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1.
J Gen Virol ; 102(8)2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34424155

RESUMO

Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein's surface. Here we used IBV propagated in embryonated hens' eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.


Assuntos
Coronavirus/fisiologia , Polissacarídeos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Alcaloides/química , Alcaloides/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células Cultivadas , Cromatografia Líquida , Biologia Computacional/métodos , Coronavirus/efeitos dos fármacos , Infecções por Coronavirus/veterinária , Regulação Viral da Expressão Gênica , Glicosilação/efeitos dos fármacos , Vírus da Bronquite Infecciosa/fisiologia , Modelos Moleculares , Conformação Molecular , Peso Molecular , Testes de Neutralização , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polissacarídeos/química , Doenças das Aves Domésticas/virologia , Transporte Proteico , Espectrometria de Massas por Ionização por Electrospray , Glicoproteína da Espícula de Coronavírus/genética , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
2.
Viruses ; 13(5)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34063508

RESUMO

Arboviruses such as bluetongue virus (BTV) replicate in arthropod vectors involved in their transmission between susceptible vertebrate-hosts. The "classical" BTV strains infect and replicate effectively in cells of their insect-vectors (Culicoides biting-midges), as well as in those of their mammalian-hosts (ruminants). However, in the last decade, some "atypical" BTV strains, belonging to additional serotypes (e.g., BTV-26), have been found to replicate efficiently only in mammalian cells, while their replication is severely restricted in Culicoides cells. Importantly, there is evidence that these atypical BTV are transmitted by direct-contact between their mammalian hosts. Here, the viral determinants and mechanisms restricting viral replication in Culicoides were investigated using a classical BTV-1, an "atypical" BTV-26 and a BTV-1/BTV-26 reassortant virus, derived by reverse genetics. Viruses containing the capsid of BTV-26 showed a reduced ability to attach to Culicoides cells, blocking early steps of the replication cycle, while attachment and replication in mammalian cells was not restricted. The replication of BTV-26 was also severely reduced in other arthropod cells, derived from mosquitoes or ticks. The data presented identifies mechanisms and potential barriers to infection and transmission by the newly emerged "atypical" BTV strains in Culicoides.


Assuntos
Vírus Bluetongue/classificação , Vírus Bluetongue/fisiologia , Proteínas do Capsídeo/metabolismo , Replicação Viral , Animais , Artrópodes , Vírus Bluetongue/isolamento & purificação , Vírus Bluetongue/ultraestrutura , Linhagem Celular , Células Cultivadas , Interações Hospedeiro-Patógeno , Sorogrupo , Ligação Viral , Replicação Viral/efeitos dos fármacos
3.
PLoS Pathog ; 17(3): e1009464, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33780514

RESUMO

Here, we report the first complete genomes of three cultivable treponeme species from bovine digital dermatitis (DD) skin lesions, two comparative human treponemes, considered indistinguishable from bovine DD species, and a bovine gastrointestinal (GI) treponeme isolate. Key genomic differences between bovine and human treponemes implicate microbial mechanisms that enhance knowledge of how DD, a severe disease of ruminants, has emerged into a prolific, worldwide disease. Bovine DD treponemes have additional oxidative stress genes compared to nearest human-isolated relatives, suggesting better oxidative stress tolerance, and potentially explaining how bovine strains can colonize skin surfaces. Comparison of both bovine DD and GI treponemes as well as bovine pathogenic and human non-pathogenic saprophyte Treponema phagedenis strains indicates genes encoding a five-enzyme biosynthetic pathway for production of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, a rare di-N-acetylated mannuronic acid sugar, as important for pathogenesis. Bovine T. phagedenis strains further differed from human strains by having unique genetic clusters including components of a type IV secretion system and a phosphate utilisation system including phoU, a gene associated with osmotic stress survival. Proteomic analyses confirmed bovine derived T. phagedenis exhibits expression of PhoU but not the putative secretion system, whilst the novel mannuronic acid pathway was expressed in near entirety across the DD treponemes. Analysis of osmotic stress response in water identified a difference between bovine and human T. phagedenis with bovine strains exhibiting enhanced survival. This novel mechanism could enable a selective advantage, allowing environmental persistence and transmission of bovine T. phagedenis. Finally, we investigated putative outer membrane protein (OMP) ortholog families across the DD treponemes and identified several families as multi-specific adhesins capable of binding extra cellular matrix (ECM) components. One bovine pathogen specific adhesin ortholog family showed considerable serodiagnostic potential with the Treponema medium representative demonstrating considerable disease specificity (91.6%). This work has shed light on treponeme host adaptation and has identified candidate molecules for future diagnostics, vaccination and therapeutic intervention.


Assuntos
Treponema/genética , Infecções por Treponema/genética , Animais , Bovinos , DNA Bacteriano , Dermatite Digital/microbiologia , Humanos , Filogenia
4.
PLoS One ; 16(1): e0245922, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33481939

RESUMO

Optimised pre-clinical models are required for TB drug development to better predict the pharmacokinetics of anti-tuberculosis (anti-TB) drugs to shorten the time taken for novel drugs and combinations to be approved for clinical trial. Microdialysis can be used to measure unbound drug concentrations in awake freely moving animals in order to describe the pharmacokinetics of drugs in the organs as a continuous sampling technique. The aim of this work was to develop and optimise the microdialysis methodology in guinea pigs to better understand the pharmacokinetics of rifampicin in the lung. In vitro experiments were performed before progressing into in vivo studies because the recovery (concentration of the drug in the tissue fluid related to that in the collected dialysate) of rifampicin was dependent on a variety of experimental conditions. Mass spectrometry of the dialysate was used to determine the impact of flow rate, perfusion fluid and the molecular weight cut-off and membrane length of probes on the recovery of rifampicin at physiologically relevant concentrations. Following determination of probe efficiency and identification of a correlation between rifampicin concentrations in the lung and skeletal muscle, experiments were conducted to measure rifampicin in the sacrospinalis of guinea pigs using microdialysis. Lung concentrations of rifampicin were estimated from the rifampicin concentrations measured in the sacrospinalis. These studies suggest the potential usefulness of the microdialysis methodology to determine drug concentrations of selected anti-TB drugs to support new TB drug development.


Assuntos
Antituberculosos/análise , Pulmão/química , Microdiálise/métodos , Rifampina/análise , Animais , Desenvolvimento de Medicamentos , Feminino , Cobaias
5.
Am J Respir Crit Care Med ; 203(2): 192-201, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33217246

RESUMO

Rationale: In life-threatening coronavirus disease (COVID-19), corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or an independent immunopathologic process is unknown.Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses and the relationships among viral presence, inflammation, and organ injury.Methods: Tissue was acquired from 11 detailed postmortem examinations. SARS-CoV-2 organotropism was mapped by using multiplex PCR and sequencing, with cellular resolution achieved by in situ viral S (spike) protein detection. Histologic evidence of inflammation was quantified from 37 anatomic sites, and the pulmonary immune response was characterized by using multiplex immunofluorescence.Measurements and Main Results: Multiple aberrant immune responses in fatal COVID-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein between or within tissues. An arteritis was identified in the lung, which was further characterized as a monocyte/myeloid-rich vasculitis, and occurred together with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticuloendothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues.Conclusions: Tissue-specific immunopathology occurs in COVID-19, implicating a significant component of the immune-mediated, virus-independent immunopathologic process as a primary mechanism in severe disease. Our data highlight novel immunopathologic mechanisms and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma-cell responses as well as promote pathogen tolerance in COVID-19.


Assuntos
COVID-19/imunologia , Inflamação/virologia , Pulmão/imunologia , Insuficiência de Múltiplos Órgãos/virologia , SARS-CoV-2/imunologia , Idoso , Idoso de 80 Anos ou mais , Autopsia , Biópsia , COVID-19/patologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19 , Feminino , Imunofluorescência , Humanos , Inflamação/imunologia , Inflamação/patologia , Pulmão/patologia , Pulmão/virologia , Masculino , Insuficiência de Múltiplos Órgãos/imunologia , Insuficiência de Múltiplos Órgãos/patologia , SARS-CoV-2/patogenicidade , Índice de Gravidade de Doença
6.
Vet Parasitol ; 289: 109321, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33276290

RESUMO

Fasciola hepatica (the liver fluke) is a common, global parasite of livestock. It can be highly pathogenic and has health and welfare implications for infected individuals. Typically, in ruminants, infections are sub-clinical, but if undiagnosed, they can lead to significant production losses. Accurate diagnosis is crucial to identify infection. Antibody detection ELISAs are commonly used to diagnose infection due to their high sensitivity and specificity and are typically based on native fluke excretory/secretory (ES) products or cathepsin L1 (CL1), the immunodominant antigen within ES products. These tests have been developed based on the antibody response of experimentally infected animals; however, this response has not been well characterised in naturally infected animals. We compared the antibody recognition of a recombinant CL1 (rCL1) antigen and native adult fluke ES products. Whilst samples from experimentally infected animals showed strong recognition of rCL1, serum antibodies from naturally infected animals did not. These results were confirmed by peptide array. Immunoblotting sera against ES products showed that experimentally infected animals had a strong, specific response to CL1/CL2 proteins whilst antibodies from naturally infected animals recognised multiple proteins and had a variable response to CL1/CL2. Mass spectrometry of proteins separated by 2D SDS PAGE, identified several antigens recognised by serum antibodies from a naturally infected cow, including cathepsins L1, L2 and L5, glutathione S-transferase and a dihydrolipoyl dehydrogenase. Overall, these results show that the antibody response in naturally infected animals to adult fluke ES products is qualitatively different to experimentally infected animals. This suggests that a diagnostic test based on CL1 alone may not be appropriate for diagnosis of natural F. hepatica infections in sheep and cattle.


Assuntos
Anticorpos Anti-Helmínticos/sangue , Doenças dos Bovinos/parasitologia , Fasciola hepatica/imunologia , Fasciolíase/veterinária , Doenças dos Ovinos/parasitologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Bovinos , Doenças dos Bovinos/sangue , Doenças dos Bovinos/imunologia , Ensaio de Imunoadsorção Enzimática , Fasciolíase/imunologia , Fasciolíase/parasitologia , Ovinos , Doenças dos Ovinos/sangue , Doenças dos Ovinos/imunologia
7.
Viruses ; 12(10)2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33066701

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Betacoronavirus/isolamento & purificação , COVID-19 , Teste para COVID-19 , Vacinas contra COVID-19 , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , DNA Complementar/análise , DNA Complementar/genética , DNA Viral/análise , DNA Viral/genética , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Epidemiologia Molecular , Reação em Cadeia da Polimerase Multiplex , Pandemias , Pneumonia Viral/diagnóstico , RNA Viral/análise , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2 , Análise de Sequência
9.
Mol Cell Proteomics ; 19(5): 793-807, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32075873

RESUMO

The respiratory epithelium comprises polarized cells at the interface between the environment and airway tissues. Polarized apical and basolateral protein secretions are a feature of airway epithelium homeostasis. Human respiratory syncytial virus (hRSV) is a major human pathogen that primarily targets the respiratory epithelium. However, the consequences of hRSV infection on epithelium secretome polarity and content remain poorly understood. To investigate the hRSV-associated apical and basolateral secretomes, a proteomics approach was combined with an ex vivo pediatric human airway epithelial (HAE) model of hRSV infection (data are available via ProteomeXchange and can be accessed at https://www.ebi.ac.uk/pride/ with identifier PXD013661). Following infection, a skewing of apical/basolateral abundance ratios was identified for several individual proteins. Novel modulators of neutrophil and lymphocyte activation (CXCL6, CSF3, SECTM1 or CXCL16), and antiviral proteins (BST2 or CEACAM1) were detected in infected, but not in uninfected cultures. Importantly, CXCL6, CXCL16, CSF3 were also detected in nasopharyngeal aspirates (NPA) from hRSV-infected infants but not healthy controls. Furthermore, the antiviral activity of CEACAM1 against RSV was confirmed in vitro using BEAS-2B cells. hRSV infection disrupted the polarity of the pediatric respiratory epithelial secretome and was associated with immune modulating proteins (CXCL6, CXCL16, CSF3) never linked with this virus before. In addition, the antiviral activity of CEACAM1 against hRSV had also never been previously characterized. This study, therefore, provides novel insights into RSV pathogenesis and endogenous antiviral responses in pediatric airway epithelium.


Assuntos
Antivirais/metabolismo , Quimiocinas/metabolismo , Proteoma/metabolismo , Mucosa Respiratória/virologia , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Brônquios/patologia , Linhagem Celular , Criança , Células Epiteliais/patologia , Células Epiteliais/virologia , Células Caliciformes/metabolismo , Células Caliciformes/virologia , Homeostase , Humanos , Lactente , Cinética , Nasofaringe/virologia , Mucosa Respiratória/metabolismo , Vírus Sincicial Respiratório Humano/crescimento & desenvolvimento , Tropismo , Proteínas Virais/metabolismo
10.
Ticks Tick Borne Dis ; 11(1): 101299, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31542229

RESUMO

Vertically-transmitted bacterial symbionts are widespread in ticks and have manifold impacts on the epidemiology of tick-borne diseases. For instance, they may provide essential nutrients to ticks, affect vector competence, induce immune responses in vertebrate hosts, or even evolve to become vertebrate pathogens. The deer or blacklegged tick Ixodes scapularis harbours the symbiont Rickettsia buchneri in its ovarian tissues. Here we show by molecular, proteomic and imaging methods that R. buchneri is also capable of colonising the salivary glands of wild I. scapularis. This finding has important implications for the diagnosis of rickettsial infections and for pathogen-symbiont interactions in this notorious vector of Lyme borreliosis.


Assuntos
Ixodes/microbiologia , Rickettsia/fisiologia , Simbiose , Animais , Proteômica , Glândulas Salivares/diagnóstico por imagem , Glândulas Salivares/microbiologia
11.
Biochem Pharmacol ; 171: 113670, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31628910

RESUMO

Human butyrylcholinesterase (E.C. 3.1.1.8) purified from blood plasma has previously been shown to provide protection against up to five and a half times the median lethal dose of an organophosphorus nerve agent in several animal models. In this study the stoichiometric nature of the protection afforded by human butyrylcholinesterase against organophosphorus nerve agents was investigated in guinea pigs. Animals were administered human butyrylcholinesterase (26.15 mg/kg ≡ 308 nmol/kg) by the intravascular or intramuscular route. Animals were subsequently dosed with either soman or VX in accordance with a stage-wise adaptive dose design to estimate the modified median lethal dose in treated animals. Human butyrylcholinesterase (308 nmol/kg) increased the median lethal dose of soman from 154 nmol/kg to 770 nmol/kg. Comparing the molar ratio of agent molecules to enzyme active sites yielded a stoichiometric protective ratio of 2:1 for soman, likely related to the similar stereoselectivity the enzyme has compared to the toxic target, acetylcholinesterase. In contrast, human butyrylcholinesterase (308 nmol/kg) increased the median lethal dose of VX from 30 nmol/kg to 312 nmol/kg, resulting in a stoichiometric protective ratio of only 1:1, suggesting a lack of stereoselectivity for this agent.


Assuntos
Butirilcolinesterase/administração & dosagem , Substâncias para a Guerra Química/envenenamento , Agentes Neurotóxicos/envenenamento , Envenenamento/prevenção & controle , Animais , Área Sob a Curva , Butirilcolinesterase/sangue , Butirilcolinesterase/química , Substâncias para a Guerra Química/química , Cobaias , Humanos , Injeções Intramusculares , Injeções Intravenosas , Dose Letal Mediana , Masculino , Taxa de Depuração Metabólica , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacocinética , Compostos Organotiofosforados/química , Compostos Organotiofosforados/envenenamento , Soman/química , Soman/envenenamento , Estereoisomerismo
12.
Viruses ; 11(11)2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31689981

RESUMO

Recent studies have shown that transcriptomic analysis of blood samples taken from patients with acute Ebola virus disease (EVD) during the 2013-2016 West African outbreak was suggestive that a severe inflammatory response took place in acutely ill patients. The significant knowledge gained from studying the Makona variant, a cause of the largest known EVD outbreak, may be applicable to other species of ebolavirus, and other variants of the Ebola virus (EBOV) species. To investigate the ability of Makona to initiate an inflammatory response in human macrophages and characterise the host response in a similar manner to previously characterised EBOV variants, the human monocytic cell line THP-1 was differentiated into macrophage-like cells and infected with Makona. RNA-Seq and quantitative proteomics were used to identify and quantify host mRNA and protein abundance during infection. Data from infection with Reston virus (RESTV) were used as comparators to investigate changes that may be specific to, or enhanced in, Makona infection in relation to a less pathogenic species of ebolavirus.. This study found demonstrable induction of the inflammatory response, and increase in the activation state of THP-1 macrophages infected with Makona. NFκB and inflammation-associated transcripts displayed significant changes in abundance, reflective of what was observed in human patients during the 2013-2016 EBOV outbreak in West Africa, and demonstrated that transcriptomic changes found in Makona-infected cells were similar to that observed in Reston virus infection and that have been described in previous studies of other variants of EBOV.


Assuntos
Ebolavirus/isolamento & purificação , Ebolavirus/patogenicidade , Citocinas/genética , Citocinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Doença pelo Vírus Ebola/virologia , Humanos , Interferons/genética , Interferons/metabolismo , Macrófagos/imunologia , Macrófagos/virologia , Proteômica , Células THP-1
13.
Viruses ; 11(10)2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658630

RESUMO

Human respiratory syncytial virus (HRSV) is a major cause of pediatric infection and also causes disease in the elderly and those with underlying respiratory problems. There is no vaccine for HRSV and anti-viral therapeutics are not broadly applicable. To investigate the effect of HRSV biology in children, nasopharyngeal aspirates were taken from children with different viral loads and a combined high throughput RNAseq and label free quantitative proteomics approach was used to characterize the nucleic acid and proteins in these samples. HRSV proteins were identified in the nasopharyngeal aspirates from infected children, and their abundance correlated with viral load (Ct value), confirming HRSV infection. Analysis of the HRSV genome indicated that the children were infected with sub-group A virus and that minor variants in nucleotide frequency occurred in discrete clusters along the HRSV genome, and within a patient clustered distinctly within the glycoprotein gene. Data from the samples were binned into four groups; no-HRSV infection (control), high viral load (Ct < 20), medium viral load (Ct = 20-25), and low viral load (Ct > 25). Cellular proteins associated with the anti-viral response (e.g., ISG15) were identified in the nasopharyngeal aspirates and their abundance was correlated with viral load. These combined approaches have not been used before to study HRSV biology in vivo and can be readily applied to the study the variation of virus host interactions.


Assuntos
Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano/genética , Criança , Pré-Escolar , Citocinas/análise , Citocinas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Interações entre Hospedeiro e Microrganismos , Humanos , Lactente , Boca/virologia , Mucosa Nasal/virologia , Proteômica , RNA Viral/genética , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/virologia , Infecções Respiratórias/virologia , Ubiquitinas/análise , Ubiquitinas/genética , Carga Viral , Proteínas Virais/genética
14.
Artigo em Inglês | MEDLINE | ID: mdl-31555604

RESUMO

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection.


Assuntos
Interações Hospedeiro-Parasita/fisiologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Toxoplasma/fisiologia , Toxoplasma/patogenicidade , Animais , Técnicas de Cultura de Células , Colesterol , Colágeno , Modelos Animais de Doenças , Células Epiteliais/parasitologia , Células Epiteliais/patologia , Humanos , Mucosa Intestinal/diagnóstico por imagem , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL
15.
Sci Rep ; 9(1): 10757, 2019 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-31341188

RESUMO

Major urinary proteins (MUP) are the major component of the urinary protein fraction in house mice (Mus spp.) and rats (Rattus spp.). The structure, polymorphism and functions of these lipocalins have been well described in the western European house mouse (Mus musculus domesticus), clarifying their role in semiochemical communication. The complexity of these roles in the mouse raises the question of similar functions in other rodents, including the Norway rat, Rattus norvegicus. Norway rats express MUPs in urine but information about specific MUP isoform sequences and functions is limited. In this study, we present a detailed molecular characterization of the MUP proteoforms expressed in the urine of two laboratory strains, Wistar Han and Brown Norway, and wild caught animals, using a combination of manual gene annotation, intact protein mass spectrometry and bottom-up mass spectrometry-based proteomic approaches. Cluster analysis shows the existence of only 10 predicted mup genes. Further, detailed sequencing of the urinary MUP isoforms reveals a less complex pattern of primary sequence polymorphism in the rat than the mouse. However, unlike the mouse, rat MUPs exhibit added complexity in the form of post-translational modifications, including the phosphorylation of Ser4 in some isoforms, and exoproteolytic trimming of specific isoforms. Our results raise the possibility that urinary MUPs may have different roles in rat chemical communication than those they play in the house mouse. Shotgun proteomics data are available via ProteomExchange with identifier PXD013986.


Assuntos
Proteínas/genética , Ratos/genética , Animais , Feminino , Masculino , Polimorfismo Genético , Proteínas/metabolismo , Proteinúria/genética , Proteômica , Ratos/metabolismo , Ratos Wistar , Fatores Sexuais , Sistema Urinário/metabolismo
16.
Parasitology ; 146(14): 1773-1784, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31190665

RESUMO

Filarial nematodes possess glutathione transferases (GSTs), ubiquitous enzymes with the potential to detoxify xenobiotic and endogenous substrates, and modulate the host immune system, which may aid worm infection establishment, maintenance and survival in the host. Here we have identified and characterized a σ class glycosylated GST (OoGST1), from the cattle-infective filarial nematode Onchocerca ochengi, which is homologous (99% amino acid identity) with an immunodominant GST and potential vaccine candidate from the human parasite, O. volvulus, (OvGST1b). Onchocerca ochengi native GSTs were purified using a two-step affinity chromatography approach, resolved by 2D and 1D SDS-PAGE and subjected to enzymic deglycosylation revealing the existence of at least four glycoforms. A combination of lectin-blotting and mass spectrometry (MS) analyses of the released N-glycans indicated that OoGST1 contained mainly oligomannose Man5GlcNAc2 structure, but also hybrid- and larger oligommanose-type glycans in a lower proportion. Furthermore, purified OoGST1 showed prostaglandin synthase activity as confirmed by Liquid Chromatography (LC)/MS following a coupled-enzyme assay. This is only the second reported and characterized glycosylated GST and our study highlights its potential role in host-parasite interactions and use in the study of human onchocerciasis.


Assuntos
Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Onchocerca/enzimologia , Onchocerca/genética , Oncocercose/veterinária , Sequência de Aminoácidos , Animais , Bovinos/parasitologia , Doenças dos Bovinos/parasitologia , Cromatografia de Afinidade , Cromatografia Líquida , Feminino , Glicosilação , Espectrometria de Massas , Onchocerca volvulus/enzimologia , Onchocerca volvulus/genética , Oncocercose/parasitologia , Polissacarídeos/química , Prostaglandina-Endoperóxido Sintases/metabolismo , Estrutura Terciária de Proteína
17.
Dis Model Mech ; 12(3)2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30814064

RESUMO

Paneth cells are key epithelial cells that provide an antimicrobial barrier and maintain integrity of the small-intestinal stem cell niche. Paneth cell abnormalities are unfortunately detrimental to gut health and are often associated with digestive pathologies such as Crohn's disease or infections. Similar alterations are observed in individuals with impaired autophagy, a process that recycles cellular components. The direct effect of autophagy impairment on Paneth cells has not been analysed. To investigate this, we generated a mouse model lacking Atg16l1 specifically in intestinal epithelial cells, making these cells impaired in autophagy. Using three-dimensional intestinal organoids enriched for Paneth cells, we compared the proteomic profiles of wild-type and autophagy-impaired organoids. We used an integrated computational approach combining protein-protein interaction networks, autophagy-targeted proteins and functional information to identify the mechanistic link between autophagy impairment and disrupted pathways. Of the 284 altered proteins, 198 (70%) were more abundant in autophagy-impaired organoids, suggesting reduced protein degradation. Interestingly, these differentially abundant proteins comprised 116 proteins (41%) that are predicted targets of the selective autophagy proteins p62, LC3 and ATG16L1. Our integrative analysis revealed autophagy-mediated mechanisms that degrade key proteins in Paneth cell functions, such as exocytosis, apoptosis and DNA damage repair. Transcriptomic profiling of additional organoids confirmed that 90% of the observed changes upon autophagy alteration have effects at the protein level, not on gene expression. We performed further validation experiments showing differential lysozyme secretion, confirming our computationally inferred downregulation of exocytosis. Our observations could explain how protein-level alterations affect Paneth cell homeostatic functions upon autophagy impairment.This article has an associated First Person interview with the joint first authors of the paper.


Assuntos
Autofagia , Intestinos/fisiologia , Organoides/citologia , Organoides/metabolismo , Celulas de Paneth/metabolismo , Proteômica , Transcriptoma/genética , Animais , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Exocitose , Feminino , Masculino , Camundongos Endogâmicos C57BL , Proteólise , Reprodutibilidade dos Testes
18.
Cell Tissue Res ; 375(2): 409-424, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30259138

RESUMO

The in vitro 3D culture of intestinal epithelium is a valuable resource in the study of its function. Organoid culture exploits stem cells' ability to regenerate and produce differentiated epithelium. Intestinal organoid models from rodent or human tissue are widely available whereas large animal models are not. Livestock enteric and zoonotic diseases elicit significant morbidity and mortality in animal and human populations. Therefore, livestock species-specific models may offer novel insights into host-pathogen interactions and disease responses. Bovine and porcine jejunum were obtained from an abattoir and their intestinal crypts isolated, suspended in Matrigel, cultured, cryopreserved and resuscitated. 'Rounding' of crypts occurred followed by budding and then enlargement of the organoids. Epithelial cells were characterised using immunofluorescent staining and confocal microscopy. Organoids were successfully infected with Toxoplasma gondii or Salmonella typhimurium. This 3D organoid model offers a long-term, renewable resource for investigating species-specific intestinal infections with a variety of pathogens.


Assuntos
Técnicas de Cultura de Células/métodos , Mucosa Intestinal/metabolismo , Animais , Bovinos , Diferenciação Celular , Criopreservação , Gado , Camundongos Endogâmicos C57BL , Organoides/metabolismo , Fenótipo , Salmonella typhimurium/fisiologia , Suínos , Sobrevivência de Tecidos , Toxoplasma/fisiologia
19.
Gigascience ; 7(12)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30445460

RESUMO

Background: Trombidid mites have a unique life cycle in which only the larval stage is ectoparasitic. In the superfamily Trombiculoidea ("chiggers"), the larvae feed preferentially on vertebrates, including humans. Species in the genus Leptotrombidium are vectors of a potentially fatal bacterial infection, scrub typhus, that affects 1 million people annually. Moreover, chiggers can cause pruritic dermatitis (trombiculiasis) in humans and domesticated animals. In the Trombidioidea (velvet mites), the larvae feed on other arthropods and are potential biological control agents for agricultural pests. Here, we present the first trombidid mites genomes, obtained both for a chigger, Leptotrombidium deliense, and for a velvet mite, Dinothrombium tinctorium. Results: Sequencing was performed using Illumina technology. A 180 Mb draft assembly for D. tinctorium was generated from two paired-end and one mate-pair library using a single adult specimen. For L. deliense, a lower-coverage draft assembly (117 Mb) was obtained using pooled, engorged larvae with a single paired-end library. Remarkably, both genomes exhibited evidence of ancient lateral gene transfer from soil-derived bacteria or fungi. The transferred genes confer functions that are rare in animals, including terpene and carotenoid synthesis. Thirty-seven allergenic protein families were predicted in the L. deliense genome, of which nine were unique. Preliminary proteomic analyses identified several of these putative allergens in larvae. Conclusions: Trombidid mite genomes appear to be more dynamic than those of other acariform mites. A priority for future research is to determine the biological function of terpene synthesis in this taxon and its potential for exploitation in disease control.


Assuntos
Alérgenos/genética , Transferência Genética Horizontal/genética , Genoma , Ácaros/genética , Metabolismo Secundário/genética , Alquil e Aril Transferases/classificação , Alquil e Aril Transferases/genética , Alérgenos/imunologia , Animais , Proteínas de Artrópodes/análise , Proteínas de Artrópodes/classificação , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Bactérias/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Cromatografia Líquida de Alta Pressão , Proteínas Fúngicas/classificação , Proteínas Fúngicas/genética , Fungos/genética , Larva/genética , Ácaros/classificação , Ácaros/crescimento & desenvolvimento , Opsinas/classificação , Opsinas/genética , Filogenia , Proteínas e Peptídeos Salivares/classificação , Proteínas e Peptídeos Salivares/genética , Espectrometria de Massas em Tandem , Trombiculidae/classificação , Trombiculidae/genética
20.
Nat Commun ; 9(1): 2635, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29980663

RESUMO

Pseudomonas aeruginosa colonises the upper airway of cystic fibrosis (CF) patients, providing a reservoir of host-adapted genotypes that subsequently establish chronic lung infection. We previously experimentally-evolved P. aeruginosa in a murine model of respiratory tract infection and observed early-acquired mutations in pmrB, encoding the sensor kinase of a two-component system that promoted establishment and persistence of infection. Here, using proteomics, we show downregulation of proteins involved in LPS biosynthesis, antimicrobial resistance and phenazine production in pmrB mutants, and upregulation of proteins involved in adherence, lysozyme resistance and inhibition of the chloride ion channel CFTR, relative to wild-type strain LESB65. Accordingly, pmrB mutants are susceptible to antibiotic treatment but show enhanced adherence to airway epithelial cells, resistance to lysozyme treatment, and downregulate host CFTR expression. We propose that P. aeruginosa pmrB mutations in CF patients are subject to an evolutionary trade-off, leading to enhanced colonisation potential, CFTR inhibition, and resistance to host defences, but also to increased susceptibility to antibiotics.


Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Evolução Biológica , Interações Hospedeiro-Patógeno , Pseudomonas aeruginosa/metabolismo , Fatores de Transcrição/metabolismo , Células A549 , Adaptação Fisiológica/efeitos dos fármacos , Animais , Anti-Infecciosos/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Contagem de Colônia Microbiana , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulação para Baixo , Células Epiteliais/metabolismo , Fímbrias Bacterianas/efeitos dos fármacos , Fímbrias Bacterianas/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Testes de Sensibilidade Microbiana , Modelos Biológicos , Movimento , Muramidase/metabolismo , Mutação/genética , Análise de Componente Principal , Proteômica , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação
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