Resumen de Estudio metagenómico de la comunidad de virus y de su interacción con la microbiota en la cavidad bucal humana
Viruses are key players regulating microbial ecosystems. Exploration of viral assemblages is now possible thanks to the development of metagenomics, the most powerful tool for studying viral ecology. Unfortunately, several sources of bias lead to the misrepresentation of certain viruses within metagenomics workflows. The oral cavity is a major portal of entry for human viruses, but it is dominated by highly personalized and time-persistent bacteriophage assemblages. Most of them follow lysogenic life cycles, deploying complex strategies to manage bacterial homeostasis. Although bacterial dysbiosis underlies common oral pathologies, the cause of these bacteria replacements remains obscure, and it is theorized that bacteriophages might play an important role.
In this thesis, we assessed the bias induced by viral enrichment and random amplification methods on mock assemblages of DNA viruses and human saliva viromes, using qPCR and deep sequencing. We observed that low-force centrifugation, 0.45μm filtration and iodixanol cushions preserved the original composition of nuclease-protected viral genomes. Comparison of unamplified and randomly amplified saliva viromes revealed that multiple displacement amplification induced stochastic bias from picograms of DNA template, but systematic bias from nanograms. This systematic bias was mainly due to under-amplification of sequences with extreme %GC, a negative bias shared with PCR-based methods that showed, in addition, high-coverage peaks in sequences with low linguistic complexity. Ordination plots of contig profiles showed overlapping of related amplified and unamplified saliva viromes and strong separation from unrelated saliva viromes. This result suggests that random amplification bias has a minor impact on beta diversity studies.
In order to gain insight into the diversity of viruses in the oral cavity, and their contribution to maintain healthy bacterial communities, we addressed the largest metagenomic study of viruses reported to date for this ecosystem: 43 Gbp. Oral viromes were dominated by bacteriophages of the Caudovirales order, with a small percentage of eukaryotic viruses, including four new human viruses of the Anelloviridae and Papillomaviridae families. 444 nearly full-length viral genomes, grouped in 31 megaclusters, were poorly related to those in the databases. However, some of the most abundant and ubiquitous megaclusters were also found in other published oral viromes. Bacteria communities differs substantially between dental plaque and oral mucosa, whereas this difference was at the limit of the statistical significance for virus assemblages. No differences in the microbiomes and viromes were found between healthy individuals and patients affected by caries or recurrent aphthous stomatitis.
Bacteriophage host-prediction by five consistent bioinformatic approaches revealed that Actinobacteria was the most frequent host in the oral cavity, in spite of its fourth position in 16S metagenomes of the same samples. Clustering of bacteriophages that infect the same phylum supports the importance of virus-host coevolution, and suggests that the host phylum is better taxonomic criteria than virus morphology for classification within the Caudovirales order. The identification of hundreds of lysins encoded by oral bacteriophages, together with their host-range prediction, might boost enzibiotics research as an alternative to antibiotics for restoring healthy oral microbiota.
