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Staphylococcus aureus (S. aureus) is often the cause of mastitis problems in dairy herds and causes great economic losses. In this study, isolates from a dairy herd with a known S. aureus mastitis problem were examined by means of molecular methods (spa typing, PFGE, and DNA microarray) to investigate their epidemiological relationship and the success of intervention measures. The investigated dairy farm has a herd size of 60 cows and uses a fully automated milking system for milk production. A S. aureus strain, which contaminated the automated milking system and was subsequently spread among the herd through the latter, was suspected to be the origin of the mastitis problem within the herd. Thanks to the applied molecular methods, the common origin of the S. aureus isolates from the collected milk and swab samples could be shown. By culling chronically infected cows, optimising dry cow management and ensuring reliable intermediate cluster disinfection, the bulk milk somatic cell count improved.
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Industria Lechera/instrumentación , Mastitis Bovina/microbiología , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/aislamiento & purificación , Animales , Bovinos , Recuento de Células/veterinaria , Compuestos Cromogénicos , Industria Lechera/normas , Electroforesis en Gel de Campo Pulsado/veterinaria , Femenino , Mastitis Bovina/prevención & control , Leche/citología , Leche/microbiología , Leche/normas , Análisis de Secuencia por Matrices de Oligonucleótidos/veterinaria , Infecciones Estafilocócicas/etiología , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus/clasificación , Staphylococcus aureus/genéticaRESUMEN
INTRODUCTION: Liver segment definition due to Couinaud is the basis for localisation of focal liver lesions in imaging, in the follow-up or for planning operations. A literature review shows variety in segment definition and the frontier between segment II and III in the left liver lobe, in the course of the portal vein level and in variations of liver veins. The aim of this study is to demonstrate liver segment anatomy in sonography compared to anatomic preparations and the literature. This leads to a proposal for a unique nomenclature and illustration. MATERIAL AND METHODS: 152 liver healthy persons (77 F, 75 M, mean age 63.3 years (18 - 91 years) were examined with standardised abdominal ultrasound in longitudinal, transversal and axis planes. (Angle) measurements were taken to define the left hepatic vein (Fissura sinistra), the Ramus umbilicalis of the portal vein (Fissura umbilicalis), the portal vein level and the amount and variations of the liver veins. RESULTS: The left hepatic vein was found with a mean angle of 24° (0 - 70°) left to the median axis, the Pars umbilicalis of the portal vein wasalmost strictly in the mid axis. The portal vein level was located with a mean angle of 61° (5 - 110°) right to the median with no variations of the two main branches. 27 (18 %) out of the remaining 151 patients showed variations of the liver veins: 7 × (4.6 %) a doubled mid hepatic vein, 12 × (8 %) a doubled left hepatic vein, 4 × (2.7 %) 3 left liver veins were found with a short (≤ 1 cm) common trunk, 1 × each (0.7 %) four left liver veins with a short common trunk, one trifurcation of the mid hepatic vein, one doubled right liver vein and one common trunk (2 cm) of all 3 main liver veins leading to the inferior V. cava. DISCUSSION: The surgical functional liver segment definition by Couinaud is the basis for localisation of focal liver lesions. The frontier between segment II and III is mainly described as a horizontal plane in the literature. The course of the left liver vein (fissura sinistra) has a mean angle of 24° left to the median and not like the umbilical fissure, which is found almost strictly in the median plane. The left hepatic vein(s), their course and liver vein variations are well demonstrated by sonography (99.3 % in this study). Anatomic landmarks as well as variations and a unique nomenclature should be well known and considered in the localisation of focal liver lesions, their feeding vessels and liver segment anatomy.
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Interpretación de Imagen Asistida por Computador/métodos , Hígado/anatomía & histología , Hígado/diagnóstico por imagen , Modelos Anatómicos , Ultrasonografía/métodos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Adulto JovenRESUMEN
Sirtuin2 (Sirt2) with its NAD+-dependent deacetylase and defatty-acylase activities plays a central role in the regulation of specific cellular functions. Dysregulation of Sirt2 activity has been associated with the pathogenesis of many diseases, thus making Sirt2 a promising target for pharmaceutical intervention. Herein, we present new high affinity Sirt2 selective Sirtuin-Rearranging Ligands (SirReals) that inhibit both Sirt2-dependent deacetylation and defatty-acylation in vitro and in cells. We show that simultaneous inhibition of both Sirt2 activities results in strongly reduced levels of the oncoprotein c-Myc and an inhibition of cancer cell migration. Furthermore, we describe the development of a NanoBRET-based assay for Sirt2, thereby providing a method to study cellular target engagement for Sirt2 in a straightforward and accurately quantifiable manner. Applying this assay, we could confirm cellular Sirt2 binding of our new Sirt2 inhibitors and correlate their anticancer effects with their cellular target engagement.
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Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.
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Microscopía por Crioelectrón/métodos , Substitución por Congelación/métodos , Mycobacterium smegmatis/ultraestructura , Fijación del Tejido/métodos , Artefactos , Pared Celular/ultraestructura , Citoplasma/ultraestructura , ADN Bacteriano/ultraestructura , Resinas Epoxi , Microscopía Electrónica de Transmisión/métodos , Microtomía , Mycobacterium smegmatis/efectos de la radiación , Temperatura , Rayos UltravioletaRESUMEN
UNLABELLED: Enuresis nocturna is regularly treated by desmopressin, a vasopressin analog. Its side effects, notably neurological, are fortunately rare. We comment on 5 enuretic children on desmopressin who suffered from hyponatremic encephalopathy (natremia 115-127, median 117 mmol/l). RESULTS: Side effects appeared at therapeutic doses (10-40 mg/d intranasal). An excessive fluid intake at night was often noted, leading to a dilutional hyponatremia. This may be due to a lack of correct information to the parents. These children presented after a period of warning symptoms, such as headache, vomiting and altered consciousness. Parents could have sought earlier medical attention if they had been informed about these symptoms. CONCLUSION: In the absence of fluid restriction, severe hyponatremia can occur in enuretic children on desmopressin. It is therefore mandatory for the prescribing doctor to adequately inform patients and parents to limit fluids at night when desmopressin is used, and seek medical help quickly if any sign of intracranial hypertension appears.
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Fármacos Antidiuréticos/efectos adversos , Coma/inducido químicamente , Confusión/inducido químicamente , Desamino Arginina Vasopresina/efectos adversos , Enuresis/tratamiento farmacológico , Hiponatremia/inducido químicamente , Convulsiones/etiología , Administración Intranasal , Fármacos Antidiuréticos/administración & dosificación , Niño , Preescolar , Coma/diagnóstico , Desamino Arginina Vasopresina/administración & dosificación , Ingestión de Líquidos , Femenino , Escala de Coma de Glasgow , Humanos , Hiponatremia/complicaciones , Hipertensión Intracraneal/inducido químicamente , Masculino , Factores de TiempoRESUMEN
The recombinantly expressed protein indoleglycerol phosphate synthase from the hyperthermophilic bacterium Thermotoga maritima (tIGPS) was purified and characterized with respect to oligomerization state, catalytic properties and thermostability. This enzyme from the biosynthetic pathway of tryptophan is a monomer in solution. In contrast to IGPS from the hyperthermophilic archaeon Sulfolobus solfataricus, tIGPS shows high catalytic activity at room temperature and only weak product inhibition. In order to test the hypothesis that salt bridges in a critical context contribute to the high thermostability of tIGPS, two solvent-exposed salt bridges were selected, based on its three-dimensional structure, for individual disruption by site-directed mutagenesis. The first salt bridge fixes the N terminus to the core of the protein, and the second serves as a clamp between helices alpha1 and alpha8, which are widely separated in sequence but adjacent in the (betaalpha)8-barrel. Kinetics of irreversible heat inactivation reveal that the salt bridge crosslinking helices alpha1 and alpha8 stabilizes tIGPS more strongly than that tethering the N terminus.
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Thermotoga maritima/enzimología , Triptófano Sintasa/metabolismo , Secuencia de Bases , Biopolímeros , Catálisis , Clonación Molecular , Cartilla de ADN , Estabilidad de Enzimas , Calor , Cinética , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Solventes , Triptófano Sintasa/genética , Triptófano Sintasa/aislamiento & purificaciónRESUMEN
Indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus is a monomeric enzyme with the common (beta/alpha)8-fold. Recently, its three-dimensional structure was solved in an orthorhombic crystal form, grown by using 1.3 M ammonium sulfate as precipitating agent. Here we describe the X-ray structure analysis of two new crystal forms of this enzyme that were obtained at medium and low ionic strength, respectively. Hexagonal crystals with space group P3(1)21 and cell dimensions a = 62.4 A, b = 62.4 A, c = 122.9 A, gamma = 120 degrees grew in 0.1 M Mes buffer at pH 6.0 with 30% polyethylene glycol monomethylether as precipitant and 0.2 M ammonium sulfate as co-precipitant. A second crystal form with space group P2(1)2(1)2(1) and cell constants a = 62.6 A, b = 74.0 A, c = 74.2 A was obtained using polyethylene glycol and ethylene glycol as precipitants in 0.1 M Mes buffer at pH 6.5. Both structures were solved by molecular replacement and refined at 2.5 A and 2.0 A resolution, respectively. Although the global folds are almost identical, alternative conformations are observed in flexible loop regions, mostly stabilized by crystal contacts. In none of the three crystal forms is the so-called phosphate binding site empty, suggesting that this position has high affinity for anions with tetrahedrally arranged oxygen atoms. Differences in ionic strength of the crystallization buffer have only minor effects on number and specificity of intramolecular salt bridges. The crystal packing, on the other hand, seems to be influenced by the ionic strength of the solvent, since the number of intermolecular salt bridges in the low ionic strength crystal forms is significantly higher.
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Indol-3-Glicerolfosfato Sintasa/química , Sulfolobus/enzimología , Sitios de Unión , Cristalografía por Rayos X , Concentración Osmolar , Fosfatos/metabolismo , Conformación Proteica , Programas Informáticos , TemperaturaRESUMEN
A series of studies demonstrated a possible correlation between eye-blink rate and central dopamine activity. The hypothesis has been put forward that the antidepressant effect of sleep deprivation (SD) is mediated by an enhanced dopamine release resulting in an amphetaminelike action of SD. Therefore, the blink rates of 12 drug-naive patients with major depression and 12 healthy controls were compared before and after SD and before and after 2.5 mg bromocriptine as a dopaminergic challenge. The main result of the study was that the depressed patients had a significantly higher increase of blinking after SD both with and without a dopaminergic challenge. Basal eye-blink rate was not different in nonretarded major depression patients compared to controls. Sleep deprivation increased blink rate in depression patients but not in controls, and the increase was proportional to improvements in depressive state after sleep deprivation. Bromocriptine did not increase blink rate 1 hour after application. This result is consistent with the hypothesis that antidepressant SD acts through dopamine release, although it is not conclusive, because other neurotransmitters like acetylcholine may be involved in the regulation of blinking.
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Bromocriptina/farmacología , Trastorno Depresivo/fisiopatología , Dopamina/metabolismo , Movimientos Oculares/efectos de los fármacos , Privación de Sueño , Adulto , Humanos , Masculino , Persona de Mediana EdadRESUMEN
PURPOSE: To describe a German family with clinical and genetic evidence of autosomal dominant North Carolina macular dystrophy. METHODS: Twenty-six individuals from a five-generation family from northern Germany were investigated clinically. In addition, we performed genetic linkage analyses using polymorphic markers from proximal 6q. RESULTS: The affected family members showed clinical abnormalities consistent with North Carolina macular dystrophy including multiple drusen, choroidal neovascularization in one patient, and geographic atrophy in elderly patients. The DNA analyses demonstrated significant linkage to the North Carolina macular dystrophy locus on chromosome 6q14-q16.2. CONCLUSION: Our findings provide strong evidence of a German pedigree with an autosomal dominant macular dystrophy manifesting with clinical abnormalities consistent with North Carolina macular dystrophy.
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Cromosomas Humanos Par 6/genética , Genes Dominantes/genética , Degeneración Macular/genética , Adolescente , Adulto , Niño , Preescolar , ADN/análisis , Femenino , Ligamiento Genético/genética , Marcadores Genéticos , Alemania/etnología , Humanos , Escala de Lod , Degeneración Macular/etnología , Degeneración Macular/patología , Masculino , North Carolina , Linaje , Reacción en Cadena de la PolimerasaRESUMEN
OBJECTIVE: The incidence of the serotonin syndrome or serotonin-syndrome-like side effects during treatment with the selective serotonin reuptake inhibitor (SSRI) fluvoxamine should be evaluated. METHOD: 200 inpatients treated for the first time with fluvoxamine were prospectively evaluated for the occurrence of a serotonin syndrome over a period of 8200 treatment days. Retrospective follow-up data of outpatient treatment covered 8891 days. RESULTS: No full-blown serotonin syndrome occurred, but 3 patients developed a reversible change of mental status with insomnia, agitation, confusion and incoherent thoughts without other symptoms of the serotonin syndrome. CONCLUSIONS: It is concluded that the occurrence of a potentially lethal serotonin syndrome is rare in fluvoxamine treatment psychosis-like syndromes as a side effect of serotonergic stimulation might occur. In the investigated sample the rate was 0.006-0.04 per 100 treatment days.
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Antidepresivos de Segunda Generación/efectos adversos , Fluvoxamina/efectos adversos , Psicosis Inducidas por Sustancias/epidemiología , Serotoninérgicos/efectos adversos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Estudios RetrospectivosRESUMEN
In rational drug design the study of protein-ligand-interactions is one of the most important approaches to get knowledge of SAR. On this study N2-Phenylthioguanines were synthesized by Schiemann reaction from thioguanine followed by a substitution of the fluorine by aniline-derivatives. The activity of these HSV1 TK inhibitors was determined by kinetic measurements of thymidine phosphorylation. The N2-Phenylthioguanines gave the same activity as the oxoanalogues. Interaction energies between thymidine and HSV1 TK were measured by microcalorimetry. Results of the measurement showed negative delta G and delta H values which indicates that the binding of the natural substrate occurs spontaneously and is enthalpy driven.
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Guanina/análogos & derivados , Guanina/química , Proteínas/química , Calorimetría , Diseño de Fármacos , Guanina/síntesis química , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Químicos , Simplexvirus/efectos de los fármacos , Espectrofotometría Infrarroja , Relación Estructura-Actividad , Timidina Quinasa/antagonistas & inhibidoresAsunto(s)
Isomerasas Aldosa-Cetosa/aislamiento & purificación , Isomerasas Aldosa-Cetosa/metabolismo , Indol-3-Glicerolfosfato Sintasa/aislamiento & purificación , Indol-3-Glicerolfosfato Sintasa/metabolismo , Thermotoga maritima/enzimología , Triptófano/biosíntesis , Isomerasas Aldosa-Cetosa/genética , Cromatografía/métodos , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Clonación Molecular/métodos , Cristalización , Durapatita , Estabilidad de Enzimas , Escherichia coli , Calor , Indol-3-Glicerolfosfato Sintasa/genética , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Termodinámica , Thermotoga maritima/genéticaRESUMEN
The closely related bacterial pathogens Neisseria gonorrhoeae (gonococci, GC) and N. meningitidis (meningococci, MC) initiate infection at human mucosal epithelia. Colonization begins at apical epithelial surfaces with a multistep adhesion cascade, followed by invasion of the host cell, intracellular persistence, transcytosis, and exit. These activities are modulated by the interaction of a panoply of virulence factors with their cognate host cell receptors, and signals are sent from pathogen to host and host to pathogen at multiple stages of the adhesion cascade. Recent advances place us on the verge of understanding the colonization process at a molecular level of detail. In this review we describe the Neisseria virulence factors in the context of epithelial cell biology, placing special emphasis on the signaling functions of type IV pili, pilus-based twitching motility, and the Opa and Opc outermembrane adhesin/invasin proteins. We also summarize what is known about bacterial intracellular trafficking and growth. With the accelerated integration of tools from cell biology, biochemistry, biophysics, and genomics, experimentation in the next few years should bring unprecedented insights into the interactions of Neisseriae with their host.
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Membrana Celular/microbiología , Neisseria gonorrhoeae/fisiología , Neisseria meningitidis/fisiología , Animales , Células Epiteliales/microbiología , Humanos , Neisseria gonorrhoeae/patogenicidad , Neisseria meningitidis/patogenicidadRESUMEN
Attachment of piliated Neisseria gonorrhoeae or Neisseria meningitidis cells to A431, Chang, HEC-1-B, or polarized T(84) cells triggers rearrangements of cortical microfilaments and the accumulation of phosphotyrosine-containing proteins at sites of bacterial contact. Actin stress fibers and the microtubule network remain unaltered in infected cells. The rearrangements reported here are triggered by piliated, Opa- and Opc- strains and also by nonpiliated gonococci (GC) that produce the invasion-associated OpaA protein. Thus, neisserial adhesion via either of at least two different adhesins can trigger cortical rearrangements. In contrast, these rearrangements are not triggered by nonadherent GC or meningococcal strains, by heat-killed or chloramphenicol-treated GC strains, or by Escherichia coli recombinants that adhere to cells via GC OpaA or Opal fusion proteins, suggesting that additional neisserial components are involved. Immunoblotting experiments did not detect consistent increases in the phosphorylation of specific proteins. Possible biological implications of these Neisseria-induced cortical rearrangements are discussed.
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Citoesqueleto de Actina/ultraestructura , Adhesión Bacteriana/fisiología , Proteínas de la Membrana Bacteriana Externa/genética , Fimbrias Bacterianas , Neisseria/patogenicidad , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actinas/ultraestructura , Antígenos Bacterianos/genética , Células Epiteliales , Epitelio/microbiología , Epitelio/ultraestructura , Microtúbulos/ultraestructura , Neisseria/genética , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/patogenicidad , Neisseria meningitidis/genética , Neisseria meningitidis/patogenicidad , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismoRESUMEN
Approximately 60% of patients with major depression disorder show a beneficial response to total sleep deprivation (TSD), but the positive effect of TSD is short, and naps or the following night's sleep destroy it. Various methods have been tried to stabilize the positive sleep-deprivation effect. A consecutive 1-week advance in the sleep phase stabilized mood in more than 50% of the sleep-deprivation responders. We examined 40 male patients with major depression who in addition to medical treatment took part in a phase advance study to prove possible synergistic effects. About 60% of the patients showed positive mood stabilization after 1-week of treatment when the patients were sleep-deprivation responders. These results support data from other groups.
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Ritmo Circadiano , Trastorno Depresivo/terapia , Privación de Sueño , Fases del Sueño , Adulto , Terapia Combinada , Trastorno Depresivo/psicología , Femenino , Fluvoxamina/uso terapéutico , Humanos , Masculino , Persona de Mediana Edad , Admisión del Paciente , Inventario de Personalidad , Inhibidores Selectivos de la Recaptación de Serotonina/uso terapéutico , Resultado del TratamientoRESUMEN
Twitching and social gliding motility allow many gram negative bacteria to crawl along surfaces, and are implicated in a wide range of biological functions. Type IV pili (Tfp) are required for twitching and social gliding, but the mechanism by which these filaments promote motility has remained enigmatic. Here we use laser tweezers to show that Tfp forcefully retract. Neisseria gonorrhoeae cells that produce Tfp actively crawl on a glass surface and form adherent microcolonies. When laser tweezers are used to place and hold cells near a microcolony, retractile forces pull the cells toward the microcolony. In quantitative experiments, the Tfp of immobilized bacteria bind to latex beads and retract, pulling beads from the tweezers at forces that can exceed 80 pN. Episodes of retraction terminate with release or breakage of the Tfp tether. Both motility and retraction mediated by Tfp occur at about 1 microm s(-1) and require protein synthesis and function of the PilT protein. Our experiments establish that Tfp filaments retract, generate substantial force and directly mediate cell movement.
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Fimbrias Bacterianas/fisiología , Neisseria gonorrhoeae/fisiología , MovimientoRESUMEN
A representative range of pyrimidine nucleoside analogues that are known to inhibit herpes simplex virus (HSV) replication have been used to construct receptor binding site models for the varicella-zoster virus (VZV) thymidine kinase (TK) and human TK1. Given a set of interacting ligands, superimposed in such a manner as to define a pharmacophore, the pseudoreceptor modelling technique Yak provides a means of building binding site models of macromolecules for which no three-dimensional experimental structures are available. Once the models have been evaluated by their ability to reproduce experimental binding data [Vedani et al., J. Am. Chem. Soc., 117 (1995) 4987], they can be used for predictive purposes. Calculated and experimental values of relative binding affinity are compared. Our models suggest that the substitution of one residue may be sufficient to determine ligand subtype affinity.
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Herpesvirus Humano 3/metabolismo , Modelos Moleculares , Receptores Virales/química , Receptores Virales/metabolismo , Timidina Quinasa/química , Timidina Quinasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Simulación por Computador , Herpesvirus Humano 3/química , Humanos , Ligandos , Datos de Secuencia Molecular , Estructura Molecular , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/metabolismo , Receptores Virales/genética , Especificidad por Sustrato , Termodinámica , Timidina Quinasa/genéticaRESUMEN
In a prospective clinical trial, the effect of systemic steroids on the incidence and severity of retinal fibrosis following retinal detachment surgery was tested against placebo. Several grades of retinal fibrosis of various degrees of severity were defined prior to the study. The maximum postoperative incidence of eyes with any signs of retinal fibrosis was 65% (75.4% in the placebo group, 63.3% in the steroid group). A set of fibrosis criteria was identified that had a tendency to respond to systemic steroids with a statistical significance increasing with time. The majority of these steroid-responsive criteria was of a mild degree and located at the posterior pole. The mean visual acuity was significantly lower in eyes with retinal fibrosis. The effect of systemic steroids on retinal fibrosis tested under clinical conditions was considerably weaker than that known from experimental conditions with intravitreal injections of triamcinolone acetonide.
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Prednisona/uso terapéutico , Desprendimiento de Retina/cirugía , Enfermedades de la Retina/prevención & control , Adulto , Anciano , Ensayos Clínicos como Asunto , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/prevención & control , Prednisona/administración & dosificación , Estudios ProspectivosRESUMEN
The pathogenic Neisseriae Neisseria meningitidis and Neisseria gonorrhoeae, initiate colonization by attaching to host cells using type IV pili. Subsequent adhesive interactions are mediated through the binding of other bacterial adhesins, in particular the Opa family of outer membrane proteins. Here, we have shown that pilus-mediated adhesion to host cells by either meningococci or gonococci triggers the rapid, localized formation of dramatic cortical plaques in host epithelial cells. Cortical plaques are enriched in both components of the cortical cytoskeleton and a subset of integral membrane proteins. These include: CD44v3, a heparan sulphate proteoglycan that may serve as an Opa receptor; EGFR, a receptor tyrosine kinase; CD44 and ICAM-1, adhesion molecules known to mediate inflammatory responses; f-actin; and ezrin, a component that tethers membrane components to the actin cytoskeleton. Genetic analyses reveal that cortical plaque formation is highly adhesin specific. Both pilE and pilC null mutants fail to induce cortical plaques, indicating that neisserial type IV pili are required for cortical plaque induction. Mutations in pilT, a gene required for pilus-mediated twitching motility, confer a partial defect in cortical plaque formation. In contrast to type IV pili, many other neisserial surface structures are not involved in cortical plaque induction, including Opa, Opc, glycolipid GgO4-binding adhesins, polysialic acid capsule or a particular lipooligosaccharide variant. Furthermore, it is shown that type IV pili allow gonococci to overcome the inhibitory effect of heparin, a soluble receptor analogue, on gonococcal invasion of Chang and A431 epithelial cells. These and other observations strongly suggest that type IV pili play an active role in initiating neisserial infection of the mucosal surface in vivo. The functions of type IV pili and other neisserial adhesins are discussed in the specific context of the mucosal microenvironment, and a multistep model for neisserial colonization of mucosal epithelia is proposed.