Defining the role of bacteriophage in mucosal immunity

Since their discovery over one hundred years ago, bacteriophages (phages) play a central role in the ongoing molecular biology revolution. As such, model phages like Lambda and T4 are perhaps the most genetically well-understood biological entities. Yet, critical aspects regarding the life histories of these phages, nevertheless non-model phages, remain unaddressed. This dissertation examines the life history of T4 and non-model phages. In chapter 2, the steps involved in the morphogenesis of T4-like phages were reevaluated. During the final stages of morphogenesis, T4 packages DNA into an empty head that is subsequently joined with a tail to make a mature virion. Bioinformatic analysis of one gene involved in this head-to-tail joining process, gene 4 (gp4), identified a previously unrecognized nuclease motif. Biochemical analysis confirmed the DNA-cutting capabilities of gp4. The nuclease function of gp4 is shown to be conserved in T4-like phages. In aggregate, the data contained in chapter 2 of this dissertation suggests gp4 plays a role in the termination of genome packaging to enable head-tail joining. Chapter 3 is dedicated to the analysis of non-model phages, focusing on those that contain diversity-generating retroelements (DGRs). DGRs are genetic cassettes that introduce sequence variation into target genes. Few isolated phages possess a DGR, yet metagenomic analyses reveal their presence in uncultured viral communities. In chapter 3, a survey identified 92 DGRs that were only found in phages exhibiting a temperate lifestyle. One novel temperate phage that possesses a DGR cassette targeting a gene of unknown function was isolated from Bacteroides dorei. This phage, here named Hankyphage, exhibits broad host-range and is capable of infecting at least 13 different Bacteroides species. Sequencing reads from whole-community metagenomes and viromes were recruited to the Hankyphage genome, highlighting the global distribution of Hankyphage. The results in chapter 3 suggest that targeted hypervariation by temperate phages, such as Hankyphage, may be a ubiquitous mechanism underlying phage-bacteria interaction in the human microbiome

Defining the role of bacteriophage in mucosal immunity

Since their discovery over one hundred years ago, bacteriophages (phages) play a central role in the ongoing molecular biology revolution. As such, model phages like Lambda and T4 are perhaps the most genetically well-understood biological entities. Yet, critical aspects regarding the life histories of these phages, nevertheless non-model phages, remain unaddressed. This dissertation examines the life history of T4 and non-model phages. In chapter 2, the steps involved in the morphogenesis of T4-like phages were reevaluated. During the final stages of morphogenesis, T4 packages DNA into an empty head that is subsequently joined with a tail to make a mature virion. Bioinformatic analysis of one gene involved in this head-to-tail joining process, gene 4 (gp4), identified a previously unrecognized nuclease motif. Biochemical analysis confirmed the DNA-cutting capabilities of gp4. The nuclease function of gp4 is shown to be conserved in T4-like phages. In aggregate, the data contained in chapter 2 of this dissertation suggests gp4 plays a role in the termination of genome packaging to enable head-tail joining. Chapter 3 is dedicated to the analysis of non-model phages, focusing on those that contain diversity-generating retroelements (DGRs). DGRs are genetic cassettes that introduce sequence variation into target genes. Few isolated phages possess a DGR, yet metagenomic analyses reveal their presence in uncultured viral communities. In chapter 3, a survey identified 92 DGRs that were only found in phages exhibiting a temperate lifestyle. One novel temperate phage that possesses a DGR cassette targeting a gene of unknown function was isolated from Bacteroides dorei. This phage, here named Hankyphage, exhibits broad host-range and is capable of infecting at least 13 different Bacteroides species. Sequencing reads from whole-community metagenomes and viromes were recruited to the Hankyphage genome, highlighting the global distribution of Hankyphage. The results in chapter 3 suggest that targeted hypervariation by temperate phages, such as Hankyphage, may be a ubiquitous mechanism underlying phage-bacteria interaction in the human microbiome

Cargo Genes of Tn 7 -Like Transposons Comprise an Enormous Diversity of Defense Systems, Mobile Genetic Elements, and Antibiotic Resistance Genes

Transposons are major vehicles of horizontal gene transfer that, in addition to genes directly involved in transposition, carry cargo genes. However, characterization of these genes is hampered by the difficulty of identification of transposon boundaries.</jats:p

The Missing Tailed Phages: Prediction of Small Capsid Candidates

Tailed phages are the most abundant and diverse group of viruses on the planet. Yet, the smallest tailed phages display relatively complex capsids and large genomes compared to other viruses. The lack of tailed phages forming the common icosahedral capsid architectures T = 1 and T = 3 is puzzling. Here, we extracted geometrical features from high-resolution tailed phage capsid reconstructions and built a statistical model based on physical principles to predict the capsid diameter and genome length of the missing small-tailed phage capsids. We applied the model to 3348 isolated tailed phage genomes and 1496 gut metagenome-assembled tailed phage genomes. Four isolated tailed phages were predicted to form T = 3 icosahedral capsids, and twenty-one metagenome-assembled tailed phages were predicted to form T &lt; 3 capsids. The smallest capsid predicted was a T = 4/3 &asymp; 1.33 architecture. No tailed phages were predicted to form the smallest icosahedral architecture, T = 1. We discuss the feasibility of the missing T = 1 tailed phage capsids and the implications of isolating and characterizing small-tailed phages for viral evolution and phage therapy

A diversity-generating retroelement encoded by a globally ubiquitous Bacteroides phage

Abstract Background Diversity-generating retroelements (DGRs) are genetic cassettes that selectively mutate target genes to produce hypervariable proteins. First characterized in Bordetella bacteriophage BPP-1, the DGR creates a hypervariable phage tail fiber that enables host tropism switching. Subsequent surveys for DGRs conclude that the majority identified to date are bacterial or archaeal in origin. This work examines bacteriophage and bacterial genomes for novel phage-encoded DGRs. Results This survey discovered 92 DGRs that were only found in phages exhibiting a temperate lifestyle. The majority of phage-encoded DGRs were identified as prophages in bacterial hosts from the phyla Bacteroidetes, Proteobacteria, and Firmicutes. Sequence reads from these previously unidentified prophages were present in viral metagenomes (viromes), indicating these prophages can produce functional viruses. Five phages possessed hypervariable proteins with structural similarity to the tail fiber of BPP-1, whereas the functions of the remaining DGR target proteins were unknown. A novel temperate phage that harbors a DGR cassette targeting a protein of unknown function was induced from Bacteroides dorei. This phage, here named Bacteroides dorei Hankyphage, lysogenizes 13 different Bacteroides species and was present in 34% and 21% of whole-community metagenomes and human-associated viromes, respectively. Conclusions Here, the number of known DGR-containing phages is increased from four to 92. All of these phages exhibit a temperate lifestyle, including a cosmopolitan human-associated phage. Targeted hypervariation by temperate phages may be a ubiquitous mechanism underlying phage-bacteria interaction in the human microbiome

Cystic Fibrosis Rapid Response: Translating Multi-omics Data into Clinically Relevant Information

Proper management of polymicrobial infections in patients with cystic fibrosis (CF) has extended their life span. Information about the composition and dynamics of each patient’s microbial community aids in the selection of appropriate treatment of pulmonary exacerbations. We propose the cystic fibrosis rapid response (CFRR) as a fast approach to determine viral and microbial community composition and activity during CF pulmonary exacerbations. The CFRR potential is illustrated with a case study in which a cystic fibrosis fatal exacerbation was characterized by the presence of shigatoxigenic Escherichia coli. The incorporation of the CFRR within the CF clinic could increase the life span and quality of life of CF patients.Pulmonary exacerbations are the leading cause of death in cystic fibrosis (CF) patients. To track microbial dynamics during acute exacerbations, a CF rapid response (CFRR) strategy was developed. The CFRR relies on viromics, metagenomics, metatranscriptomics, and metabolomics data to rapidly monitor active members of the viral and microbial community during acute CF exacerbations. To highlight CFRR, a case study of a CF patient is presented, in which an abrupt decline in lung function characterized a fatal exacerbation. The microbial community in the patient’s lungs was closely monitored through the multi-omics strategy, which led to the identification of pathogenic shigatoxigenic Escherichia coli (STEC) expressing Shiga toxin. This case study illustrates the potential for the CFRR to deconstruct complicated disease dynamics and provide clinicians with alternative treatments to improve the outcomes of pulmonary exacerbations and expand the life spans of individuals with CF

Metabolomics of reef benthic interactions reveals a bioactive lipid involved in coral defence.

Holobionts are assemblages of microbial symbionts and their macrobial host. As extant representatives of some of the oldest macro-organisms, corals and algae are important for understanding how holobionts develop and interact with one another. Using untargeted metabolomics, we show that non-self interactions altered the coral metabolome more than self-interactions (i.e. different or same genus, respectively). Platelet activating factor (PAF) and Lyso-PAF, central inflammatory modulators in mammals, were major lipid components of the coral holobionts. When corals were damaged during competitive interactions with algae, PAF increased along with expression of the gene encoding Lyso-PAF acetyltransferase; the protein responsible for converting Lyso-PAF to PAF. This shows that self and non-self recognition among some of the oldest extant holobionts involve bioactive lipids identical to those in highly derived taxa like humans. This further strengthens the hypothesis that major players of the immune response evolved during the pre-Cambrian

Genomic and ecological attributes of marine bacteriophages encoding bacterial virulence genes

Background Bacteriophages encode genes that modify bacterial functions during infection. The acquisition of phage-encoded virulence genes is a major mechanism for the rise of bacterial pathogens. In coral reefs, high bacterial density and lysogeny has been proposed to exacerbate reef decline through the transfer of phage-encoded virulence genes. However, the functions and distribution of these genes in phage virions on the reef remain unknown. Results Here, over 28,000 assembled viral genomes from the free viral community in Atlantic and Pacific Ocean coral reefs were queried against a curated database of virulence genes. The diversity of virulence genes encoded in the viral genomes was tested for relationships with host taxonomy and bacterial density in the environment. These analyses showed that bacterial density predicted the profile of virulence genes encoded by phages. The Shannon diversity of virulence-encoding phages was negatively related with bacterial density, leading to dominance of fewer genes at high bacterial abundances. A statistical learning analysis showed that reefs with high microbial density were enriched in viruses encoding genes enabling bacterial recognition and invasion of metazoan epithelium. Over 60% of phages could not have their hosts identified due to limitations of host prediction tools; for those which hosts were identified, host taxonomy was not an indicator of the presence of virulence genes. Conclusions This study described bacterial virulence factors encoded in the genomes of bacteriophages at the community level. The results showed that the increase in microbial densities that occurs during coral reef degradation is associated with a change in the genomic repertoire of bacteriophages, specifically in the diversity and distribution of bacterial virulence genes. This suggests that phages are implicated in the rise of pathogens in disturbed marine ecosystems

Secreted adiponectin interleukin 6 (IL-6) and leptin levels during 3T3-L1 cell adipogenesis.

<p>Culture medium samples of 3T3-L1 preadipocytes (d0) and adipocytes (d7, d21) were obtained for adipokine ELISA assay. Data are means ± SEM of the concentration (ng/mL) of each adipokine secreted to the cell medium. (A) Adiponectin (B) Interleukin 6 (IL-6) (C) Leptin. Groups were compared using the Wilcoxon signed rank test. Data are referred to day 0 *. p≤0.05, **. p≤0.01 or to day 7 Δ. p≤0.05.</p

Three-Dimensional Molecular Cartography of the Caribbean Reef-Building Coral Orbicella faveolata

All organisms host a diversity of associated viruses, bacteria, and protists, collectively defined as the holobiont. While scientific advancements have enhanced the understanding of the functional roles played by various components of the holobiont, there is a growing need to integrate multiple types of molecular data into spatially and temporally resolved frameworks. To that end, we mapped 16S and 18S rDNA metabarcoding, metatranscriptomics, and metabolomic data onto three-dimensional reconstructions of coral colonies to examine microbial diversity, microbial gene expression, and biochemistry on two colonies of the ecologically important, reef-building coral, Orbicella faveolata and their competitors (i.e., adjacent organisms interacting with the corals: fleshy algae, turf algae, hydrozoans, and other corals). Overall, no statistically significant spatial patterns were observed among the samples for any of the data types; instead, strong signatures of the macroorganismal hosts (e.g., coral, algae, hydrozoa) were detected, in the microbiome, the transcriptome, and the metabolome. The 16S rDNA analysis demonstrated higher abundance of Firmicutes in the coral microbiome than in its competitors. A single bacterial amplicon sequence variant from the genus Clostridium was found exclusively in all O. faveolata samples. In contrast to microbial taxa, a portion of the functionally annotated bacterial RNA transcripts (6.86%) and metabolites (1.95%) were ubiquitous in all coral and competitor samples. Machine learning analysis of microbial transcripts revealed elevated T7-like cyanophage-encoded photosystem II transcripts in O. faveolata samples, while sequences involved in bacterial cell division were elevated in turf algal and interface samples. Similar analysis of metabolites revealed that bacterial-produced antimicrobial and antifungal compounds were highly enriched in coral samples. This study provides insight into the spatial and biological patterning of the coral microbiome, transcriptome, and metabolome