope, of bacterial origin, was fused to an N-terminal fragment (LFn) from the lethal-factor component of the toxin, and the fusion protein was injected, together with the protective antigen (PA) component, into BALB/c mice. Here we report that PA plus LFn is capable of delivering a different epitope--OVA(257-264) from ovalbumin. Delivery was accomplished in a different mouse haplotype, H-2Kb and occurred in vitro as well as in vivo. An OVA(257-264)-specific CTL clone, GA-4, recognized EL-4 cells treated in vitro with PA plus as little as 30 fmol of the LFn-OVA(257-264) fusion protein. PA mutants attenuated in toxin self-assembly or translocation were inactive, implying that the role of PA in epitope delivery is the same as that in toxin action. Also, we showed that OVA(257-264)-specific CTL could be induced to proliferate by incubation with splenocytes treated with PA plus LFn-OVA(257-264). These findings imply that PA-LFn may serve as a general delivery vehicle for CTL epitopes in vivo and as a safe, efficient tool for the ex vivo expansion of patient-derived CTL for use in adoptive immunotherapy.

Asunto(s)

Antígenos Bacterianos , Toxinas Bacterianas/administración & dosificación , Epítopos de Linfocito T , Ovalbúmina/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Femenino , Inmunoterapia Adoptiva , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/inmunología

RESUMEN

The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor into the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (LFn; 255 amino acids) binds LF to PA, and when fused to heterologous proteins, the LFn domain delivers such proteins to the cytoplasm in the presence of PA. In the current study, we fused a 9-amino acid cytotoxic T-lymphocyte (CTL) epitope (LLO91-99) from an intracellular pathogen, Listeria monocytogenes, to LFn and measured the ability of the resulting LFn-LLO91-99 fusion protein to stimulate a CTL response against the epitope in BALB/c mice. As little as 300 fmol of fusion could stimulate a response. The stimulation was PA-dependent and occurred with the peptide fused to either the amino terminus or the carboxyl terminus of LFn. Upon challenge with L. monocytogenes, mice previously injected with LFn-LLO91-99 and PA showed a reduction of colony-forming units in spleen and liver, relative to nonimmunized control mice. These results indicate that anthrax toxin may be useful as a CTL-peptide delivery system for research and medical applications.

Asunto(s)

Carbunco , Antígenos Bacterianos , Bacillus anthracis , Toxinas Bacterianas/metabolismo , Epítopos de Linfocito T/metabolismo , Linfocitos T Citotóxicos/metabolismo , Animales , Listeria monocytogenes , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos

RESUMEN

The obligate intracellular bacterium Chlamydia trachomatis is associated with human diseases ranging from blinding trachoma to sexually acquired genital infections and the systemic disease lymphogranuloma venereum (LGV). We have previously reported the isolation and culture of protective murine cytotoxic T lymphocytes (CTL) following intraperitoneal infection with C. trachomatis serovar L2, a serotype associated with human LGV. In this report, we now demonstrate that CTL can also be primed following introduction of C. trachomatis serovar L2 into the uterus or ovarian bursa of mice. We also describe Chlamydia-specific CTL lines isolated following murine infection with a typical human urogenital isolate of C. trachomatis (serovar D) and show that such CTL can be primed by intraperitoneal, intrauterine, or intrabursal infection. Last, we demonstrate that these murine CTL lines respond to multiple serovars, recognizing and lysing cells infected with C. trachomatis serovars B, C, D, F, J, K, L2, and L3, representative of organisms causing blinding trachoma, genital infection, and LGV.

Asunto(s)

Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Enfermedades de los Genitales Femeninos/inmunología , Linfocitos T Citotóxicos/inmunología , Células 3T3 , Animales , Línea Celular , Femenino , Ratones , Ratones Endogámicos BALB C

RESUMEN

T cell responses are often an important component in immunity to organisms that replicate intracellularly. Cytotoxic T lymphocyte (CTL) recognition of peptide Ag in the context of MHC class I molecules results in lysis of infected cells and the release of cytokines including IFN-gamma. Members of the genus Chlamydia are obligate intracellular pathogens that cause blindness and sexually transmitted disease worldwide. Even though it replicates within a membrane-bound vacuole, Chlamydia trachomatis may elicit a CTL response if Chlamydia Ags are present in the cytoplasmic compartment where they can be processed for presentation and bound by MHC class I. In this study, we characterized a CTL line derived from mice infected with C. trachomatis. This CTL line is specific for, and able to lyse, Chlamydia-infected cells. The peptide epitope recognized by this CTL line is present on infected cells, and is presented to the CTL by the classical MHC class I molecule H-2 Ld. Adoptive transfer of this CTL line into an infected mouse affords protection, and this protection requires the activity of IFN-gamma.

Asunto(s)

Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/prevención & control , Chlamydia trachomatis/inmunología , Inmunoterapia Adoptiva , Linfocitos T Citotóxicos/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Cultivadas , Infecciones por Chlamydia/terapia , Pruebas Inmunológicas de Citotoxicidad , Epítopos/inmunología , Femenino , Antígenos H-2/inmunología , Interferón gamma/fisiología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Bazo/citología

RESUMEN

Bacteria of the genus Yersinia cause disease in humans ranging from enteritis and lymphadenitis to plaque. These organisms have the ability to bind to host cells and, in a complex interaction, are able to deliver a specific subset of proteins into the host cell. These proteins alter host structures and functions, and in the case of macrophages, prevent phagocytosis of the organism. In Yersinia enterocolitica, one of these proteins, Yop51, is known to be a protein tyrosine phosphatase. By using a directed approach, we have raised murine CTL that recognize a peptide epitope from Yop51 in the context of the MHC class I molecule H-2 Db. This epitope, Yop51 249-257, seems to be presented both by cells that produce Yop51 endogenously and by epithelial cells infected with virulent Y. enterocolitica. In vivo, Y. enterocolitica is believed to remain mostly in an extracellular niche. Therefore, detection of this organism by cytotoxic T cells and, possibly, resistance to disease may depend on recognition of epitopes from a subset of virulence determinants delivered into the cytoplasm of host cells by surface-bound organisms.

Asunto(s)

Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Tirosina Fosfatasas , Linfocitos T Citotóxicos/inmunología , Yersiniosis/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Secuencia de Bases , Citoplasma/inmunología , Citotoxicidad Inmunológica , Femenino , Antígenos H-2/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Péptidos/inmunología

RESUMEN

Salmonella typhimurium grown under oxygen-limiting conditions were found to enter into, elicit actin filament rearrangement in, and effect morphological changes upon HEp-2 cells within 15 min after infection. Video microscopy revealed that host cell morphological changes associated with entry began within 1 min of productive adherence. Polarized Caco-2 cell morphology was affected 40 s after infection with low-oxygen-grown S. typhimurium. Stationary-phase S. typhimurium did not elicit these phenomena within this time-period even when adherence was enhanced with the afimbial adhesin, AFA-I. Thus, environmental cues regulate S. typhimurium invasion factors, allowing for immediate entry into host cells. Additionally, actin filament rearrangement and morphological changes in the eukaryotic host cell are essential for entry and occur within minutes of infection.

Asunto(s)

Actinas/metabolismo , Adhesión Bacteriana/fisiología , Epitelio/microbiología , Oxígeno/fisiología , Salmonella typhimurium/patogenicidad , Adhesinas de Escherichia coli , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Línea Celular , Movimiento Celular/fisiología , Citoesqueleto/metabolismo , Perros , Células Epiteliales , Epitelio/ultraestructura , Humanos , Microscopía , Salmonella typhimurium/crecimiento & desarrollo , Células Tumorales Cultivadas

RESUMEN

In order to better understand the regulation of Pseudomonas aeruginosa flagellin expression we cloned the sigma factor of RNA polymerase used to transcribe the flagellin gene. It is a member of the sigma 28 class of alternative sigma factors described in several bacterial genera. Using the published sequence of the fliA gene encoding the sigma 28 from Salmonella typhimurium, we designed two oligonucleotides and, using the polymerase chain reaction, isolated the fliA gene from S. typhimurium chromosomal DNA. This heterologous probe was used in the DNA blot analysis of restriction digests of P. aeruginosa DNA. A 1.7 kb SalI-EcoRI fragment reacted with the probe and this fragment was cloned into the pBluescript vectors. The P. aeruginosa fliA gene was able to complement the motility defect of an Escherichia coli fliA mutant, but only when transcription was driven from the vector promoter. Insertional inactivation of the fliA gene with a gentamicin gene cassette rendered P. aeruginosa nonmotile and unable to express the flagellin gene. The 1.7 kb cloned fragment was sequenced and shown to contain the entire fliA gene. P. aeruginosa FliA shares 67% amino acid similarity with the homologous S. typhimurium sequence. Transcriptional analysis of the fliA gene showed that its expression was not dependent on RpoN, a sigma factor shown also to be required for flagellin synthesis. A reading frame downstream of fliA was found to encode the P. aeruginosa homologue of the enterobacterial cheY gene.

Asunto(s)

Proteínas Bacterianas/genética , Flagelina/biosíntesis , Pseudomonas aeruginosa/genética , Factor sigma/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Flagelina/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Prueba de Complementación Genética , Modelos Genéticos , Datos de Secuencia Molecular , Pseudomonas aeruginosa/metabolismo , Factor sigma/metabolismo , Transcripción Genética

RESUMEN

A sensitive detection system specific for Legionella pneumophila in water was developed. This system is based on amplification of a chromosomal DNA sequence from L. pneumophila by the polymerase chain reaction, followed by detection of the amplified product by hybridization of a radiolabeled oligodeoxynucleotide. After 35 cycles of amplification, a water sample which had been seeded with 35 CFU of L. pneumophila contained sufficient amplified DNA to be detected on dot blots. Bacteria of other genera tested did not generate positive signals under these conditions. Application of this technique to environmental water samples may help identify the natural reservoirs of nosocomial and community-acquired L. pneumophila infections.

Asunto(s)

ADN Bacteriano/análisis , Amplificación de Genes , Legionella/aislamiento & purificación , Hibridación de Ácido Nucleico , Microbiología del Agua , Southern Blotting , Sondas de ADN , Electroforesis en Gel de Agar , Heces/microbiología , Humanos , Legionella/genética , Especificidad de la Especie

RESUMEN

Surface protein mutants of the invasive Salmonella species, S. choleraesuis, were generated using the transposon TnphoA. 626 alkaline phosphatase (PhoA+) fusion mutants were identified and screened for their ability to pass through (transcytose) polarized epithelial monolayers of Madin Darby canine kidney (MDCK) cells grown on membrane filters. Forty two mutants were unable to pass through this barrier. All of these transcytosis mutants were unable to adhere to or invade MDCK monolayers, yet these mutations were not in the genes encoding type 1 pili or mannose-resistant haemagglutination (MRHA). These transcytosis mutants could be grouped into six classes. Class 1 mutants had altered lipopolysaccharide (LPS) O side-chain structures while Class 2 mutants had defects in their LPS core. Mutants belonging to Classes 5 and 6 did not decrease the transepithelial electrical resistance of polarized MDCK cell monolayers, in contrast to the parental strain and the other mutants (Classes 1, 2, 3 and 4). Mutants belonging to Class 1 were less virulent in mice, while Class 2 (defective core) and Classes 4 and 5 (normal LPS) mutant strains were avirulent in mice. Mutants from Classes 3 and 6 were as virulent in mice as S. choleraesuis. These results suggest that the ability to pass through epithelial barriers may be an important virulence characteristic of Salmonella. These data indicate that bacterial adherence, internalization and monolayer transcytosis are closely linked events. It was also demonstrated that a mutant with decreased rates of intracellular replication still passed through the monolayer at rates similar to wild-type S. choleraesuis.

Asunto(s)

Fosfatasa Alcalina/genética , Elementos Transponibles de ADN , Genes Bacterianos , Salmonella/genética , Animales , Adhesión Bacteriana , División Celular , Células Cultivadas , ADN Bacteriano/genética , Lipopolisacáridos/genética , Ratones , Mutación , Proteínas Recombinantes de Fusión/genética , Salmonella/patogenicidad , Salmonella/fisiología , Virulencia