Phage-encoded carbohydrate-interacting proteins in the human gut

In the human gastrointestinal tract, the gut mucosa and the bacterial component of the microbiota interact and modulate each other to accomplish a variety of critical functions. These include digestion aid, maintenance of the mucosal barrier, immune regulation, and production of vitamins, hormones, and other metabolites that are important for our health. The mucus lining of the gut is primarily composed of mucins, large glycosylated proteins with glycosylation patterns that vary depending on factors including location in the digestive tract and the local microbial population. Many gut bacteria have evolved to reside within the mucus layer and thus encode mucus-adhering and -degrading proteins. By doing so, they can influence the integrity of the mucus barrier and therefore promote either health maintenance or the onset and progression of some diseases. The viral members of the gut – mostly composed of bacteriophages – have also been shown to have mucus-interacting capabilities, but their mechanisms and effects remain largely unexplored. In this review, we discuss the role of bacteriophages in influencing mucosal integrity, indirectly via interactions with other members of the gut microbiota, or directly with the gut mucus via phage-encoded carbohydrate-interacting proteins. We additionally discuss how these phage-mucus interactions may influence health and disease states

Gut virome profiling identifies a widespread bacteriophage family associated with metabolic syndrome

There is significant interest in altering the course of cardiometabolic disease development via gut microbiomes. Nevertheless, the highly abundant phage members of the complex gut ecosystem -which impact gut bacteria- remain understudied. Here, we show gut virome changes associated with metabolic syndrome (MetS), a highly prevalent clinical condition preceding cardiometabolic disease, in 196 participants by combined sequencing of bulk whole genome and virus like particle communities. MetS gut viromes exhibit decreased richness and diversity. They are enriched in phages infecting Streptococcaceae and Bacteroidaceae and depleted in those infecting Bifidobacteriaceae. Differential abundance analysis identifies eighteen viral clusters (VCs) as significantly associated with either MetS or healthy viromes. Among these are a MetS-associated Roseburia VC that is related to healthy control-associated Faecalibacterium and Oscillibacter VCs. Further analysis of these VCs revealed the Candidatus Heliusviridae, a highly widespread gut phage lineage found in 90+% of participants. The identification of the temperate Ca. Heliusviridae provides a starting point to studies of phage effects on gut bacteria and the role that this plays in MetS

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Bacteriophage: from exploration to exploitation

Over the past decades, bacteriophage research has revealed the abundance of phages in nature, their morphological and genomic diversity, their influence in the regulation of microbial balance in the ecosystem and their impact on the evolution of microbial diversity. Since the 1950s, phages have also played a central role in some of the most significant fundamental discoveries in biological sciences that have been crucial for the development of molecular biology. More recently, phage research has resulted in the development of genome editing tools, and it has generated the renewed interest of using phages and phage-related products as therapeutic agents. Although major progress has been made, basic understanding on phage biology is still lacking. The number of phage genes with unknown function still largely outnumber those with established roles. Therefore, further progress depends on a deeper understanding on phage biology. The present thesis aims at developing tools to support phage research, explores the use of phages for therapeutic purposes, and expands our insights into the biology of phages. A literature review on the molecular, structural and evolutionary determinants of phage-host interaction (Chapters 1 and 2) underlines the relatively poor understanding of the subject. A great variety of structures and mechanisms of infection are being revealed, but no correlations have yet been established between these and host interaction. Furthermore, so far no evolutionary model accurately describes the coevolution of phages and bacteria. A particular interest of evolutionary studies concerns the understanding of the prevalence of broad-host range phages in natural environments, since these are rarely isolated using standard laboratory isolation procedures. Indeed, we have tried to isolate broad host range phages targeting the Escherichia coli reference collection (Chapter 3), but found narrow-host range phages to be more prevalent. Only one phage of relatively broad host range was found (S2-36s), being able to infect 14 of the 72 strains. Proteins of interest for further exploration were found, such as depolymerases and colanic acid-degrading proteins, both with potential anti-biofilm activity. The isolation procedures against the ECOR collection proved to be challenging due to the amount of strains and samples to be evaluated. Consequently, a high-throughput methodology was developed to simplify these isolation procedures (Chapter 4). By automated monitoring of cell growth in 96-well plates it is possible to use differences in optical densities (plotted as heatmaps) between cells subjected to the samples and in control conditions to screen for the presence of phages. The method revealed an accuracy of 98% and reduced the workload by 90%. The method developed can also be used to screen for broad-host range phages or to screen collections of phages for variants or cocktails that are suitable for treating bacterial infections. A discussion is provided of the advantages and limitations of phages for therapeutic applications (Chapter 5). It is suggested that phages in their natural state cannot be used in therapeutic applications. The future of phage therapy may possibly be genome engineering for tailoring of phage properties. Subsequently, the genetic modification of phage T7 was shown to improve (2-log) the capacity of the phage to resist to the strongly acidic conditions and enzymatic challenges of the gastrointestinal tract (Chapter 6). This was achieved by modifying the phage to express a signal peptide on its capsid to which phospholipids attach forming a protective coating. The removal of the phospholipid coating using phospholipase caused reversion to the pH-sensitive phenotype of the wild-type phage. In case of orally-delivered phages, this may improve the efficacy of phage therapy. Engineering of phage genomes can also support evolutionary studies and basic phage research, e.g. analyzing if a certain gene is essential. A strategy developed for the random recombination of phage genomes (Chapter 7) demonstrated that it is possible to create novel productive phages by combining elements of different phage families. The findings reveal an unexpected level of flexibility and adaptability of phage genomes to accommodate and re-arrange genetic information, reflecting the pre-existing evolutionary compatibility of genes from different phages. The method is further expected to serve as a platform for improving our understanding of phage gene function and importance, where the random recombination of a single phage genome may be the preferred approach. A different approach for the therapeutic application of phages was explored. Using phage display it was possible to identify peptides targeting claudin-low breast cancer cells (Chapter 8) and osteoarthritis cells (Chapter 9) with high levels of specificity. The peptides identified may contribute to an early detection of claudin-low breast carcinomas, and to develop more individualized therapies for both breast cancer and osteoarthritis. In summary, the work developed in this thesis has resulted in new methodologies and biological data, thereby contributing to an increased understanding of phage biology and of the opportunities for the use of phages for diagnosis and therapy.</p

Bowel Biofilms : Tipping Points between a Healthy and Compromised Gut?

Bacterial communities are known to impact human health and disease. Mixed species biofilms, mostly pathogenic in nature, have been observed in dental and gastric infections as well as in intestinal diseases, chronic gut wounds and colon cancer. Apart from the appendix, the presence of thick polymicrobial biofilms in the healthy gut mucosa is still debated. Polymicrobial biofilms containing potential pathogens appear to be an early-warning signal of developing disease and can be regarded as a tipping point between a healthy and a diseased state of the gut mucosa. Key biofilm-forming pathogens and associated molecules hold promise as biomarkers. Criteria to distinguish microcolonies from biofilms are crucial to provide clarity when reporting biofilm-related phenomena in health and disease in the gut.Peer reviewe

Cas4-Cas1 fusions drive efficient PAM selection and control CRISPR adaptation

Microbes have the unique ability to acquire immunological memories from mobile genetic invaders to protect themselves from predation. To confer CRISPR resistance, new spacers need to be compatible with a targeting requirement in the invader's DNA called the protospacer adjacent motif (PAM). Many CRISPR systems encode Cas4 proteins to ensure new spacers are integrated that meet this targeting prerequisite. Here we report that a gene fusion between cas4 and cas1 from the Geobacter sulfurreducens I-U CRISPR-Cas system is capable of introducing functional spacers carrying interference proficient TTN PAM sequences at much higher frequencies than unfused Cas4 adaptation modules. Mutations of Cas4-domain catalytic residues resulted in dramatically decreased naïve and primed spacer acquisition, and a loss of PAM selectivity showing that the Cas4 domain controls Cas1 activity. We propose the fusion gene evolved to drive the acquisition of only PAM-compatible spacers to optimize CRISPR interference.</p

Complete genome sequence of the Escherichia coli phage Ayreon

We report the whole-genome sequence of a new Escherichia coli temperate phage, Ayreon, comprising a linear double-stranded DNA (dsDNA) genome of 44,708 bp

Molecular and Evolutionary Determinants of Bacteriophage Host Range

The host range of a bacteriophage is the taxonomic diversity of hosts it can successfully infect. Host range, one of the central traits to understand in phages, is determined by a range of molecular interactions between phage and host throughout the infection cycle. While many well studied model phages seem to exhibit a narrow host range, recent ecological and metagenomics studies indicate that phages may have specificities that range from narrow to broad. There is a growing body of studies on the molecular mechanisms that enable phages to infect multiple hosts. These mechanisms, and their evolution, are of considerable importance to understanding phage ecology and the various clinical, industrial, and biotechnological applications of phage. Here we review knowledge of the molecular mechanisms that determine host range, provide a framework defining broad host range in an evolutionary context, and highlight areas for additional research.</p

Phage-encoded carbohydrate-interacting proteins in the human gut

In the human gastrointestinal tract, the gut mucosa and the bacterial component of the microbiota interact and modulate each other to accomplish a variety of critical functions. These include digestion aid, maintenance of the mucosal barrier, immune regulation, and production of vitamins, hormones, and other metabolites that are important for our health. The mucus lining of the gut is primarily composed of mucins, large glycosylated proteins with glycosylation patterns that vary depending on factors including location in the digestive tract and the local microbial population. Many gut bacteria have evolved to reside within the mucus layer and thus encode mucus-adhering and -degrading proteins. By doing so, they can influence the integrity of the mucus barrier and therefore promote either health maintenance or the onset and progression of some diseases. The viral members of the gut – mostly composed of bacteriophages – have also been shown to have mucus-interacting capabilities, but their mechanisms and effects remain largely unexplored. In this review, we discuss the role of bacteriophages in influencing mucosal integrity, indirectly via interactions with other members of the gut microbiota, or directly with the gut mucus via phage-encoded carbohydrate-interacting proteins. We additionally discuss how these phage-mucus interactions may influence health and disease states