RESUMEN
There is a lack of insight into the basic mechanisms by which Bordetella pertussis adapts to the local host environment during infection. We analyzed B. pertussis gene expression in the upper and lower airways of mice and compared this to SO4-induced in vitro Bvg-regulated gene transcription. Approximately 30% of all genes were differentially expressed between in vitro and in vivo conditions. This included several novel potential vaccine antigens that were exclusively expressed in vivo. Significant differences in expression profile and metabolic pathways were identified between the upper versus the lower airways, suggesting distinct antigenic profiles. We found high-level expression of several Bvg-repressed genes during infection, and mouse vaccination experiments using purified protein fractions from both Bvg- and Bvg+ cultures demonstrated protection against intranasal B. pertussis challenge. This study provides novel insights into the in vivo adaptation of B. pertussis and may facilitate the improvement of pertussis vaccines.
Asunto(s)
Bordetella pertussis/patogenicidad , Sistema Respiratorio/microbiología , Tos Ferina/microbiología , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Bordetella pertussis/genética , Femenino , Regulación Bacteriana de la Expresión Génica/genética , Ratones , Ratones Endogámicos BALB C , Factores de Transcripción/genéticaRESUMEN
The pneumococcal capsular serotype is an important determinant of complement resistance and invasive disease potential, but other virulence factors have also been found to contribute. Pneumococcal surface protein C (PspC), a highly variable virulence protein that binds complement factor H to evade C3 opsonization, is divided into two subgroups: choline-bound subgroup I and LPxTG-anchored subgroup II. The prevalence of different PspC subgroups in invasive pneumococcal disease (IPD) and functional differences in complement evasion are unknown. The prevalence of PspC subgroups in IPD isolates was determined in a collection of 349 sequenced strains of Streptococcus pneumoniae isolated from adult patients. pspC deletion mutants and isogenic pspC switch mutants were constructed to study differences in factor H binding and complement evasion in relation to capsule thickness. Subgroup I pspC was far more prevalent in IPD isolates than subgroup II pspC The presence of capsule was associated with a greater ability of bound factor H to reduce complement opsonization. Pneumococcal subgroup I PspC bound significantly more factor H and showed more effective complement evasion than subgroup II PspC in isogenic encapsulated pneumococci. We conclude that variation in the PspC subgroups, independent of capsule serotypes, affects pneumococcal factor H binding and its ability to evade complement deposition.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas del Sistema Complemento/inmunología , Genotipo , Infecciones Neumocócicas/inmunología , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/inmunología , Anciano , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Proteínas del Sistema Complemento/metabolismo , Femenino , Humanos , Evasión Inmune , Masculino , Persona de Mediana Edad , Tipificación Molecular , Mutación , Infecciones Neumocócicas/epidemiología , Prevalencia , Serogrupo , Virulencia/genética , Factores de Virulencia/genéticaRESUMEN
PlsX is an acyl-acyl carrier protein (ACP):phosphate transacylase that interconverts the two acyl donors in Gram-positive bacterial phospholipid synthesis. The deletion of plsX in Staphylococcus aureus results in a requirement for both exogenous fatty acids and de novo type II fatty acid biosynthesis. Deletion of plsX (SP0037) in Streptococcus pneumoniae did not result in an auxotrophic phenotype. The ΔplsX S. pneumoniae strain was refractory to myristic acid-dependent growth arrest, and unlike the wild-type strain, was susceptible to fatty acid synthesis inhibitors in the presence of exogenous oleate. The ΔplsX strain contained longer chain saturated fatty acids imparting a distinctly altered phospholipid molecular species profile. An elevated pool of 18- and 20-carbon saturated fatty acids was detected in the ΔplsX strain. A S. pneumoniae thioesterase (TesS, SP1408) hydrolyzed acyl-ACPâ in vitro, and the ΔtesS ΔplsX double knockout strain was a fatty acid auxotroph. Thus, the TesS thioesterase hydrolyzed the accumulating acyl-ACP in the ΔplsX strain to liberate fatty acids that were activated by fatty acid kinase to bypass a requirement for extracellular fatty acid. This work identifies tesS as the gene responsible for the difference in exogenous fatty acid growth requirement of the ΔplsX strains of S. aureus and S. pneumoniae.
Asunto(s)
Proteínas Bacterianas/genética , Ácidos Grasos/metabolismo , Eliminación de Secuencia , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/genética , Tioléster Hidrolasas/metabolismo , Proteína Transportadora de Acilo/metabolismo , Secuencia de Bases , Ácidos Grasos/biosíntesis , Ácidos Grasos/genética , Técnicas de Inactivación de Genes , Ácido Mirístico/metabolismo , Fenotipo , Fosfolípidos/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Streptococcus pneumoniae/metabolismo , Tioléster Hidrolasas/genéticaRESUMEN
The complement system is an important innate defence mechanism, and the ability to resist complement-mediated killing is considered a key virulence trait of the respiratory tract pathogen M. catarrhalis. We studied the molecular basis of complement resistance by transcriptional profiling and Tn-seq, a genome-wide negative-selection screenings technology. Exposure of M. catarrhalis to human serum resulted in increased expression of 84 genes and reduced expression of 134 genes, among which genes encoding ABC transporter systems and surface proteins UspA1 and McaP. By subjecting a â¼ 15 800 transposon mutant library to serum, mutants of 53 genes were negatively selected, including the key complement-resistance factor uspA2H. Validation with directed mutants confirmed Tn-seq phenotypes of uspA2H and 11 newly identified genes, with mutants of MCR_0424, olpA, MCR_1483, and dsbB most severely attenuated. Detailed analysis showed that both components of the disulphide bond formation (DSB) system, DsbB and DsbA, were required for complement-resistance in multiple isolates, and fulfil a critical role in evasion of IgG-dependent classical pathway-mediated killing. Lipooligosaccharide (LOS) structure and membrane stability were severely affected in ΔdsbA strains, suggesting a pivotal role for the DSB system in LOS structure safeguarding and membrane stability maintenance.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Disulfuros/metabolismo , Moraxella catarrhalis/enzimología , Moraxella catarrhalis/inmunología , Oxidorreductasas/metabolismo , Factores de Virulencia/metabolismo , Actividad Bactericida de la Sangre , Perfilación de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Moraxella catarrhalis/genética , Moraxella catarrhalis/metabolismo , Mutagénesis Insercional , Oxidorreductasas/genética , Análisis de Secuencia de ADN , Factores de Virulencia/genéticaRESUMEN
Acellular pertussis (aP) booster vaccines are central to pertussis immunization programs, although their effectiveness varies. The Bacille Calmette-Guérin (BCG) vaccine is a prototype inducer of trained immunity, which enhances immune responses to subsequent infections or vaccinations. While previous clinical studies have demonstrated that trained immunity can protect against heterologous infections, its effect on aP vaccines in humans is unknown. We conducted a clinical study in order to determine the immunological effects of trained immunity on pertussis vaccination. Healthy female volunteers were randomly assigned to either receive BCG followed by a booster dose of tetanus-diphteria-pertussis inactivated polio vaccine (Tdap-IPV) 3 months later (BCG-trained), BCG + Tdap-IPV concurrently, or Tdap-IPV followed by BCG 3 months later. Primary outcomes were pertussis-specific humoral, T- and B-cell responses and were quantified at baseline of Tdap-IPV vaccination and 2 weeks thereafter. As a secondary outcome in the BCG-trained cohort, ex vivo leukocyte responses were measured in response to unrelated stimuli before and after BCG vaccination. BCG vaccination 3 months prior to, but not concurrent with, Tdap-IPV improves pertussis-specific Th1-cell and humoral responses, and also increases total memory B cell responses. These responses were correlated with enhanced IL-6 and IL-1ß production at the baseline of Tdap-IPV vaccination in the BCG-trained cohort. Our study demonstrates that prior BCG vaccination potentiates immune responses to pertussis vaccines and that biomarkers of trained immunity are the most reliable correlates of those responses.
RESUMEN
Type I IFNs, such as interferon alpha and interferon beta, are key regulators of the adaptive immune response during infectious diseases. Type I IFNs are induced upon infection, bind interferon α/ß receptors on T-cells and activate intracellular pathways. The activating and inhibitory consequences of type I IFN-signaling are determined by cell type and cellular environment. The neonatal immune system is associated with increased vulnerability to infectious diseases which could partly be explained by an immature CD4+ T-cell compartment. Here, we show low IFN-ß-mediated inhibition of CD4+ T-cell proliferation, phosphorylation of retinoblastoma protein and cytokine production in human newborns compared to adults. In addition, both naïve and total newborn CD4+ T-cells are unable to induce the cell-cycle inhibitor p21 upon exposure to IFN-ß in contrast to adults. The distinct IFN-ß-signaling in newborns provides novel insights into T cell functionality and regulation of T cell-dependent inflammation during early life immune responses.
Asunto(s)
Inmunidad Adaptativa/fisiología , Linfocitos T CD4-Positivos/inmunología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/deficiencia , Interferón beta/metabolismo , Transducción de Señal/inmunología , Inmunidad Adaptativa/efectos de los fármacos , Adulto , Factores de Edad , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Sangre Fetal/citología , Sangre Fetal/inmunología , Citometría de Flujo , Humanos , Separación Inmunomagnética , Recién Nacido , Cultivo Primario de Células , Receptor de Interferón alfa y beta/antagonistas & inhibidores , Receptor de Interferón alfa y beta/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and it has infected over 100 million people in less than a year. There is a clear need for effective antiviral drugs to complement current preventive measures, including vaccines. In this study, we demonstrate that berberine and obatoclax, two broad-spectrum antiviral compounds, are effective against multiple isolates of SARS-CoV-2. Berberine, a plant-derived alkaloid, inhibited SARS-CoV-2 at low micromolar concentrations and obatoclax, which was originally developed as an anti-apoptotic protein antagonist, was effective at sub-micromolar concentrations. Time-of-addition studies indicated that berberine acts on the late stage of the viral life cycle. In agreement, berberine mildly affected viral RNA synthesis, but it strongly reduced infectious viral titers, leading to an increase in the particle-to-pfu ratio. In contrast, obatoclax acted at the early stage of the infection, which is in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells that were cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of specific sets of cytokines and chemokines. Moreover, both obatoclax and berberine inhibited SARS-CoV-2 replication in these primary target cells. We propose berberine and obatoclax as potential antiviral drugs against SARS-CoV-2 that could be considered for further efficacy testing.
Asunto(s)
Antivirales/farmacología , Berberina/farmacología , Indoles/farmacología , Pirroles/farmacología , SARS-CoV-2/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Adolescente , Animales , COVID-19/virología , Células Cultivadas , Chlorocebus aethiops , Células Epiteliales/virología , Humanos , Masculino , ARN Viral/genética , SARS-CoV-2/fisiología , Células VeroRESUMEN
Accurate differentiation between pneumococci and other viridans streptococci is essential given their differences in clinical significance. However, classical phenotypic tests are often inconclusive, and many examples of atypical reactions have been reported. In this study, we applied various phenotypic and genotypic methods to discriminate between a collection of 12 streptococci isolated from the upper respiratory tract of HIV-seropositive individuals in 1998 and 1999. Conventional phenotypic characterization initially classified these streptococci as Streptococcus pneumoniae, as they were all sensitive to optochin and were all bile soluble. However, they did not agglutinate with anti-pneumococcal capsular antibodies and were also far more resistant to antimicrobial agents than typeable pneumococci isolated in the same period. Genotypic characterization of these isolates and control isolates by both multilocus sequence analysis (MLSA) and comparative genomic hybridization (CGH) showed that only a single isolate was genetically considered to be a true S. pneumoniae isolate, and that the remaining 11 non-typable isolates were indeed distinct from true pneumococci. Of these, 10 most closely resembled a subgroup of Streptococcus mitis isolates genetically, while one strain was identified as a Streptococcus pseudopneumoniae isolate. CGH also showed that a considerable part of the proposed pneumococcal core genome, including many of the known pneumococcal virulence factors, was conserved in the non-typable isolates. Sequencing of part of the 16S rRNA gene and investigation for the presence of ply by PCR corroborated these results. In conclusion, our findings confirm the close relationship between streptococci of the Mitis group, and show that both MLSA and CGH enable pneumococci to be distinguished from other Mitis group streptococci.
Asunto(s)
Genoma Bacteriano , Seropositividad para VIH/microbiología , Infecciones Estreptocócicas/microbiología , Streptococcus/clasificación , Técnicas de Tipificación Bacteriana , Hibridación Genómica Comparativa , ADN Bacteriano/genética , Farmacorresistencia Bacteriana , Humanos , Fenotipo , Filogenia , ARN Ribosómico 16S/genética , Infecciones del Sistema Respiratorio/microbiología , Streptococcus/genética , Streptococcus/aislamiento & purificaciónRESUMEN
While performing surveillance studies in Oeiras, Portugal, designed to describe the impact of pneumococcal conjugate vaccine on colonization, we observed an increase from 0.7% in 2003 to 5% in 2006 in the prevalence of penicillin resistance (MIC of 2 to 6 mg/liter) among presumptively identified pneumococcal isolates. Although 15 of the 22 penicillin-resistant isolates obtained in 2006 were optochin resistant, they were bile soluble and thus considered to be bona fide pneumococci. This study aimed to clarify the nature of these isolates by using a combination of phenotypic and genotypic approaches that included routine strategies for pneumococcal identification, multilocus sequence analysis (MLSA), and comparative genomic hybridization (CGH). By MLSA, all isolates were classified as "streptococci of the mitis group" that, however, were distinct from typical Streptococcus pneumoniae or Streptococcus mitis. A single isolate was identified as Streptococcus pseudopneumoniae. CGH confirmed these findings and further indicated that a considerable part of the proposed pneumococcal core genome is conserved in these isolates, including several pneumococcal virulence genes (e.g., pavA, spxB, cbpE, and cbpD). These results suggest that among pneumococci and closely related streptococci, universal unique phenotypic and genetic properties that could aid species identification are virtually impossible to define. In pneumococcal colonization studies, when atypical strains are found, MLSA and CGH are informative tools that can be used to complement routine tests. In our study, after correct identification of the penicillin-resistant true pneumococci, we found that penicillin resistance levels among pneumococci remained stable from 2003 to 2006.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple , Infecciones Estreptocócicas/epidemiología , Infecciones Estreptocócicas/microbiología , Streptococcus/clasificación , Streptococcus/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Preescolar , Hibridación Genómica Comparativa , Secuencia Conservada , Dermatoglifia del ADN , ADN Bacteriano/genética , Genotipo , Humanos , Fenotipo , Portugal/epidemiología , Prevalencia , Análisis de Secuencia de ADN , Streptococcus/efectos de los fármacos , Streptococcus mitis/clasificación , Streptococcus pneumoniae/clasificación , Factores de Virulencia/genéticaRESUMEN
Bordetella pertussis vaccine escape mutants that lack expression of the pertussis antigen pertactin (Prn) have emerged in vaccinated populations in the last 10-20 years. Additionally, clinical isolates lacking another acellular pertussis (aP) vaccine component, filamentous hemagglutinin (FHA), have been found sporadically. Here, we show that both whole-cell pertussis (wP) and aP vaccines induced protection in the lungs of mice, but that the wP vaccine was more effective in nasal clearance. Importantly, bacterial populations isolated from the lungs shifted to an FHA-negative phenotype due to frameshift mutations in the fhaB gene. Loss of FHA expression was strongly selected for in Prn-deficient strains in the lungs following aP but not wP vaccination. The combined loss of Prn and FHA led to complete abrogation of bacterial surface binding by aP-induced serum antibodies. This study demonstrates vaccine- and anatomical site-dependent adaptation of B. pertussis and has major implications for the design of improved pertussis vaccines.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Bordetella pertussis/fisiología , Vacunas contra Difteria, Tétanos y Tos Ferina Acelular/inmunología , Hemaglutininas/metabolismo , Factores de Virulencia de Bordetella/metabolismo , Animales , Anticuerpos Antibacterianos/inmunología , Bordetella pertussis/inmunología , Regulación de la Expresión Génica , Pulmón/metabolismo , Pulmón/microbiología , Ratones , Vacunación , Tos Ferina/metabolismo , Tos Ferina/patología , Tos Ferina/prevención & controlRESUMEN
Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders with a strong genetic etiology. Cytogenetic abnormalities have been detected in 5-10% of the patients with autism. In this study, we present the clinical, cytogenetic and array-comparative genomic hybridization (array-CGH) evaluation of a 13-year-old male with severe developmental delay, facial dysmorphic features, autism and self mutilation. The patient was found to carry a de novo duplication of chromosome region 8p21 of minimally 6.14 and maximally 6.58 Mb as ascertained by bacterial artificial chromosome (BAC)-based array-CGH. Hitherto, only a few patients with autism with cytogenetically visible duplications involving the chromosome 8p21 region have been described, but the extent of these duplications has not been determined at the molecular level. This represents the smallest rearrangement of chromosomal region 8p21 as yet found in a patient with autism. For 11 of the 36 genes with known functions located within this duplication clear transcription in the brain was found. Of those the STMN4 and DPYSL2 genes are the most likely candidate genes to be involved in neuronal development, and, if altered in gene-dosage, in the autistic phenotype of our patient.
Asunto(s)
Trastorno Autístico/genética , Trastorno Autístico/psicología , Aberraciones Cromosómicas , Cromosomas Humanos Par 8 , Duplicación de Gen , Automutilación/genética , Adolescente , Trastorno Autístico/diagnóstico , Hibridación Genómica Comparativa , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/fisiopatología , Discapacidades del Desarrollo/psicología , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/fisiopatología , Discapacidad Intelectual/psicología , Masculino , Automutilación/fisiopatología , Automutilación/psicologíaRESUMEN
The Wolf-Hirschhorn syndrome (WHS (MIM 194190)), which is characterized by growth delay, mental retardation, epilepsy, facial dysmorphisms, and midline fusion defects, shows extensive phenotypic variability. Several of the proposed mutational and epigenetic mechanisms in this and other chromosomal deletion syndromes fail to explain the observed phenotypic variability. To explain the complex phenotype of a patient with WHS and features reminiscent of Wolfram syndrome (WFS (MIM 222300)), we performed extensive clinical evaluation and classical and molecular cytogenetic (GTG banding, FISH and array-CGH) and WFS1 gene mutation analyses. We detected an 8.3 Mb terminal deletion and an adjacent 2.6 Mb inverted duplication in the short arm of chromosome 4, which encompasses a gene associated with WFS (WFS1). In addition, a nonsense mutation in exon 8 of the WFS1 gene was found on the structurally normal chromosome 4. The combination of the 4p deletion with the WFS1 point mutation explains the complex phenotype presented by our patient. This case further illustrates that unmasking of hemizygous recessive mutations by chromosomal deletions represents an additional explanation for the phenotypic variability observed in chromosomal deletion disorders.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 4/genética , Heterocigoto , Proteínas de la Membrana/genética , Síndrome de Wolf-Hirschhorn/genética , Preescolar , Codón sin Sentido/genética , Femenino , Homocigoto , Humanos , Lactante , Recién Nacido , Masculino , Fenotipo , Mutación Puntual/genéticaRESUMEN
We report on an 8(1)/(2)-year-old girl with severe pre- and postnatal growth retardation, congenital heart malformation, facial asymmetry, oculocutaneous albinism without misrouting and subluxation of the radial heads. Her intelligence was in the low normal range. By GTG-banding a deletion of band 15q26 was found. Array-CGH, using a 3783 BAC array, revealed a segmental monosomy of the 15(q26.2-->qter) region, which was narrowed down to a 6.87Mb deletion by using the Illumina Infinium 317 K SNP array system, and subsequently confirmed by fluorescence in situ hybridisation (FISH) analysis. The deletion appeared to have arisen de novo. The IGF1R (insulin-like growth factor 1 receptor) and the NR2F2 genes were situated within, but the OCA2 (oculocutaneous albinism II) gene (formerly called the P gene) was located outside the deleted region. Clinical findings in our patient were compared with previously reported cases carrying terminal deletions of 15q26.2. This allowed us to expand the clinical phenotype of terminal 15q26.2 deletions and to indicate candidate genes for several phenotypic features.
Asunto(s)
Albinismo Oculocutáneo/genética , Deleción Cromosómica , Cromosomas Humanos Par 15/genética , Trastornos del Crecimiento/genética , Albinismo Oculocutáneo/patología , Factor de Transcripción COUP II/genética , Niño , Femenino , Trastornos del Crecimiento/patología , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Proteínas de Transporte de Membrana/genética , Receptores de Somatomedina/genéticaRESUMEN
Keyhole limpet hemocyanin (KLH) is used as an immunogenic neo-antigen for various clinical applications and during vaccine development. For advanced monitoring of KLH-based interventions, we developed a flow cytometry-based assay for the ex vivo detection, phenotyping and isolation of KLH-specific B cells. As proof-of-principle, we analyzed 10 melanoma patients exposed to KLH during anti-cancer dendritic cell vaccination. Our assay demonstrated sensitive and specific detection of KLH-specific B cells in peripheral blood and KLH-specific B cell frequencies strongly correlated with anti-KLH serum antibody titers. Profiling of B cell subsets over the vaccination course revealed that KLH-specific B cells matured from naïve to class-switched memory B cells, confirming the prototypic B cell response to a neo-antigen. We conclude that flow-cytometric detection and in-depth phenotyping of KLH-specific B cells is specific, sensitive, and scalable. Our findings provide novel opportunities to monitor KLH-specific immune responses and serve as a blueprint for the development of new flow-cytometric protocols.
Asunto(s)
Adyuvantes Inmunológicos/química , Subgrupos de Linfocitos B/inmunología , Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/trasplante , Hemocianinas/química , Melanoma/terapia , Neoplasias Cutáneas/terapia , Anticuerpos/sangre , Subgrupos de Linfocitos B/clasificación , Subgrupos de Linfocitos B/patología , Rastreo Celular/métodos , Células Dendríticas/química , Células Dendríticas/citología , Células Dendríticas/inmunología , Ensayo de Immunospot Ligado a Enzimas , Citometría de Flujo , Humanos , Memoria Inmunológica , Inmunofenotipificación , Melanoma/inmunología , Melanoma/patología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Vacunación/métodosRESUMEN
Streptococcus pneumoniae is responsible for an estimated 1.6 million deaths worldwide every year. While rapid detection and timely treatment with appropriate antibiotics is preferred, this is often difficult due to the amount of time that detection with blood cultures takes. In this study, a novel quantitative PCR assay for the detection of Streptococcus pneumoniae was developed. To identify novel targets, we analysed the pneumococcal genome for unique, repetitive DNA sequences. This approach identified comX, which is conserved and present in duplicate copies in Streptococcus pneumoniae but not in other bacterial species. Comparison with lytA, the current 'gold standard' for detection by quantitative PCR, demonstrated an analytic specificity of 100% for both assays on a panel of 10 pneumococcal and 18 non-pneumococcal isolates, but a reduction of 3.5 quantitation cycle values (± 0.23 sem), resulting in an increased analytical detection rate of comX. We validated our assay on DNA extracted from the serum of 30 bacteraemic patients who were blood culture positive for Streptococcus pneumoniae and 51 serum samples that were culture positive for other bacteria. This resulted in a similar clinical sensitivity between the comX and lytA assays (47%) and in a diagnostic specificity of 98.2 and 100% for the lytA and comX assays, respectively. In conclusion, we have developed a novel quantitative PCR assay with increased analytical sensitivity for the detection of Streptococcus pneumoniae, which may be used to develop a rapid bedside test for the direct detection of Streptococcus pneumoniae in clinical specimens.
Asunto(s)
Bacteriemia/microbiología , Proteínas Bacterianas/genética , Infecciones Neumocócicas/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Streptococcus pneumoniae/aislamiento & purificación , Factores de Transcripción/genética , Bacteriemia/diagnóstico , Femenino , Humanos , Masculino , Infecciones Neumocócicas/diagnóstico , Sensibilidad y Especificidad , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/genéticaRESUMEN
OBJECT: Hemangioblastomas (HBs) occur sporadically or as a manifestation of von Hippel-Lindau (VHL) disease. In the majority of VHL-related HBs, inactivation of the VHL tumor suppressor gene (TSG), which is located on chromosome 3p25-26, is found. The VHL gene is assumed to be involved also in the development of sporadic HBs. In a previous study of chromosomal aberrations of sporadic HBs, multiple chromosomal imbalances were found in the majority of tumors. The aim of this study was to analyze further both sporadic HBs and VHL-related HBs to determine if these histopathologically identical tumors have a different genetic background. METHODS: Sixteen sporadic HBs and seven VHL-related HBs were identified by clinical criteria and analyzed. Comparative genomic hybridization was used to screen for chromosomal imbalances throughout the entire HB genome. Additionally, mutation analysis of the VHL gene was performed using direct sequencing. Loss of chromosome 3 and multiple other chromosomal imbalances were found in the sporadic HBs, although only one imbalance, a loss of chromosome 3, was detected in the seven VHL-related HBs. Somatic VHL gene mutations were found in one third of sporadic HBs, whereas a mutation of the VHL gene was detected in all VHL-related HBs. CONCLUSIONS: These results indicate that the molecular mechanisms underlying sporadic HBs and VHL-related HBs are different. Inactivation of the VHL gene is probably not the most important event in the tumorigenesis of sporadic HBs. Other mechanisms of inhibition of VHL protein function, or inactivation of other TSGs, on chromosome 3p or on other chromosomes, might be important in the development of sporadic HBs.
Asunto(s)
Neoplasias Cerebelosas/genética , Heterogeneidad Genética , Hemangioblastoma/genética , Ligasas/genética , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Enfermedad de von Hippel-Lindau/genética , Adulto , Anciano , Aberraciones Cromosómicas , Cromosomas Humanos Par 3 , Análisis Mutacional de ADN/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Hibridación de Ácido Nucleico , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Since Streptococcus pneumoniae transmits through droplet spread, this respiratory tract pathogen may be able to survive in saliva. Here, we show that saliva supports survival of clinically relevant S. pneumoniae strains for more than 24 h in a capsule-independent manner. Moreover, saliva induced growth of S. pneumoniae in growth-permissive conditions, suggesting that S. pneumoniae is well adapted for uptake of nutrients from this bodily fluid. By using Tn-seq, a method for genome-wide negative selection screening, we identified 147 genes potentially required for growth and survival of S. pneumoniae in saliva, among which genes predicted to be involved in cell envelope biosynthesis, cell transport, amino acid metabolism, and stress response predominated. The Tn-seq findings were validated by testing a panel of directed gene deletion mutants for their ability to survive in saliva under two testing conditions: at room temperature without CO2, representing transmission, and at 37 °C with CO2, representing in-host carriage. These validation experiments confirmed that the plsX gene and the amiACDEF and aroDEBC operons, involved in respectively fatty acid metabolism, oligopeptide transport, and biosynthesis of aromatic amino acids play an important role in the growth and survival of S. pneumoniae in saliva at 37 °C. In conclusion, this study shows that S. pneumoniae is well-adapted for growth and survival in human saliva and provides a genome-wide list of genes potentially involved in adaptation. This notion supports earlier evidence that S. pneumoniae can use human saliva as a vector for transmission.
Asunto(s)
Proteínas Bacterianas/genética , Genoma Bacteriano/genética , Infecciones Neumocócicas/genética , Infecciones Neumocócicas/mortalidad , Saliva/microbiología , Streptococcus pneumoniae/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Humanos , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidad , Tasa de SupervivenciaRESUMEN
Moraxella catarrhalis is a mucosal pathogen that causes childhood otitis media and exacerbations of chronic obstructive pulmonary disease in adults. During the course of infection, M. catarrhalis needs to adhere to epithelial cells of different host niches such as the nasopharynx and lungs, and consequently, efficient adhesion to epithelial cells is considered an important virulence trait of M. catarrhalis. By using Tn-seq, a genome-wide negative selection screenings technology, we identified 15 genes potentially required for adherence of M. catarrhalis BBH18 to pharyngeal epithelial Detroit 562 and lung epithelial A549 cells. Validation with directed deletion mutants confirmed the importance of aroA (3-phosphoshikimate 1-carboxyvinyl-transferase), ecnAB (entericidin EcnAB), lgt1 (glucosyltransferase), and MCR_1483 (outer membrane lipoprotein) for cellular adherence, with ΔMCR_1483 being most severely attenuated in adherence to both cell lines. Expression profiling of M. catarrhalis BBH18 during adherence to Detroit 562 cells showed increased expression of 34 genes in cell-attached versus planktonic bacteria, among which ABC transporters for molybdate and sulfate, while reduced expression of 16 genes was observed. Notably, neither the newly identified genes affecting adhesion nor known adhesion genes were differentially expressed during adhesion, but appeared to be constitutively expressed at a high level. Profiling of the transcriptional response of Detroit 562 cells upon adherence of M. catarrhalis BBH18 showed induction of a panel of pro-inflammatory genes as well as genes involved in the prevention of damage of the epithelial barrier. In conclusion, this study provides new insight into the molecular interplay between M. catarrhalis and host epithelial cells during the process of adherence.
Asunto(s)
Células Epiteliales/microbiología , Interacciones Huésped-Patógeno , Moraxella catarrhalis/fisiología , Sistema Respiratorio/citología , Adhesión Bacteriana , Línea Celular , Eliminación de Gen , Genes Bacterianos/genética , Genómica , Humanos , Moraxella catarrhalis/genética , Transcripción GenéticaRESUMEN
To determine the phenotypic significance of copy number changes (CNCs) in the human genome, we performed genome-wide segmental aneuploidy profiling by BAC-based array-CGH of 278 unrelated patients with multiple congenital abnormalities and mental retardation (MCAMR) and in 48 unaffected family members. In 20 patients, we found de novo CNCs composed of multiple consecutive probes. Of the 125 probes making up these probably pathogenic CNCs, 14 were also found as single CNCs in other patients and 5 in healthy individuals. Thus, these CNCs are not by themselves pathogenic. Almost one out of five patients and almost one out of six healthy individuals in our study cohort carried a gain or a loss for any one of the recently discovered microdeletion/microduplication loci, whereas seven patients and one healthy individual showed losses or gains for at least two different loci. The pathogenic burden resulting from these CNCs may be limited as they were found with similar frequencies among patients and healthy individuals (P=0.165; Fischer's exact test), and several individuals showed CNCs at multiple loci. CNCs occurring specifically in our study cohort were enriched for components of the glutamate receptor family (GRIA2, GRIA4, GRIK2 and GRIK4) and genes encoding proteins involved in guiding cell localization during development (ATP1A2, GIRK3, GRIA2, KCNJ3, KCNJ10, KCNK17 and KCNK5). This indicates that disease cohort-specific compilations of CNCs may aid in identifying loci, genes and biological processes that contribute to the phenotype of patients.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Discapacidad Intelectual/genética , Receptores de Glutamato/genética , Niño , Cromosomas Artificiales Bacterianos/genética , Estudios de Cohortes , Hibridación Genómica Comparativa , Sitios Genéticos/genética , Humanos , Patrón de Herencia/genética , Transporte de ProteínasRESUMEN
A 10-year-old boy with vermis hypoplasia, dilatation of the fourth ventricle, enlarged cisterna magna and aplasia of the corpus callosum, consistent with the Dandy-Walker complex (DWC), and slight facial dysmorphisms, severe motor and mental retardation is presented. By combining data obtained by karyotyping, array-CGH, FISH, and multiplex ligation-mediated probe amplification (MLPA) we identified a 5 Mb deletion of the 1q44 --> qter region resulting from a paternal t(1;20)(q44;q13.33). This smallest 1q44 deletion reported so far, enabled us to significantly narrow down the number of candidate genes for the DWC in this region. Since the ZNF124 transcription factor is strongly expressed in the fetal brain it may represent a candidate gene for the DWC at 1q44.