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1.
Sci Rep ; 13(1): 9403, 2023 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-37296186

RESUMO

Respiratory syncytial virus (RSV) is a leading cause of serious and even fatal acute lower respiratory tract infections in infants and in the elderly. Potent RSV neutralization has been achieved by antibodies that selectively bind the prefusion form of the viral fusion (F) protein. We hypothesised that similar potent neutralization could be achieved using F protein targeting aptamers. Aptamers have yet to reach their translational potential for therapeutics or diagnostics due to their short half-life and limited range of target-aptamer interactions; these shortcomings can, however, be ameliorated by application of amino acid-like side chain holding nucleotides. In this study, a stabilized version of the prefusion RSV F protein was targeted by aptamer selection using an oligonucleotide library holding a tryptophan-like side chain. This process resulted in aptamers that bound the F protein with high affinity and differentiated between its pre- and postfusion conformation. Identified aptamers inhibited viral infection of lung epithelial cells. Moreover, introduction of modified nucleotides extended aptamer half-lives. Our results suggest that targeting aptamers to the surface of viruses could yield effective drug candidates, which could keep pace with the continuously evolving pathogens.


Assuntos
Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Humanos , Idoso , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Triptofano , Anticorpos Neutralizantes , Anticorpos Antivirais , Pulmão , Células Epiteliais , Oligonucleotídeos , Proteínas Virais de Fusão
2.
Microbiol Spectr ; 10(3): e0005022, 2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35678554

RESUMO

The nasopharyngeal commensal Streptococcus pneumoniae can become invasive and cause metastatic infection. This requires the pneumococcus to have the ability to adapt, grow, and reside in diverse host environments. Therefore, we studied whether the likelihood of severe disease manifestations was related to pneumococcal growth kinetics. For 383 S. pneumoniae blood isolates and 25 experimental mutants, we observed highly reproducible growth curves in nutrient-rich medium. The derived growth features were lag time, maximum growth rate, maximum density, and stationary-phase time before lysis. First, the pathogenicity of each growth feature was probed by comparing isolates from patients with and without marked preexisting comorbidity. Then, growth features were related to the propensity of causing severe manifestations of invasive pneumococcal disease (IPD). A high maximum bacterial density was the most pronounced pathogenic growth feature, which was also an independent predictor of 30-day mortality (P = 0.03). Serotypes with an epidemiologically higher propensity for causing meningitis displayed a relatively high maximum density (P < 0.005) and a short stationary phase (P < 0.005). Correspondingly, isolates from patients diagnosed with meningitis showed an especially high maximum density and short stationary phase compared to isolates from the same serotype that had caused uncomplicated bacteremic pneumonia. In contrast, empyema-associated strains were characterized by a relatively long lag phase (P < 0.0005), and slower growth (P < 0.005). The course and dissemination of IPD may partly be attributable to the pneumococcal growth features involved. If confirmed, we should tailor the prevention and treatment strategies for the different infection sites that can complicate IPD. IMPORTANCE Streptococcus pneumoniae is a leading infectious cause of deaths worldwide. To understand the course and outcome of pneumococcal infection, most research has focused on the host and its response to contain bacterial growth. However, bacterial epidemiology suggest that certain pneumococcal serotypes are particularly prone to causing complicated infections. Therefore, we took the bacterial point of view, simply examining in vitro growth features for hundreds of pneumococcal blood isolates. Their growth curves were very reproducible. Certain poles of pneumococcal growth features were indeed associated with specific clinical manifestations like meningitis or pleural empyema. This indicates that bacterial growth style potentially affects the progression of infection. Further research on bacterial growth and adaptation to different host environments may therefore provide key insight into pathogenesis of complicated invasive disease. Such knowledge could lead to more tailored vaccine targets or therapeutic approaches to reduce the million deaths that are caused by pneumococcal disease every year.


Assuntos
Meningite , Infecções Pneumocócicas , Humanos , Lactente , Infecções Pneumocócicas/microbiologia , Vacinas Pneumocócicas , Sorogrupo , Sorotipagem , Streptococcus pneumoniae
3.
NPJ Vaccines ; 7(1): 21, 2022 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-35177621

RESUMO

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.

4.
Front Immunol ; 12: 652965, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33912177

RESUMO

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.


Assuntos
Imunidade Adaptativa/fisiologia , Linfócitos T CD4-Positivos/imunologia , Inibidor de Quinase Dependente de Ciclina p21/deficiência , Interferon beta/metabolismo , Transdução de Sinais/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Adulto , Fatores Etários , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Sangue Fetal/citologia , Sangue Fetal/imunologia , Citometria de Fluxo , Humanos , Separação Imunomagnética , Recém-Nascido , Cultura Primária de Células , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
Viruses ; 13(2)2021 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-33670363

RESUMO

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.


Assuntos
Antivirais/farmacologia , Berberina/farmacologia , Indóis/farmacologia , Pirróis/farmacologia , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Adolescente , Animais , COVID-19/virologia , Células Cultivadas , Chlorocebus aethiops , Células Epiteliais/virologia , Humanos , Masculino , RNA Viral/genética , SARS-CoV-2/fisiologia , Células Vero
6.
PLoS One ; 15(8): e0237394, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32822419

RESUMO

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.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Bordetella pertussis/fisiologia , Vacinas contra Difteria, Tétano e Coqueluche Acelular/imunologia , Hemaglutininas/metabolismo , Fatores de Virulência de Bordetella/metabolismo , Animais , Anticorpos Antibacterianos/imunologia , Bordetella pertussis/imunologia , Regulação da Expressão Gênica , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos , Vacinação , Coqueluche/metabolismo , Coqueluche/patologia , Coqueluche/prevenção & controle
7.
Astrobiology ; 20(11): 1353-1362, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32391711

RESUMO

The last decade has witnessed a renewed interest in space exploration. Public and private institutions are investing considerable effort toward the direct exploration of the Moon and Mars, as well as more distant bodies in the solar system. Both automated and human-crewed spacecraft are being considered in these efforts. As inevitable fellow travelers on the bodies of astronauts, spaceships, or equipment, terrestrial microorganisms will undoubtedly come into contact with extraterrestrial environments, despite stringent decontamination. These microorganisms could eventually adapt and grow in their new habitats, where they might potentially recolonize and lead to the infection of the human space travelers. In this article, we demonstrate that clinically relevant bacterial species found in the environment are able to grow in minimal media with sugar compounds identified in extraterrestrial carbon sources. As a surrogate model, we used carbohydrates previously isolated from carbonaceous meteorites. The bacteria underwent an adaptation process that caused structural modifications in the cell envelope that sparked changes in pathogenic potential, both in vitro and in vivo. Understanding the adaptation of microorganisms exposed to extraterrestrial environments, with subsequent changes in their immunogenicity and virulence, requires a comprehensive analysis of such scenarios to ensure the safety of major space expeditions in the decades to come.


Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/imunologia , Carboidratos , Meio Ambiente Extraterreno , Marte , Meteoroides , Voo Espacial , Astronave
8.
PLoS Pathog ; 16(1): e1008153, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31999804

RESUMO

Human space travel is on the verge of visiting Mars and, in the future, even more distant places in the solar system. These journeys will be also made by terrestrial microorganisms (hitchhiking on the bodies of astronauts or on scientific instruments) that, upon arrival, will come into contact with new planetary environments, despite the best measures to prevent contamination. These microorganisms could potentially adapt and grow in the new environments and subsequently recolonize and infect astronauts. An even more challenging situation would be if truly alien microorganisms will be present on these solar system bodies: What will be their pathogenic potential, and how would our immune host defenses react? It will be crucial to anticipate these situations and investigate how the immune system of humans might cope with modified terrestrial or alien microbes. We propose several scenarios that may be encountered and how to respond to these challenges.


Assuntos
Equipamentos e Provisões/microbiologia , Interações Hospedeiro-Patógeno , Sistema Imunitário/imunologia , Astronautas , Exobiologia , Meio Ambiente Extraterreno , Humanos , Voo Espacial , Astronave
9.
J Infect Dis ; 217(12): 1987-1996, 2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29528444

RESUMO

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.


Assuntos
Bordetella pertussis/patogenicidade , Sistema Respiratório/microbiologia , Coqueluche/microbiologia , Animais , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Bordetella pertussis/genética , Feminino , Regulação Bacteriana da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Transcrição/genética
10.
Infect Immun ; 86(4)2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29378798

RESUMO

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.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas do Sistema Complemento/imunologia , Genótipo , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Idoso , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Proteínas do Sistema Complemento/metabolismo , Feminino , Humanos , Evasão da Resposta Imune , Masculino , Pessoa de Meia-Idade , Tipagem Molecular , Mutação , Infecções Pneumocócicas/epidemiologia , Prevalência , Sorogrupo , Virulência/genética , Fatores de Virulência/genética
11.
Sci Rep ; 7: 43486, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28344338

RESUMO

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.


Assuntos
Adjuvantes Imunológicos/química , Subpopulações de Linfócitos B/imunologia , Vacinas Anticâncer/uso terapêutico , Células Dendríticas/transplante , Hemocianinas/química , Melanoma/terapia , Neoplasias Cutâneas/terapia , Anticorpos/sangue , Subpopulações de Linfócitos B/classificação , Subpopulações de Linfócitos B/patologia , Rastreamento de Células/métodos , Células Dendríticas/química , Células Dendríticas/citologia , Células Dendríticas/imunologia , ELISPOT , Citometria de Fluxo , Humanos , Memória Imunológica , Imunofenotipagem , Melanoma/imunologia , Melanoma/patologia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Vacinação/métodos
12.
J Med Microbiol ; 65(2): 129-136, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26628261

RESUMO

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.


Assuntos
Bacteriemia/microbiologia , Proteínas de Bactérias/genética , Infecções Pneumocócicas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Streptococcus pneumoniae/isolamento & purificação , Fatores de Transcrição/genética , Bacteriemia/diagnóstico , Feminino , Humanos , Masculino , Infecções Pneumocócicas/diagnóstico , Sensibilidade e Especificidade , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/genética
13.
Mol Microbiol ; 96(1): 28-41, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25534847

RESUMO

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-ACP 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.


Assuntos
Proteínas de Bactérias/genética , Ácidos Graxos/metabolismo , Deleção de Sequência , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/genética , Tioléster Hidrolases/metabolismo , Proteína de Transporte de Acila/metabolismo , Sequência de Bases , Ácidos Graxos/biossíntese , Ácidos Graxos/genética , Técnicas de Inativação de Genes , Ácido Mirístico/metabolismo , Fenótipo , Fosfolipídeos/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Streptococcus pneumoniae/metabolismo , Tioléster Hidrolases/genética
14.
PLoS One ; 9(2): e89541, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24586856

RESUMO

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.


Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano/genética , Infecções Pneumocócicas/genética , Infecções Pneumocócicas/mortalidade , Saliva/microbiologia , Streptococcus pneumoniae/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Humanos , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidade , Taxa de Sobrevida
15.
Mol Microbiol ; 91(3): 522-37, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24344868

RESUMO

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 ∼ 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.


Assuntos
Proteínas do Sistema Complemento/imunologia , Dissulfetos/metabolismo , Moraxella catarrhalis/enzimologia , Moraxella catarrhalis/imunologia , Oxirredutases/metabolismo , Fatores de Virulência/metabolismo , Atividade Bactericida do Sangue , Perfilação da Expressão Gênica , Humanos , Dados de Sequência Molecular , Moraxella catarrhalis/genética , Moraxella catarrhalis/metabolismo , Mutagênese Insercional , Oxirredutases/genética , Análise de Sequência de DNA , Fatores de Virulência/genética
16.
PLoS One ; 8(8): e72193, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23936538

RESUMO

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.


Assuntos
Células Epiteliais/microbiologia , Interações Hospedeiro-Patógeno , Moraxella catarrhalis/fisiologia , Sistema Respiratório/citologia , Aderência Bacteriana , Linhagem Celular , Deleção de Genes , Genes Bacterianos/genética , Genômica , Humanos , Moraxella catarrhalis/genética , Transcrição Gênica
17.
BMC Genomics ; 14: 74, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23375113

RESUMO

BACKGROUND: Tuberculosis (TB) continues to cause a high toll of disease and death among children worldwide. The diagnosis of childhood TB is challenged by the paucibacillary nature of the disease and the difficulties in obtaining specimens. Whereas scientific and clinical research efforts to develop novel diagnostic tools have focused on TB in adults, childhood TB has been relatively neglected. Blood transcriptional profiling has improved our understanding of disease pathogenesis of adult TB and may offer future leads for diagnosis and treatment. No studies applying gene expression profiling of children with TB have been published so far. RESULTS: We identified a 116-gene signature set that showed an average prediction error of 11% for TB vs. latent TB infection (LTBI) and for TB vs. LTBI vs. healthy controls (HC) in our dataset. A minimal gene set of only 9 genes showed the same prediction error of 11% for TB vs. LTBI in our dataset. Furthermore, this minimal set showed a significant discriminatory value for TB vs. LTBI for all previously published adult studies using whole blood gene expression, with average prediction errors between 17% and 23%. In order to identify a robust representative gene set that would perform well in populations of different genetic backgrounds, we selected ten genes that were highly discriminative between TB, LTBI and HC in all literature datasets as well as in our dataset. Functional annotation of these genes highlights a possible role for genes involved in calcium signaling and calcium metabolism as biomarkers for active TB. These ten genes were validated by quantitative real-time polymerase chain reaction in an additional cohort of 54 Warao Amerindian children with LTBI, HC and non-TB pneumonia. Decision tree analysis indicated that five of the ten genes were sufficient to classify 78% of the TB cases correctly with no LTBI subjects wrongly classified as TB (100% specificity). CONCLUSIONS: Our data justify the further exploration of our signature set as biomarkers for potential childhood TB diagnosis. We show that, as the identification of different biomarkers in ethnically distinct cohorts is apparent, it is important to cross-validate newly identified markers in all available cohorts.


Assuntos
Perfilação da Expressão Gênica , Indígenas Norte-Americanos/genética , Tuberculose/etnologia , Tuberculose/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Genômica , Humanos , Lactente , Masculino , Reprodutibilidade dos Testes
18.
J Clin Microbiol ; 48(1): 238-46, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19906899

RESUMO

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.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Infecções Estreptocócicas/epidemiologia , Infecções Estreptocócicas/microbiologia , Streptococcus/classificação , Streptococcus/isolamento & purificação , Técnicas de Tipagem Bacteriana , Pré-Escolar , Hibridização Genômica Comparativa , Sequência Conservada , Impressões Digitais de DNA , DNA Bacteriano/genética , Genótipo , Humanos , Fenótipo , Portugal/epidemiologia , Prevalência , Análise de Sequência de DNA , Streptococcus/efeitos dos fármacos , Streptococcus mitis/classificação , Streptococcus pneumoniae/classificação , Fatores de Virulência/genética
19.
Microbiology (Reading) ; 156(Pt 3): 838-848, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19959577

RESUMO

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.


Assuntos
Genoma Bacteriano , Soropositividade para HIV/microbiologia , Infecções Estreptocócicas/microbiologia , Streptococcus/classificação , Técnicas de Tipagem Bacteriana , Hibridização Genômica Comparativa , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Humanos , Fenótipo , Filogenia , RNA Ribossômico 16S/genética , Infecções Respiratórias/microbiologia , Streptococcus/genética , Streptococcus/isolamento & purificação
20.
Eur J Hum Genet ; 18(1): 39-46, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19623214

RESUMO

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.


Assuntos
Variações do Número de Cópias de DNA/genética , Deficiência Intelectual/genética , Receptores de Glutamato/genética , Criança , Cromossomos Artificiais Bacterianos/genética , Estudos de Coortes , Hibridização Genômica Comparativa , Loci Gênicos/genética , Humanos , Padrões de Herança/genética , Transporte Proteico
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