RESUMO
Wastewater surveillance is a proven method for tracking community spread and prevalence of some infectious viral diseases. A primary concentration step is often used to enrich viral particles from wastewater prior to subsequent viral quantification and/or sequencing. Here, we present a simple procedure for concentrating viruses from wastewater using bacterial biofilm protein nanofibers known as curli fibers. Through simple genetic engineering, we produced curli fibers functionalized with single-domain antibodies (also known as nanobodies) specific for the coat protein of the model virus bacteriophage MS2. Using these modified fibers in a simple spin-down protocol, we demonstrated efficient concentration of MS2 in both phosphate-buffered saline (PBS) and in the wastewater matrix. Additionally, we produced nanobody-functionalized curli fibers capable of binding the spike protein of SARS-CoV-2, showing the versatility of the system. Our concentration protocol is simple to implement, can be performed quickly under ambient conditions, and requires only components produced through bacterial culture. We believe this technology represents an attractive alternative to existing concentration methods and warrants further research and optimization for field-relevant applications.
RESUMO
Bacterial cellulose (BC) has excellent material properties and can be produced sustainably through simple bacterial culture, but BC-producing bacteria lack the extensive genetic toolkits of model organisms such as Escherichia coli (E. coli). Here, a simple approach is reported for producing highly programmable BC materials through incorporation of engineered E. coli. The acetic acid bacterium Gluconacetobacter hansenii is cocultured with engineered E. coli in droplets of glucose-rich media to produce robust cellulose capsules, which are then colonized by the E. coli upon transfer to selective lysogeny broth media. It is shown that the encapsulated E. coli can produce engineered protein nanofibers within the cellulose matrix, yielding hybrid capsules capable of sequestering specific biomolecules from the environment and enzymatic catalysis. Furthermore, capsules are produced which can alter their own bulk physical properties through enzyme-induced biomineralization. This novel system uses a simple fabrication process, based on the autonomous activity of two bacteria, to significantly expand the functionality of BC-based living materials.
Assuntos
Celulose/biossíntese , Escherichia coli/metabolismo , Bioengenharia , Cápsulas , Técnicas de Cocultura , Meios de Cultura , Gluconacetobacter/metabolismo , Nanofibras/químicaRESUMO
Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes that are crucial for the function of an organism will be depleted of such variants in natural populations, whereas non-essential genes will tolerate their accumulation. However, predicted loss-of-function variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes1. Here we describe the aggregation of 125,748 exomes and 15,708 genomes from human sequencing studies into the Genome Aggregation Database (gnomAD). We identify 443,769 high-confidence predicted loss-of-function variants in this cohort after filtering for artefacts caused by sequencing and annotation errors. Using an improved model of human mutation rates, we classify human protein-coding genes along a spectrum that represents tolerance to inactivation, validate this classification using data from model organisms and engineered human cells, and show that it can be used to improve the power of gene discovery for both common and rare diseases.
Assuntos
Exoma/genética , Genes Essenciais/genética , Variação Genética/genética , Genoma Humano/genética , Adulto , Encéfalo/metabolismo , Doenças Cardiovasculares/genética , Estudos de Coortes , Bases de Dados Genéticas , Feminino , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Mutação com Perda de Função/genética , Masculino , Taxa de Mutação , Pró-Proteína Convertase 9/genética , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
Many of the sequenced bacterial and archaeal genomes encode regions of viral provenance. Yet, not all of these regions encode bona fide viruses. Gene transfer agents (GTAs) are thought to be former viruses that are now maintained in genomes of some bacteria and archaea and are hypothesized to enable exchange of DNA within bacterial populations. In Alphaproteobacteria, genes homologous to the "head-tail" gene cluster that encodes structural components of the Rhodobacter capsulatus GTA (RcGTA) are found in many taxa, even if they are only distantly related to Rhodobacter capsulatus. Yet, in most genomes available in GenBank RcGTA-like genes have annotations of typical viral proteins, and therefore are not easily distinguished from their viral homologs without additional analyses. Here, we report a "support vector machine" classifier that quickly and accurately distinguishes RcGTA-like genes from their viral homologs by capturing the differences in the amino acid composition of the encoded proteins. Our open-source classifier is implemented in Python and can be used to scan homologs of the RcGTA genes in newly sequenced genomes. The classifier can also be trained to identify other types of GTAs, or even to detect other elements of viral ancestry. Using the classifier trained on a manually curated set of homologous viruses and GTAs, we detected RcGTA-like "head-tail" gene clusters in 57.5% of the 1,423 examined alphaproteobacterial genomes. We also demonstrated that more than half of the in silico prophage predictions are instead likely to be GTAs, suggesting that in many alphaproteobacterial genomes the RcGTA-like elements remain unrecognized.
Assuntos
Alphaproteobacteria/genética , Prófagos/genética , Máquina de Vetores de Suporte , Alphaproteobacteria/classificação , Genes Bacterianos , Genes Virais , Genoma BacterianoRESUMO
Curli are amyloid proteins that are assembled into extracellular polymeric fibers by bacteria during biofilm formation. The beta-sheet-rich protein CsgA, the primary structural component of the fibers, is secreted through dedicated machinery and self-assembles into cell-anchored fibers many times longer than the cell. Here, we have developed an in situ fluorescence assay for curli production that exploits the fluorescent properties of Congo red (CR) dye when bound to amyloid, allowing for rapid and robust curli quantification. We initially evaluated three amyloid-binding dyes for the fluorescent detection of curli in bacterial culture and found only Congo red compatible with in situ quantification. We further characterized the fluorescent properties of the dye directly in bacterial culture and calibrated the fluorescence using purified CsgA protein. We then used the Congo red assay to rapidly develop and characterize inducible curli-producing constructs in both an MC4100-derived lab strain of Escherichia coli and a derivative of the probiotic strain E. coli Nissle. This technique can be used to evaluate curli production in a minimally invasive manner using a range of equipment, simplifying curli quantification and the development of novel engineered curli systems.IMPORTANCE Curli are proteins produced by many bacteria as a structural component of biofilms, and they have recently emerged as a platform for fabrication of biological materials. Curli fibers are very robust and resistant to degradation, and the curli subunits can tolerate many protein fusions, facilitating the biosynthesis of novel functional materials. A serious bottleneck in the development of more sophisticated engineered curli systems is the rapid quantification of curli production by the bacteria. In this work we address this issue by developing a technique to monitor curli production directly in bacterial cultures, allowing for rapid curli quantification in a manner compatible with many powerful high-throughput techniques that can be used to engineer complex biological material systems.
Assuntos
Proteínas Amiloidogênicas/química , Vermelho Congo/química , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas Amiloidogênicas/metabolismo , Biofilmes/crescimento & desenvolvimento , Escherichia coli/química , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fluorescência , Coloração e RotulagemRESUMO
The clinical interpretation of genetic variants has come to rely heavily on reference population databases such as the Exome Aggregation Consortium (ExAC) database. Pathogenic variants in genes associated with severe, pediatric-onset, highly penetrant, autosomal dominant conditions are assumed to be absent or rare in these databases. Exome sequencing of a 6-year-old female patient with seizures, developmental delay, dysmorphic features, and failure to thrive identified an ASXL1 variant previously reported as causative of Bohring-Opitz syndrome (BOS). Surprisingly, the variant was observed seven times in the ExAC database, presumably in individuals without BOS. Although the BOS phenotype fit, the presence of the variant in reference population databases introduced ambiguity in result interpretation. Review of the literature revealed that acquired somatic mosaicism of ASXL1 variants (including pathogenic variants) during hematopoietic clonal expansion can occur with aging in healthy individuals. We examined all ASXL1 truncating variants in the ExAC database and determined most are likely somatic. Failure to consider somatic mosaicism may lead to the inaccurate assumption that conditions like BOS have reduced penetrance, or the misclassification of potentially pathogenic variants.
Assuntos
Craniossinostoses/diagnóstico , Craniossinostoses/genética , Estudos de Associação Genética , Mutação em Linhagem Germinativa , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Mutação , Proteínas Repressoras/genética , Idoso , Idoso de 80 Anos ou mais , Alelos , Substituição de Aminoácidos , Pré-Escolar , Bases de Dados Genéticas , Fácies , Feminino , Estudos de Associação Genética/métodos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC). This catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of predicted protein-truncating variants, with 72% of these genes having no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human 'knockout' variants in protein-coding genes.