An intron endonuclease facilitates interference competition between coinfecting viruses.

Introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. In this work, we studied intron-encoded homing endonuclease gp210 in bacteriophage ΦPA3 and found that it contributes to viral competition by interfering with the replication of a coinfecting phage, ΦKZ. We show that gp210 targets a specific sequence in ΦKZ, which prevents the assembly of progeny viruses. This work demonstrates how a homing endonuclease can be deployed in interference competition among viruses and provide a relative fitness advantage. Given the ubiquity of homing endonucleases, this selective advantage likely has widespread evolutionary implications in diverse plasmid and viral competition as well as virus-host interactions.

More evolvable bacteriophages better suppress their host.

The number of multidrug-resistant strains of bacteria is increasing rapidly, while the number of new antibiotic discoveries has stagnated. This trend has caused a surge in interest in bacteriophages as anti-bacterial therapeutics, in part because there is near limitless diversity of phages to harness. While this diversity provides an opportunity, it also creates the dilemma of having to decide which criteria to use to select phages. Here we test whether a phage's ability to coevolve with its host (evolvability) should be considered and how this property compares to two previously proposed criteria: fast reproduction and thermostability. To do this, we compared the suppressiveness of three phages that vary by a single amino acid yet differ in these traits such that each strain maximized two of three characteristics. Our studies revealed that both evolvability and reproductive rate are independently important. The phage most able to suppress bacterial populations was the strain with high evolvability and reproductive rate, yet this phage was unstable. Phages varied due to differences in the types of resistance evolved against them and their ability to counteract resistance. When conditions were shifted to exaggerate the importance of thermostability, one of the stable phages was most suppressive in the short-term, but not over the long-term. Our results demonstrate the utility of biological therapeutics' capacities to evolve and adjust in action to resolve complications like resistance evolution. Furthermore, evolvability is a property that can be engineered into phage therapeutics to enhance their effectiveness.

Asesino: a nucleus-forming phage that lacks PhuZ.

As nucleus-forming phages become better characterized, understanding their unifying similarities and unique differences will help us understand how they occupy varied niches and infect diverse hosts. All identified nucleus-forming phages fall within the proposed Chimalliviridae family and share a core genome of 68 unique genes including chimallin, the major nuclear shell protein. A well-studied but non-essential protein encoded by many nucleus-forming phages is PhuZ, a tubulin homolog which aids in capsid migration, nucleus rotation, and nucleus positioning. One clade that represents 24% of all currently known chimalliviruses lacks a PhuZ homolog. Here we show that Erwinia phage Asesino, one member of this PhuZ-less clade, shares a common overall replication mechanism with other characterized nucleus-forming phages despite lacking PhuZ. We show that Asesino replicates via a phage nucleus that encloses phage DNA and partitions proteins in the nuclear compartment and cytoplasm in a manner similar to previously characterized nucleus-forming phages. Consistent with a lack of PhuZ, however, we did not observe active positioning or rotation of the phage nucleus within infected cells. These data show that some nucleus-forming phages have evolved to replicate efficiently without PhuZ, providing an example of a unique variation in the nucleus-based replication pathway.

Viral Receptor-Binding Protein Evolves New Function through Mutations That Cause Trimer Instability and Functional Heterogeneity.

When proteins evolve new activity, a concomitant decrease in stability is often observed because the mutations that confer new activity can destabilize the native fold. In the conventional model of protein evolution, reduced stability is considered a purely deleterious cost of molecular innovation because unstable proteins are prone to aggregation and are sensitive to environmental stressors. However, recent work has revealed that nonnative, often unstable protein conformations play an important role in mediating evolutionary transitions, raising the question of whether instability can itself potentiate the evolution of new activity. We explored this question in a bacteriophage receptor-binding protein during host-range evolution. We studied the properties of the receptor-binding protein of bacteriophage λ before and after host-range evolution and demonstrated that the evolved protein is relatively unstable and may exist in multiple conformations with unique receptor preferences. Through a combination of structural modeling and in vitro oligomeric state analysis, we found that the instability arises from mutations that interfere with trimer formation. This study raises the intriguing possibility that protein instability might play a previously unrecognized role in mediating host-range expansions in viruses.

Design, optimization, and inference of multiphasic decay of infectious virus particles.

The loss of virus particles is typically considered to arise from a first-order kinetic process. Signals of deviations from this exponential decay are often de-prioritized. Here, we propose methods to evaluate if a design is adequate to evaluate evidence for multiphasic virus particle decay and to optimize the sampling times of decay experiments, accounting for uncertainties in viral kinetics. First, we evaluate 1500 synthetic scenarios of biphasic decays, with varying decay rates and initial proportions of subpopulations. Robust inference of multiphasic decay is more likely when the faster decaying subpopulation predominates insofar as early samples are taken to resolve the faster decay rate. Overall, we find that design optimization leads to a better precision of estimation while reducing the number of samples. It helps to estimate adequately the fastest decay in 54% of situations vs. 41% using a non-optimized design. We then apply these methods to infer multiple decay rates associated with the decay of ΦD9, an evolved isolate derived from phage Φ21. A pilot experiment confirmed that ΦD9 decay is multiphasic, but was unable to resolve the rate or proportion of the fast decay subpopulation(s). We then applied optimal design methods to propose new ΦD9 sampling times. Using this strategy, we were able to robustly estimate both decay rates and their respective subpopulations. Notably, we conclude that the vast majority (94%) of the population decays at a rate 16-fold higher than a slow decaying population. Altogether, these results provide methods to quantitatively estimate heterogeneity in viral decay.

Bacterial resistance response and resource availability mediate viral coexistence.

Viruses that infect bacteria, known as bacteriophages or phages, are the most prevalent entities on Earth. Their genetic diversity in nature is well documented, and members of divergent lineages can be found sharing the same ecological niche. This viral diversity can be influenced by a number of factors, including productivity, spatial structuring of the environment, and host-range trade-offs. Rapid evolution is also known to promote diversity by buffering ecological systems from extinction. There is, however, little known about the impact of coevolution on the maintenance of viral diversity within a microbial community. To address this, we developed a 4 species experimental system where two bacterial hosts, a generalist and a specialist phage, coevolved in a spatially homogenous environment over time. We observed the persistence of both viruses if the resource availability was sufficiently high. This coexistence occurred in the absence of any detectable host-range trade-offs that are costly for generalists and thus known to promote viral diversity. However, the coexistence was lost if two bacteria were not permitted to evolve alongside the phages or if two phages coevolved with a single bacterial host. Our findings indicate that a host's resistance response in mixed-species communities plays a significant role in maintaining viral diversity in the environment.

Competition-driven eco-evolutionary feedback reshapes bacteriophage lambda's fitness landscape and enables speciation.

A major challenge in evolutionary biology is explaining how populations navigate rugged fitness landscapes without getting trapped on local optima. One idea illustrated by adaptive dynamics theory is that as populations adapt, their newly enhanced capacities to exploit resources alter fitness payoffs and restructure the landscape in ways that promote speciation by opening new adaptive pathways. While there have been indirect tests of this theory, to our knowledge none have measured how fitness landscapes deform during adaptation, or test whether these shifts promote diversification. Here, we achieve this by studying bacteriophage [Formula: see text], a virus that readily speciates into co-existing receptor specialists under controlled laboratory conditions. We use a high-throughput gene editing-phenotyping technology to measure [Formula: see text]'s fitness landscape in the presence of different evolved-[Formula: see text] competitors and find that the fitness effects of individual mutations, and their epistatic interactions, depend on the competitor. Using these empirical data, we simulate [Formula: see text]'s evolution on an unchanging landscape and one that recapitulates how the landscape deforms during evolution. [Formula: see text] heterogeneity only evolves in the shifting landscape regime. This study provides a test of adaptive dynamics, and, more broadly, shows how fitness landscapes dynamically change during adaptation, potentiating phenomena like speciation by opening new adaptive pathways.

Inferring strain-level mutational drivers of phage-bacteria interaction phenotypes.

The enormous diversity of bacteriophages and their bacterial hosts presents a significant challenge to predict which phages infect a focal set of bacteria. Infection is largely determined by complementary -and largely uncharacterized- genetics of adsorption, injection, and cell take-over. Here we present a machine learning (ML) approach to predict phage-bacteria interactions trained on genome sequences of and phenotypic interactions amongst 51 Escherichia coli strains and 45 phage λ strains that coevolved in laboratory conditions for 37 days. Leveraging multiple inference strategies and without a priori knowledge of driver mutations, this framework predicts both who infects whom and the quantitative levels of infections across a suite of 2,295 potential interactions. The most effective ML approach inferred interaction phenotypes from independent contributions from phage and bacteria mutations, predicting phage host range with 86% mean classification accuracy while reducing the relative error in the estimated strength of the infection phenotype by 40%. Further, transparent feature selection in the predictive model revealed 18 of 176 phage λ and 6 of 18 E. coli mutations that have a significant influence on the outcome of phage-bacteria interactions, corroborating sites previously known to affect phage λ infections, as well as identifying mutations in genes of unknown function not previously shown to influence bacterial resistance. While the genetic variation studied was limited to a focal, coevolved phage-bacteria system, the method's success at recapitulating strain-level infection outcomes provides a path forward towards developing strategies for inferring interactions in non-model systems, including those of therapeutic significance.

Upper Bound on the Mutational Burden Imposed by a CRISPR-Cas9 Gene-Drive Element.

CRISPR-Cas9 gene drives (CCGDs) are powerful tools for genetic control of wild populations, useful for eradication of disease vectors, conservation of endangered species and other applications. However, Cas9 alone and in a complex with gRNA can cause double-stranded DNA breaks at off-target sites, which could increase the mutational load and lead to loss of heterozygosity (LOH). These undesired effects raise potential concerns about the long-term evolutionary safety of CCGDs, but the magnitude of these effects is unknown. To estimate how the presence of a CCGD or a Cas9 alone in the genome affects the rates of LOH events and de novo mutations, we carried out a mutation accumulation experiment in yeast Saccharomyces cerevisiae. Despite its substantial statistical power, our experiment revealed no detectable effect of CCGD or Cas9 alone on the genome-wide rates of mutations or LOH events, suggesting that these rates are affected by less than 30%. Nevertheless, we found that Cas9 caused a slight but significant shift towards more interstitial and fewer terminal LOH events, and the CCGD caused a significant difference in the distribution of LOH events on Chromosome V. Taken together, our results show that these genetic elements impose a weak and likely localized additional mutational burden in the yeast model. Although the mutagenic effects of CCGDs need to be further evaluated in other systems, our results suggest that the effect of CCGDs on off-target mutation rates and genetic diversity may be acceptable.

Rapid bacteria-phage coevolution drives the emergence of multiscale networks.

Interactions between species catalyze the evolution of multiscale ecological networks, including both nested and modular elements that regulate the function of diverse communities. One common assumption is that such complex pattern formation requires spatial isolation or long evolutionary timescales. We show that multiscale network structure can evolve rapidly under simple ecological conditions without spatial structure. In just 21 days of laboratory coevolution, Escherichia coli and bacteriophage Φ21 coevolve and diversify to form elaborate cross-infection networks. By measuring ~10,000 phage-bacteria infections and testing the genetic basis of interactions, we identify the mechanisms that create each component of the multiscale pattern. Our results demonstrate how multiscale networks evolve in parasite-host systems, illustrating Darwin's idea that simple adaptive processes can generate entangled banks of ecological interactions.

A mobile intron facilitates interference competition between co-infecting viruses.

Mobile introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. Here we studied a mobile intron in bacteriophage ΦPA3 and found its homing endonuclease gp210 contributes to viral competition by interfering with the virogenesis of co-infecting phage ΦKZ. We show that gp210 targets a specific sequence in its competitor ΦKZ, preventing the assembly of progeny viruses. This work reports the first demonstration of how a mobile intron can be deployed to engage in interference competition and provide a reproductive advantage. Given the ubiquity of introns, this selective advantage likely has widespread evolutionary implications in nature.

Competition-driven eco-evolutionary feedback reshapes bacteriophage lambda's fitness landscape and enables speciation.

A major challenge in evolutionary biology is explaining how populations navigate rugged fitness landscapes without getting trapped on local optima. One idea illustrated by adaptive dynamics theory is that as populations adapt, their newly enhanced capacities to exploit resources alter fitness payoffs and restructure the landscape in ways that promote speciation by opening new adaptive pathways. While there have been indirect tests of this theory, none have measured how fitness landscapes deform during adaptation, or test whether these shifts promote diversification. Here, we achieve this by studying bacteriophage λ, a virus that readily speciates into co-existing receptor specialists under controlled laboratory conditions. We used a high-throughput gene editing-phenotyping technology to measure λ's fitness landscape in the presence of different evolved-λ competitors and found that the fitness effects of individual mutations, and their epistatic interactions, depend on the competitor. Using these empirical data, we simulated λ's evolution on an unchanging landscape and one that recapitulates how the landscape deforms during evolution. λ heterogeneity only evolved in the shifting landscape regime. This study provides a test of adaptive dynamics, and, more broadly, shows how fitness landscapes dynamically change during adaptation, potentiating phenomena like speciation by opening new adaptive pathways.

Quality Improvement to Reduce High-Flow Nasal Cannula Overuse in Children With Bronchiolitis.

BACKGROUND: High-flow nasal cannula oxygen therapy (HFNC) is increasingly used to treat bronchiolitis. However, HFNC has not reduced time on supplemental oxygen, length of stay (LOS), or ICU admission. Our objective was to reduce HFNC use in children admitted for bronchiolitis from 41% to 20% over 2 years. METHODS: Using quality improvement methods, our multidisciplinary team formulated key drivers, including standardization of HFNC use, effective communication, knowledgeable staff, engaged providers and families, data transparency, and high-value care focus. Interventions included: (1) standardized HFNC initiation criteria, (2) staff education, (3) real-time feedback to providers, (4) a script for providers to use with families about expectations during admission, (5) team huddle for patients admitted on HFNC to discuss necessity, and (6) distribution of a bronchiolitis toolkit. We used statistical process control charts to track the percentage of children with bronchiolitis who received HFNC. Data were compared with a comparison institution not actively involved in quality improvement work around HFNC use to ensure improvements were not secondary to the COVID-19 pandemic alone. RESULTS: Over 10 months of interventions, we saw a decrease in HFNC use for patients admitted with bronchiolitis from 41% to 22%, which was sustained for >12 months. There was no change in HFNC use at the comparison institution. The overall mean LOS for children with bronchiolitis decreased from 60 to 45 hours. CONCLUSIONS: We successfully reduced HFNC use in children with bronchiolitis, improving delivery of high-value and evidence-based care. This reduction was associated with a 25% decrease in LOS.

Fecal virome transplantation is sufficient to alter fecal microbiota and drive lean and obese body phenotypes in mice.

The gastrointestinal microbiome plays a significant role in modulating numerous host processes, including metabolism. Prior studies show that when mice receive fecal transplants from obese donors on high-fat diets (HFD) (even when recipient mice are fed normal diets after transplantation), they develop obese phenotypes, demonstrating the prominent role that gut microbiota play in determining lean and obese phenotypes. While much of the credit has been given to gut bacteria, the impact of gut viruses on these phenotypes is understudied. To address this shortcoming, we gavaged mice with viromes isolated from donors fed HFD or normal chow over a 4-week study. By characterizing the gut bacterial biota via 16S rRNA amplicon sequencing and measuring mouse weights over time, we demonstrate that transplanted viruses affect the gut bacterial community, as well as weight gain/loss. Notably, mice fed chow but gavaged with HFD-derived viromes gained more weight than their counterparts receiving chow-derived viromes. The converse was also true: mice fed HFD but gavaged with chow-derived viromes gained less weight than their counterparts receiving HFD-derived viromes. Results were replicated in two independent experiments and phenotypic changes were accompanied by significant and identifiable differences in the fecal bacterial biota. Due to methodological limitations, we were unable to identify the specific bacterial strains responsible for respective phenotypic changes. This study confirms that virome-mediated perturbations can alter the fecal microbiome in vivo and indicates that such perturbations are sufficient to drive lean and obese phenotypes in mice.

Canonical Host-Pathogen Trade-Offs Subverted by Mutations with Dual Benefits.

AbstractHost-parasite coevolution is expected to drive the evolution of genetic diversity because the traits used in arms races-namely, host range and parasite resistance-are hypothesized to trade off with traits used in resource competition. We therefore tested data for several trade-offs among 93 isolates of bacteriophage λ and 51 Escherichia coli genotypes that coevolved during a laboratory experiment. Surprisingly, we found multiple trade-ups (positive trait correlations) but little evidence of several canonical trade-offs. For example, some bacterial genotypes evaded a trade-off between phage resistance and absolute fitness, instead evolving simultaneous improvements in both traits. This was surprising because our experimental design was predicted to expose resistance-fitness trade-offs by culturing E. coli in a medium where the phage receptor, LamB, is also used for nutrient acquisition. On reflection, LamB mediates not one but many trade-offs, allowing for more complex trait interactions than just pairwise trade-offs. Here, we report that mathematical reasoning and laboratory data highlight how trade-ups should exist whenever an evolutionary system exhibits multiple interacting trade-offs. Does this mean that coevolution should not promote genetic diversity? No, quite the contrary. We deduce that whenever positive trait correlations are observed in multidimensional traits, other traits may trade off and so provide the right circumstances for diversity maintenance. Overall, this study reveals that there are predictive limits when data account only for pairwise trait correlations, and it argues that a wider range of circumstances than previously anticipated can promote genetic and species diversity.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were still to be determined. Here, we show that phages encoding the major phage nucleus protein chimallin share 72 conserved genes encoded within seven gene blocks. Of these, 21 core genes are unique to nucleus-forming phage, and all but one of these genes encode proteins of unknown function. We propose that these phages comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryoelectron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication are conserved among diverse chimalliviruses and reveal variations on this replication mechanism. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were unknown. By studying phages that encode the major phage nucleus protein chimallin, including previously sequenced yet uncharacterized phages, we discovered that chimallin-encoding phages share a set of 72 highly conserved genes encoded within seven distinct gene blocks. Of these, 21 core genes are unique to this group, and all but one of these unique genes encode proteins of unknown function. We propose that phages with this core genome comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryo-electron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication encoded in the core genome are conserved among diverse chimalliviruses, and reveal that non-core components can confer intriguing variations on this replication mechanism. For instance, unlike previously studied nucleus-forming phages, RAY doesn't degrade the host genome, and its PhuZ homolog appears to form a five-stranded filament with a lumen. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Fecal virome transplantation is sufficient to alter fecal microbiota and drive lean and obese body phenotypes in mice.

Background: The gastrointestinal microbiome plays a significant role in numerous host processes and has an especially large impact on modulating the host metabolism. Prior studies have shown that when mice receive fecal transplants from obese donors that were fed high-fat diets (HFD) (even when recipient mice are fed normal diets after transplantation), they develop obese phenotypes. These studies demonstrate the prominent role that the gut microbiota play in determining lean and obese phenotypes. While much of the credit has been given to gut bacteria, studies have not measured the impact of gut viruses on these phenotypes. To address this shortcoming, we gavaged mice with viromes isolated from donors fed HFD or normal chow. By characterizing the mice’s gut bacterial biota and weight-gain phenotypes over time, we demonstrate that viruses can shape the gut bacterial community and affect weight gain or loss. Results: We gavaged mice longitudinally over 4 weeks while measuring their body weights and collecting fecal samples for 16S rRNA amplicon sequencing. We evaluated mice that were fed normal chow or high-fat diets, and gavaged each group with either chow-derived fecal viromes, HFD-derived fecal viromes, or phosphate buffered saline controls. We found a significant effect of gavage type, where mice fed chow but gavaged with HFD-derived viromes gained significantly more weight than their counterparts receiving chow-derived viromes. The converse was also true: mice fed HFD but gavaged with chow-derived viromes gained significantly less weight than their counterparts receiving HFD-derived viromes. These results were replicated in two separate experiments and the phenotypic changes were accompanied by significant and identifiable differences in the fecal bacterial biota. Notably, there were differences in Lachnospirales and Clostridia in mice fed chow but gavaged with HFD-derived fecal viromes, and in Peptostreptococcales, Oscillospirales, and Lachnospirales in mice fed HFD but gavaged with chow-derived fecal viromes. Due to methodological limitations, we were unable to identify specific bacterial species or strains that were responsible for respective phenotypic changes. Conclusions: This study confirms that virome-mediated perturbations can alter the fecal microbiome in an in vivo model and indicates that such perturbations are sufficient to drive lean and obese phenotypes in mice.