Mucosal surfaces are a main entry point for pathogens and the principal sites of defense against infection. Both bacteria and phage are associated with this mucus. Here we show that phageto-bacteria ratios were increased, relative to the adjacent environment, on all mucosal surfaces sampled, ranging from cnidarians to humans. In vitro studies of tissue culture cells with and without surface mucus demonstrated that this increase in phage abundance is mucus dependent and protects the underlying epithelium from bacterial infection. Enrichment of phage in mucus occurs via binding interactions between mucin glycoproteins and Ig-like protein domains exposed on phage capsids. In particular, phage Ig-like domains bind variable glycan residues that coat the mucin glycoprotein component of mucus. Metagenomic analysis found these Ig-like proteins present in the phages sampled from many environments, particularly from locations adjacent to mucosal surfaces. Based on these observations, we present the bacteriophage adherence to mucus model that provides a ubiquitous, but non-host-derived, immunity applicable to mucosal surfaces. The model suggests that metazoan mucosal surfaces and phage coevolve to maintain phage adherence. This benefits the metazoan host by limiting mucosal bacteria, and benefits the phage through more frequent interactions with bacterial hosts. The relationships shown here suggest a symbiotic relationship between phage and metazoan hosts that provides a previously unrecognized antimicrobial defense that actively protects mucosal surfaces.symbiosis | host-pathogen | virus | immunoglobulin | immune system

Microbial viruses can control host abundances via density-dependent lytic predator-prey dynamics. Less clear is how temperate viruses, which coexist and replicate with their host, influence microbial communities. Here we show that virus-like particles are relatively less abundant at high host densities. This suggests suppressed lysis where established models predict lytic dynamics are favoured. Meta-analysis of published viral and microbial densities showed that this trend was widespread in diverse ecosystems ranging from soil to freshwater to human lungs. Experimental manipulations showed viral densities more consistent with temperate than lytic life cycles at increasing microbial abundance. An analysis of 24 coral reef viromes showed a relative increase in the abundance of hallmark genes encoded by temperate viruses with increased microbial abundance. Based on these four lines of evidence, we propose the Piggyback-the-Winner model wherein temperate dynamics become increasingly important in ecosystems with high microbial densities; thus 'more microbes, fewer viruses'.

The species composition and metabolic potential of microbial and viral communities are predictable and stable for most ecosystems. This apparent stability contradicts theoretical models as well as the viral-microbial dynamics observed in simple ecosystems, both of which show Kill-the-Winner behavior causing cycling of the dominant taxa. Microbial and viral metagenomes were obtained from four human-controlled aquatic environments at various time points separated by one day to 41 year. These environments were maintained within narrow geochemical bounds and had characteristic species composition and metabolic potentials at all time points. However, underlying this stability were rapid changes at the fine-grained level of viral genotypes and microbial strains. These results suggest a model wherein functionally redundant microbial and viral taxa are cycling at the level of viral genotypes and virus-sensitive microbial strains. Microbial taxa, viral taxa, and metabolic function persist over time in stable ecosystems and both communities fluctuate in a Kill-the-Winner manner at the level of viral genotypes and microbial strains.

Significance Microbial communities associated with coral reefs influence the health and sustenance of keystone benthic organisms (e.g., coral holobionts). The present study investigated the community structure and metabolic potential of microbes inhabiting coral reefs located across an extensive area in the central Pacific. We found that the taxa present correlated strongly with the percent coverage of corals and algae, while community metabolic potential correlated best with geographic location. These findings are inconsistent with prevailing biogeographic models of microbial diversity (e.g., distance decay) and metabolic potential (i.e., similar functional profiles regardless of phylogenetic variability). Based on these findings, we propose that the primary carbon sources determine community structure and that local biogeochemistry determines finer-scale metabolic function.

Bacteriophages (phages) defend mucosal surfaces against bacterial infections. However, their complex interactions with their bacterial hosts and with the mucus-covered epithelium remain mostly unexplored. Our previous work demonstrated that T4 phage with Hoc proteins exposed on their capsid adhered to mucin glycoproteins and protected mucus-producing tissue culture cells in vitro. On this basis, we proposed our bacteriophage adherence to mucus (BAM) model of immunity. Here, to test this model, we developed a microfluidic device (chip) that emulates a mucosal surface experiencing constant fluid flow and mucin secretion dynamics. Using mucus-producing human cells and Escherichia coli in the chip, we observed similar accumulation and persistence of mucus-adherent T4 phage and nonadherent T4Δhoc phage in the mucus. Nevertheless, T4 phage reduced bacterial colonization of the epithelium >4,000-fold compared with T4Δhoc phage. This suggests that phage adherence to mucus increases encounters with bacterial hosts by some other mechanism. Phages are traditionally thought to be completely dependent on normal diffusion, driven by random Brownian motion, for host contact. We demonstrated that T4 phage particles displayed subdiffusive motion in mucus, whereas T4Δhoc particles displayed normal diffusion. Experiments and modeling indicate that subdiffusive motion increases phage-host encounters when bacterial concentration is low. By concentrating phages in an optimal mucus zone, subdiffusion increases their host encounters and antimicrobial action. Our revised BAM model proposes that the fundamental mechanism of mucosal immunity is subdiffusion resulting from adherence to mucus. These findings suggest intriguing possibilities for engineering phages to manipulate and personalize the mucosal microbiome.BAM | virus | mucus | subdiffusion | search strategy I n all animals, mucosal surfaces provide critical immunological services by both protecting against invading bacterial pathogens and supporting large communities of commensal microorganisms (1, 2). Being exposed to the environment, mucosal surfaces are also the infection sites for many important bacterial diseases, including acute diarrhea and cystic fibrosis in humans. This, combined with their accessibility, make mucosal surfaces attractive venues for phage therapy; that is, the use of bacteriophages (phages) to treat and clear bacterial infections (3,4). Clinical success so far has been erratic (5). The complexities and dynamics of the mucus layer are rarely considered, and the activity of phages therein is mostly unknown. Not surprisingly, phages effective in vitro do not consistently reduce mucosal bacterial host levels in vivo (6, 7). An understanding of the interactions between phages and their bacterial hosts within the relevant physiological environment is critical for consistent success of phage therapy applications.The multilayered mucus is composed primarily of gel-forming mucin glycoproteins that are continually secreted by the underlying epithelium (8). The mucin...

Viruses are the most abundant and the most diverse life form. In this meta-analysis we estimate that there are 4.80×10 phages on Earth. Further, 97% of viruses are in soil and sediment-two underinvestigated biomes that combined account for only ∼2.5% of publicly available viral metagenomes. The majority of the most abundant viral sequences from all biomes are novel. Our analysis drawing on all publicly available viral metagenomes observed a mere 257,698 viral genotypes on Earth-an unrealistically low number-which attests to the current paucity of viral metagenomic data. Further advances in viral ecology and diversity call for a shift of attention to previously ignored major biomes and careful application of verified methods for viral metagenomic analysis.

Background Samples collected from CF patient airways often contain large amounts of host-derived nucleic acids that interfere with recovery and purification of microbial and viral nucleic acids. This study describes metagenomic and metatranscriptomic methods that address these issues. Methods Microbial and viral metagenomes, and microbial metatranscriptomes, were successfully prepared from sputum samples from five adult CF patients. Results Contaminating host DNA was dramatically reduced in the metagenomes. Each CF patient presented a unique microbiome; in some Pseudomonas aeruginosa was replaced by other opportunistic bacteria. Even though the taxonomic composition of the microbiomes are very different, the metabolic potentials encoded by the community are very similar. The viral communities were dominated by phages that infect major CF pathogens. The metatranscriptomes reveal differential expression of encoded metabolic potential with changing health status. Conclusions Microbial and viral metagenomics combined with microbial transcriptomics characterize the dynamic polymicrobial communities found in CF airways, revealing both the taxa present and their current metabolic activities. These approaches can facilitate the development of individualized treatment plans and novel therapeutic approaches.

and 7 Rainbow Rock, Ocean View, Hawaii Current therapy for cystic fibrosis (CF) focuses on minimizing the microbial community and the host's immune response through the aggressive use of airway clearance techniques, broad-spectrum antibiotics, and treatments that break down the pervasive endobronchial biofilm. Antibiotic selection is typically based on the susceptibility of individual microbial strains to specific antibiotics in vitro. Often this approach cannot accurately predict medical outcomes because of factors both technical and biological. Recent cultureindependent assessments of the airway microbial and viral communities demonstrated that the CF airway infection is considerably more complex and dynamic than previously appreciated. Understanding the ecological and evolutionary pressures that shape these communities is critically important for the optimal use of current therapies (in both the choice of therapy and timing of administration) and the development of newer strategies. The climax-attack model (CAM) presented here, grounded in basic ecological principles, postulates the existence of two major functional communities. The attack community consists of transient viral and microbial populations that induce strong innate immune responses. The resultant intense immune response creates microenvironments that facilitate the establishment of a climax community that is slower-growing and inherently resistant to antibiotic therapy. Newer methodologies, including sequence-based metagenomic analysis, can track not only the taxonomic composition but also the metabolic capabilities of these changing viral and microbial communities over time. Collecting this information for CF airways will enable the mathematical modeling of microbial community dynamics during disease progression. The resultant understanding of airway communities and their effects on lung physiology will facilitate the optimization of CF therapies.