CrAssphages are an extensive and ubiquitous family of tailed bacteriophages, predicted to infect bacteria of the order Bacteroidales. Despite being found in ~50% of individuals and representing up to ...90% of human gut viromes, members of this viral family have never been isolated in culture and remain understudied. Here, we report the isolation of a CrAssphage (ΦCrAss001) from human faecal material. This bacteriophage infects the human gut symbiont Bacteroides intestinalis, confirming previous in silico predictions of the likely host. DNA sequencing demonstrates that the bacteriophage genome is circular, 102 kb in size, and has unusual structural traits. In addition, electron microscopy confirms that ΦcrAss001 has a podovirus-like morphology. Despite the absence of obvious lysogeny genes, ΦcrAss001 replicates in a way that does not disrupt proliferation of the host bacterium, and is able to maintain itself in continuous host culture during several weeks.
CrAssphages represent the most abundant virus in the human gut microbiota, but the lack of available genome sequences for comparison has kept them enigmatic. Recently, sequence-based classification ...of distantly related crAss-like phages from multiple environments was reported, leading to a proposed familial-level taxonomic group. Here, we assembled the metagenomic sequencing reads from 702 human fecal virome/phageome samples and analyzed 99 complete circular crAss-like phage genomes and 150 contigs ≥70 kb. In silico comparative genomics and taxonomic analysis enabled a classification scheme of crAss-like phages from human fecal microbiomes into four candidate subfamilies composed of ten candidate genera. Laboratory analysis was performed on fecal samples from an individual harboring seven distinct crAss-like phages. We achieved crAss-like phage propagation in ex vivo human fecal fermentations and visualized short-tailed podoviruses by electron microscopy. Mass spectrometry of a crAss-like phage capsid protein could be linked to metagenomic sequencing data, confirming crAss-like phage structural annotations.
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•Screening of human fecal metagenomic samples reveals 249 crAss-like phage genomes•The crAss-like phages were classified into 4 subfamilies composed of 10 candidate genera•A crAss-like phage was propagated in ex vivo human fecal fermentations•Short-tailed phage virions could be visualized by electron microscopy
CrAssphage is the most abundant human gut-associated virus. Guerin et al. identify 249 crAss-like phage genomes and classify them into four subfamilies and ten candidate genera that differ among human populations. These in silico predictions are combined with ex vivo propagations, electron microscopy imaging, and mass spectrometry detection.
The human gut microbiome is often described as the collection of bacteria, archaea, fungi, protists, and viruses associated with an individual, with no acknowledgement of the plasmid constituents. ...However, like viruses, plasmids are autonomous intracellular replicating entities that can influence the genotype and phenotype of their host and mediate trans-kingdom interactions. Plasmids are frequently noted as vehicles for horizontal gene transfer and for spreading antibiotic resistance, yet their multifaceted contribution to mutualistic and antagonistic interactions within the human microbiome and impact on human health is overlooked. In this review, we highlight the importance of plasmids and their biological properties as overlooked components of microbiomes. Subsequent human microbiome studies should include dedicated analyses of plasmids, particularly as a holistic understanding of human–microbial interactions is required before effective and safe interventions can be implemented to improve human well-being.
Streptococcus mutans is one of the principal agents of caries formation mainly, because of its ability to form biofilms at the tooth surface. Bacteriophages (phages) are promising antimicrobial ...agents that could be used to prevent or treat caries formation by S. mutans. The aim of this study was to isolate new S. mutans phages and to characterize their antimicrobial properties. A new phage, ɸAPCM01, was isolated from a human saliva sample. Its genome was closely related to the only two other available S. mutans phage genomes, M102 and M102AD. ɸAPCM01 inhibited the growth of S. mutans strain DPC6143 within hours in broth and in artificial saliva at multiplicity of infections as low as 2.5x10-5. In the presence of phage ɸAPCM01 the metabolic activity of a S. mutans biofilm was reduced after 24 h of contact and did not increased again after 48 h, and the live cells in the biofilm decreased by at least 5 log cfu/ml. Despite its narrow host range, this newly isolated S. mutans phage exhibits promising antimicrobial properties.
Not all viruses associated with humans cause disease. Non-pathogenic human-infecting viruses are predicted as important for immune system induction and preparation. Phages that infect bacteria are ...the most abundant predators of the human microbial ecosystem, promoting and maintaining bacterial diversity. Metagenomic analyses of the human gut virome and microbiome are unravelling the intricate predator-prey dynamics of phage-bacteria co-existence, co-evolution, and their interplay with the human host. While most adults harbour a distinctly individualistic and persistent community of virulent phages, new-borns are dominated by temperate phages heavily influenced by environmental exposures. The future development of microbiome-based interventions, therapeutics, and manipulation, will require a greater understanding of the human microbiome and the virome.
To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in ...cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.
We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.
For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of 'liquid biopsies' to better model drug discovery.
Recent studies have demonstrated that the human gut is populated by complex, highly individual and stable communities of viruses, the majority of which are bacteriophages. While disease-specific ...alterations in the gut phageome have been observed in IBD, AIDS and acute malnutrition, the human gut phageome remains poorly characterised. One important obstacle in metagenomic studies of the human gut phageome is a high level of discrepancy between results obtained by different research groups. This is often due to the use of different protocols for enriching virus-like particles, nucleic acid purification and sequencing. The goal of the present study is to develop a relatively simple, reproducible and cost-efficient protocol for the extraction of viral nucleic acids from human faecal samples, suitable for high-throughput studies. We also analyse the effect of certain potential confounding factors, such as storage conditions, repeated freeze-thaw cycles, and operator bias on the resultant phageome profile. Additionally, spiking of faecal samples with an exogenous phage standard was employed to quantitatively analyse phageomes following metagenomic sequencing. Comparative analysis of phageome profiles to bacteriome profiles was also performed following 16S rRNA amplicon sequencing.
Faecal phageome profiles exhibit an overall greater individual specificity when compared to bacteriome profiles. The phageome and bacteriome both exhibited moderate change when stored at + 4 °C or room temperature. Phageome profiles were less impacted by multiple freeze-thaw cycles than bacteriome profiles, but there was a greater chance for operator effect in phageome processing. The successful spiking of faecal samples with exogenous bacteriophage demonstrated large variations in the total viral load between individual samples.
The faecal phageome sequencing protocol developed in this study provides a valuable additional view of the human gut microbiota that is complementary to 16S amplicon sequencing and/or metagenomic sequencing of total faecal DNA. The protocol was optimised for several confounding factors that are encountered while processing faecal samples, to reduce discrepancies observed within and between research groups studying the human gut phageome. Rapid storage, limited freeze-thaw cycling and spiking of faecal samples with an exogenous phage standard are recommended for optimum results.
Establishing a diverse gut microbiota after birth is being increasingly recognised as important for preventing illnesses later in life. It is well established that bacterial diversity rapidly ...increases post-partum; however, few studies have examined the infant gut virome/phageome during this developmental period. We performed a metagenomic analysis of 20 infant faecal viromes at one year of age to determine whether spontaneous vaginal delivery (SVD) or caesarean section (CS) influenced viral composition. We find that birth mode results in distinctly different viral communities, with SVD infants having greater viral and bacteriophage diversity. We demonstrate that CrAssphage is acquired early in life, both in this cohort and two others, although no difference in birth mode is detected. A previous study has shown that bacterial OTU's (operational taxonomic units) identified in the same infants could not discriminate between birth mode at 12 months of age. Therefore, our results indicate that vertical transmission of viral communities from mother to child may play a role in shaping the early life microbiome, and that birth mode should be considered when studying the early life gut virome.
RNA Phage Biology in a Metagenomic Era Callanan, Julie; Stockdale, Stephen R; Shkoporov, Andrey ...
Viruses,
07/2018, Volume:
10, Issue:
7
Journal Article
Peer reviewed
Open access
The number of novel bacteriophage sequences has expanded significantly as a result of many metagenomic studies of phage populations in diverse environments. Most of these novel sequences bear little ...or no homology to existing databases (referred to as the "viral dark matter"). Also, these sequences are primarily derived from DNA-encoded bacteriophages (phages) with few RNA phages included. Despite the rapid advancements in high-throughput sequencing, few studies enrich for RNA viruses, i.e., target viral rather than cellular fraction and/or RNA rather than DNA via a reverse transcriptase step, in an attempt to capture the RNA viruses present in a microbial communities. It is timely to compile existing and relevant information about RNA phages to provide an insight into many of their important biological features, which should aid in sequence-based discovery and in their subsequent annotation. Without comprehensive studies, the biological significance of RNA phages has been largely ignored. Future bacteriophage studies should be adapted to ensure they are properly represented in phageomic studies.
The human gut microbiome consists of bacteria, archaea, eukaryotes, and viruses. The gut viruses are relatively underexplored. Here, we longitudinally analyzed the gut virome composition in 11 ...healthy adults: its stability, variation, and the effect of a gluten-free diet. Using viral enrichment and a de novo assembly-based approach, we demonstrate the quantitative dynamics of the gut virome, including dsDNA, ssDNA, dsRNA, and ssRNA viruses. We observe highly divergent individual viral communities, carrying on an average 2,143 viral genomes, 13.1% of which were present at all 3 time points. In contrast to previous reports, the Siphoviridae family dominates over Microviridae in studied individual viromes. We also show individual viromes to be stable at the family level but to vary substantially at the genera and species levels. Finally, we demonstrate that lower initial diversity of the human gut virome leads to a more pronounced effect of the dietary intervention on its composition.
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•Viral communities of the human gut are highly divergent across individuals•Lower initial viral diversity is associated with greater virome response to diet•Combining virome datasets increases the number of identified viruses per sample
Garmaeva et al. explore the influence of a gluten-free diet on the gut virome and microbiome. They observe high variability of gut viral communities across individuals and identify a strong effect of the diet on the virome composition in individuals with lower initial viral diversity.
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