A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodeling hijacked phage coat proteins into small capsids.

Phage satellites commonly remodel capsids they hijack from the phages they parasitize, but only a few mechanisms regulating the change in capsid size have been reported. Here, we investigated how a satellite from Vibrio cholerae, phage-inducible chromosomal island-like element (PLE), remodels the capsid it has been predicted to steal from the phage ICP1 (Netter et al., 2021). We identified that a PLE-encoded protein, TcaP, is both necessary and sufficient to form small capsids during ICP1 infection. Interestingly, we found that PLE is dependent on small capsids for efficient transduction of its genome, making it the first satellite to have this requirement. ICP1 isolates that escaped TcaP-mediated remodeling acquired substitutions in the coat protein, suggesting an interaction between these two proteins. With a procapsid-like particle (PLP) assembly platform in Escherichia coli, we demonstrated that TcaP is a bona fide scaffold that regulates the assembly of small capsids. Further, we studied the structure of PLE PLPs using cryogenic electron microscopy and found that TcaP is an external scaffold that is functionally and somewhat structurally similar to the external scaffold, Sid, encoded by the unrelated satellite P4 (Kizziah et al., 2020). Finally, we showed that TcaP is largely conserved across PLEs. Together, these data support a model in which TcaP directs the assembly of small capsids comprised of ICP1 coat proteins, which inhibits the complete packaging of the ICP1 genome and permits more efficient packaging of replicated PLE genomes.

Cryo-EM structure of a Shigella podophage reveals a hybrid tail and novel decoration proteins.

There is a paucity of high-resolution structures of phages infecting Shigella, a human pathogen and a serious threat to global health. HRP29 is a Shigella podophage belonging to the Autographivirinae family, and has very low sequence identity to other known phages. Here, we resolved the structure of the entire HRP29 virion by cryo-EM. Phage HRP29 has a highly unusual tail that is a fusion of a T7-like tail tube and P22-like tailspikes mediated by interactions from a novel tailspike adaptor protein. Understanding phage tail structures is critical as they mediate hosts interactions. Furthermore, we show that the HRP29 capsid is stabilized by two novel, and essential decoration proteins, gp47 and gp48. Only one high resolution structure is currently available for Shigella podophages. The presence of a hybrid tail and an adapter protein suggests that it may be a product of horizontal gene transfer, and may be prevalent in other phages.

The phage fought the cells, and the phage won: a satellite symposium at the ASV 2023 annual meeting.

This satellite symposium was focused on the molecular arms race between bacteria and their predators, the bacteriophages: who's the friend and who's the foe? This Gem recounts highlights of the talks and presents food for thought and additional reflections on the current state of the field.

A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodeling hijacked phage coat proteins into small capsids.

Phage satellites commonly remodel capsids they hijack from the phages they parasitize, but only a few mechanisms regulating the change in capsid size have been reported. Here, we investigated how a satellite from Vibrio cholerae, PLE, remodels the capsid it has been predicted to steal from the phage ICP1 (1). We identified that a PLE-encoded protein, TcaP, is both necessary and sufficient to form small capsids during ICP1 infection. Interestingly, we found that PLE is dependent on small capsids for efficient transduction of its genome, making it the first satellite to have this requirement. ICP1 isolates that escaped TcaP-mediated remodeling acquired substitutions in the coat protein, suggesting an interaction between these two proteins. With a procapsid-like-particle (PLP) assembly platform in Escherichia coli, we demonstrated that TcaP is a bona fide scaffold that regulates the assembly of small capsids. Further, we studied the structure of PLE PLPs using cryogenic electron microscopy and found that TcaP is an external scaffold, that is functionally and somewhat structurally similar to the external scaffold, Sid, encoded by the unrelated satellite P4 (2). Finally, we showed that TcaP is largely conserved across PLEs. Together, these data support a model in which TcaP directs the assembly of small capsids comprised of ICP1 coat proteins, which inhibits the complete packaging of the ICP1 genome and permits more efficient packaging of replicated PLE genomes.

Phage defence by deaminase-mediated depletion of deoxynucleotides in bacteria.

Vibrio cholerae biotype El Tor is perpetuating the longest cholera pandemic in recorded history. The genomic islands VSP-1 and VSP-2 distinguish El Tor from previous pandemic V. cholerae strains. Using a co-occurrence analysis of VSP genes in >200,000 bacterial genomes we built gene networks to infer biological functions encoded in these islands. This revealed that dncV, a component of the cyclic-oligonucleotide-based anti-phage signalling system (CBASS) anti-phage defence system, co-occurs with an uncharacterized gene vc0175 that we rename avcD for anti-viral cytodine deaminase. We show that AvcD is a deoxycytidylate deaminase and that its activity is post-translationally inhibited by a non-coding RNA named AvcI. AvcID and bacterial homologues protect bacterial populations against phage invasion by depleting free deoxycytidine nucleotides during infection, thereby decreasing phage replication. Homologues of avcD exist in all three domains of life, and bacterial AvcID defends against phage infection by combining traits of two eukaryotic innate viral immunity proteins, APOBEC and SAMHD1.

Host Range Expansion of Shigella Phage Sf6 Evolves through Point Mutations in the Tailspike.

The first critical step in a virus's infection cycle is attachment to its host. This interaction is precise enough to ensure the virus will be able to productively infect the cell, but some flexibility can be beneficial to enable coevolution and host range switching or expansion. Bacteriophage Sf6 utilizes a two-step process to recognize and attach to its host Shigella flexneri. Sf6 first recognizes the lipopolysaccharide (LPS) of S. flexneri and then binds outer membrane protein (Omp) A or OmpC. This phage infects serotype Y strains but can also form small, turbid plaques on serotype 2a2; turbid plaques appear translucent rather than transparent, indicating greater survival of bacteria. Reduced plating efficiency further suggested inefficient infection. To examine the interactions between Sf6 and this alternate host, phages were experimentally evolved using mixed populations of S. flexneri serotypes Y and 2a2. The recovered mutants could infect serotype 2a2 with greater efficiency than the ancestral Sf6, forming clear plaques on both serotypes. All mutations mapped to two distinct regions of the receptor-binding tailspike protein: (i) adjacent to the LPS binding site near the N terminus; and (ii) at the distal, C-terminal tip of the protein. Although we anticipated interactions between the Sf6 tailspike and 2a2 O-antigen to be weak, LPS of this serotype appears to inhibit infection through strong binding of particles, effectively removing them from the environment. The mutations of the evolved strains reduce the inhibitory effect by either reducing electrostatic interactions with the O-antigen or increasing reliance on the Omp secondary receptors. IMPORTANCE Viruses depend on host cells to propagate themselves. In mixed populations and communities of host cells, finding these susceptible host cells may have to be balanced with avoiding nonhost cells. Alternatively, being able to infect new cell types can increase the fitness of the virus. Many bacterial viruses use a two-step process to identify their hosts, binding first to an LPS receptor and then to a host protein. For Shigella virus Sf6, the tailspike protein was previously known to bind the LPS receptor. Genetic data from this work imply the tailspike also binds to the protein receptor. By experimentally evolving Sf6, we also show that point mutations in this protein can dramatically affect the binding of one or both receptors. This may provide Sf6 flexibility in identifying host cells and the ability to rapidly alter its host range under selective pressure.

Shigella viruses Sf22 and KRT47 require outer membrane protein C for infection.

Viruses rely on hosts for their replication: thus, a critical step in the infection process is identifying a suitable host cell. Bacterial viruses, known as bacteriophages or phages, often use receptor binding proteins to discriminate between susceptible and non-susceptible hosts. By being able to evade predation, bacteria with modified or deleted receptor-encoding genes often undergo positive selection during growth in the presence of phage. Depending on the specific receptor(s) a phage uses, this may subsequently affect the bacteria's ability to form biofilms, its resistance to antibiotics, pathogenicity, or its phenotype in various environments. In this study, we characterize the interactions between two T4-like phages, Sf22 and KRT47, and their host receptor S. flexneri outer membrane protein C (OmpC). Results indicate that these phages use a variety of surface features on the protein, and that complete resistance most frequently occurs when hosts delete the ompC gene in full, encode premature stop codons to prevent OmpC synthesis, or eliminate specific regions encoding exterior loops.

Structure and physiology of giant DNA viruses.

Although giant viruses have existed for millennia and possibly exerted great evolutionary influence in their environment. Their presence has only been noticed by virologists recently with the discovery of Acanthamoeba polyphaga mimivirus in 2003. Its virion with a diameter of 500 nm and its genome larger than 1 Mpb shattered preconceived standards of what a virus is and triggered world-wide prospection studies. Thanks to these investigations many giant virus families were discovered, each with its own morphological peculiarities and genomes ranging from 0.4 to 2.5 Mpb that possibly encode more than 400 viral proteins. This review aims to present the morphological diversity, the different aspects observed in host-virus interactions during replication, as well as the techniques utilized during their investigation.

Modeling the Effects of Calcium Overload on Mitochondrial Ultrastructural Remodeling.

Mitochondrial cristae are dynamic invaginations of the inner membrane and play a key role in its metabolic capacity to produce ATP. Structural alterations caused by either genetic abnormalities or detrimental environmental factors impede mitochondrial metabolic fluxes and lead to a decrease in their ability to meet metabolic energy requirements. While some of the key proteins associated with mitochondrial cristae are known, very little is known about how the inner membrane dynamics are involved in energy metabolism. In this study, we present a computational strategy to understand how cristae are formed using a phase-based separation approach of both the inner membrane space and matrix space, which are explicitly modeled using the Cahn-Hilliard equation. We show that cristae are formed as a consequence of minimizing an energy function associated with phase interactions which are subject to geometric boundary constraints. We then extended the model to explore how the presence of calcium phosphate granules, entities that form in calcium overload conditions, exert a devastating inner membrane remodeling response that reduces the capacity for mitochondria to produce ATP. This modeling approach can be extended to include arbitrary geometrical constraints, the spatial heterogeneity of enzymes, and electrostatic effects to mechanize the impact of ultrastructural changes on energy metabolism.

Large metabolic rewiring from small genomic changes between strains of Shigella flexneri.

The instability of Shigella genomes has been described, but how this instability causes phenotypic differences within the Shigella flexneri species is largely unknown and likely variable. We describe herein the genome of S. flexneri strain PE577, originally a clinical isolate, which exhibits several phenotypic differences compared to the model strain 2457T. Like many previously described strains of S. flexneri, PE577 lacks discernible, functional CRISPR and restriction-modification systems. Its phenotypic differences when compared to 2457T include lower transformation efficiency, higher oxygen sensitivity, altered carbon metabolism, and greater susceptibility to a wide variety of lytic bacteriophage isolates. Since relatively few Shigella phages have been isolated on 2457T or the previously characterized strain M90T, developing a more universal model strain for isolating and studying Shigella phages is critical to understanding both phages and phage-host interactions. In addition to phage biology, the genome sequence of PE577 was used to generate and test hypotheses of how pseudogenes in this strain-whether interrupted by degraded prophages, transposases, frameshifts, or point mutations-have led to metabolic rewiring compared to the model strain 2457T. Results indicate that PE577 can utilise the less-efficient pyruvate oxidase/acetyl-CoA synthetase (PoxB/Acs) pathway to produce acetyl-CoA, while strain 2457T cannot due to a nonsense mutation in acs, rendering it a pseudogene in this strain. Both strains also utilize pyruvate-formate lyase to oxidize formate but cannot survive with this pathway alone, possibly because a component of the formate-hydrogen lyase (fdhF) is a pseudogene in both strains.Importance Shigella causes millions of dysentery cases worldwide, primarily affecting children under five years old. Despite active research in developing vaccines and new antibiotics, relatively little is known about the variation of physiology or metabolism across multiple isolates. In this work, we investigate two strains of S. flexneri that share 98.9% genetic identity but exhibit drastic differences in metabolism, ultimately affecting the growth of the two strains. Results suggest additional strains within the S. flexneri species utilize different metabolic pathways to process pyruvate. Metabolic differences between these closely-related isolates suggest an even wider variety of differences in growth across S. flexneri and Shigella in general. Exploring this variation further may assist the development or application of vaccines and therapeutics to combat Shigella infections.

The mitochondrial permeability transition phenomenon elucidated by cryo-EM reveals the genuine impact of calcium overload on mitochondrial structure and function.

Mitochondria have a remarkable ability to uptake and store massive amounts of calcium. However, the consequences of massive calcium accumulation remain enigmatic. In the present study, we analyzed a series of time-course experiments to identify the sequence of events that occur in a population of guinea pig cardiac mitochondria exposed to excessive calcium overload that cause mitochondrial permeability transition (MPT). By analyzing coincident structural and functional data, we determined that excessive calcium overload is associated with large calcium phosphate granules and inner membrane fragmentation, which explains the extent of mitochondrial dysfunction. This data also reveals a novel mechanism for cyclosporin A, an inhibitor of MPT, in which it preserves cristae despite the presence of massive calcium phosphate granules in the matrix. Overall, these findings establish a mechanism of calcium-induced mitochondrial dysfunction and the impact of calcium regulation on mitochondrial structure and function.

Students' use of chemistry core ideas to explain the structure and stability of DNA.

Students tend to think of their science courses as isolated and unrelated to each other, making it difficult for them to see connections across disciplines. In addition, many existing science assessments target rote memorization and algorithmic problem-solving skills. Here, we describe the development, implementation, and evaluation of an activity aimed to help students integrate knowledge across introductory chemistry and biology courses. The activity design and evaluation of students' responses were guided by the Framework for K-12 Science Education as the understanding of core ideas and crosscutting concepts and the development of scientific practices are essential for students at all levels. In this activity, students are asked to use their understanding of noncovalent interactions to explain (a) why the boiling point differs for two pure substances (chemistry phenomenon) and (b) why temperature and base pair composition affects the stability of DNA (biological phenomenon). The activity was implemented at two different institutions (N = 441) in both introductory chemistry and biology courses. Students' overall performance suggests that they can provide sophisticated responses that incorporate their understanding of noncovalent interactions and energy to explain the chemistry phenomenon, but have difficulties integrating the same knowledge to explain the biological phenomenon. Our findings reinforce the notion that students should be provided with opportunities in the classroom to purposefully practice and support the use and integration of knowledge from multiple disciplines. Students' evaluations of the activity indicated that they found it to be interesting and helpful for making connections across disciplines.

Promotion of CTL epitope presentation by a nanoparticle with environment-responsive stability and phagolysosomal escape capacity.

Vaccines that induce cytotoxic T lymphocyte (CTL)-mediated immune responses constitute an important class of medical tools to fend off diseases like infections and malignancy. Epitope peptides, as a format of CTL vaccines, are being tested preclinically and clinically. To elicit CTL responses, epitope vaccines go through an epitope presentation pathway in dendritic cells (DCs) that has multiple bottleneck steps and hence is inefficient. Here, we report the development of a strategy to overcome one of these barriers, phagolysosomal escape in DCs. First, we furnished a previously established carrier-an immune-tolerant elastin-like polypeptide nanoparticle (iTEP NP)-with the peptides that are derived from the DNA polymerase of herpes simplex virus 1 (Pol peptides). Pol peptides were reported to facilitate phagolysosomal escape. In this study, while we found that Pol peptides promoted the CTL epitope presentation; we also discovered Pol peptides disrupted the formation of the iTEP NP. Thus, we engineered a series of new iTEPs and identified several iTEPs that could accommodate Pol peptides and maintain their NP structure at the same time. We next optimized one of these NPs so that its stability is responsive to its redox environment. This environment-responsive NP further strengthened the CTL epitope presentation and CTL responses. Lastly, we revealed how this NP and Pol peptides utilized biological cues of phagolysosomes to realize phagolysosomal escape and epitope release. In summary, we developed iTEP NP carriers with a new phagolysosomal escape function. These carriers, with their priorly incorporated functions, resolve three bottleneck issues in the CTL epitope presentation pathway: vaccine uptake, phagolysosomal escape, and epitope release.

Ecology, Structure, and Evolution of Shigella Phages.

Numerous bacteriophages-viruses of bacteria, also known as phages-have been described for hundreds of bacterial species. The Gram-negative Shigella species are close relatives of Escherichia coli, yet relatively few previously described phages appear to exclusively infect this genus. Recent efforts to isolate Shigella phages have indicated these viruses are surprisingly abundant in the environment and have distinct genomic and structural properties. In addition, at least one model system used for experimental evolution studies has revealed a unique mechanism for developing faster infection cycles. Differences between these bacteriophages and other well-described model systems may mirror differences between their hosts' ecology and defense mechanisms. In this review, we discuss the history of Shigella phages and recent developments in their isolation and characterization and the structural information available for three model systems, Sf6, Sf14, and HRP29; we also provide an overview of potential selective pressures guiding both Shigella phage and host evolution.

Structural and Proteomic Characterization of the Initiation of Giant Virus Infection.

Since their discovery, giant viruses have expanded our understanding of the principles of virology. Due to their gargantuan size and complexity, little is known about the life cycles of these viruses. To answer outstanding questions regarding giant virus infection mechanisms, we set out to determine biomolecular conditions that promote giant virus genome release. We generated four infection intermediates in Samba virus (Mimivirus genus, lineage A) as visualized by cryoelectron microscopy (cryo-EM), cryoelectron tomography (cryo-ET), and scanning electron microscopy (SEM). Each of these four intermediates reflects similar morphology to a stage that occurs in vivo. We show that these genome release stages are conserved in other mimiviruses. Finally, we identified proteins that are released from Samba and newly discovered Tupanvirus through differential mass spectrometry. Our work revealed the molecular forces that trigger infection are conserved among disparate giant viruses. This study is also the first to identify specific proteins released during the initial stages of giant virus infection.

A cornucopia of Shigella phages from the Cornhusker State.

Bacteriophages are abundant in the environment, yet the vast majority have not been discovered or described. Many characterized bacteriophages infect a small subset of Enterobacteriaceae hosts. Despite its similarity to Escherichia coli, the pathogenic Shigella flexneri has relatively few known phages, which exhibit significant differences from many E. coli phages. This suggests that isolating additional Shigella phages is necessary to further explore these differences. To address questions of novelty and prevalence, high school students isolated bacteriophages on non-pathogenic strains of enteric bacteria. Results indicate that Shigella phages are abundant in the environment and continue to differ significantly from E. coli phages. Our findings suggest that Shigella-infecting members of the Ounavirinae subfamily continue to be over-represented and show surprisingly low diversity within and between sampling sites. Additionally, a podophage with distinct genomic and structural properties suggests that continued isolation on non-model species of bacteria is necessary to truly understand bacteriophage diversity.

The phage L capsid decoration protein has a novel OB-fold and an unusual capsid binding strategy.

The major coat proteins of dsDNA tailed phages (order Caudovirales) and herpesviruses form capsids by a mechanism that includes active packaging of the dsDNA genome into a precursor procapsid, followed by expansion and stabilization of the capsid. These viruses have evolved diverse strategies to fortify their capsids, such as non-covalent binding of auxiliary 'decoration' (Dec) proteins. The Dec protein from the P22-like phage L has a highly unusual binding strategy that distinguishes between nearly identical three-fold and quasi-three-fold sites of the icosahedral capsid. Cryo-electron microscopy and three-dimensional image reconstruction were employed to determine the structure of native phage L particles. NMR was used to determine the structure/dynamics of Dec in solution. The NMR structure and the cryo-EM density envelope were combined to build a model of the capsid-bound Dec trimer. Key regions that modulate the binding interface were verified by site-directed mutagenesis.

Shigella Phages Isolated during a Dysentery Outbreak Reveal Uncommon Structures and Broad Species Diversity.

In 2016, Michigan experienced the largest outbreak of shigellosis, a type of bacillary dysentery caused by Shigella spp., since 1988. Following this outbreak, we isolated 16 novel Shigella-infecting bacteriophages (viruses that infect bacteria) from environmental water sources. Most well-known bacteriophages infect the common laboratory species Escherichia coli and Salmonella enterica, and these phages have built the foundation of molecular and bacteriophage biology. Until now, comparatively few bacteriophages were known to infect Shigella spp., which are close relatives of E. coli We present a comprehensive analysis of these phages' host ranges, genomes, and structures, revealing genome sizes and capsid properties that are shared by very few previously described phages. After sequencing, a majority of the Shigella phages were found to have genomes of an uncommon size, shared by only 2% of all reported phage genomes. To investigate the structural implications of this unusual genome size, we used cryo-electron microscopy to resolve their capsid structures. We determined that these bacteriophage capsids have similarly uncommon geometry. Only two other viruses with this capsid structure have been described. Since most well-known bacteriophages infect Escherichia or Salmonella, our understanding of bacteriophages has been limited to a subset of well-described systems. Continuing to isolate phages using nontraditional strains of bacteria can fill gaps that currently exist in bacteriophage biology. In addition, the prevalence of Shigella phages during a shigellosis outbreak may suggest a potential impact of human health epidemics on local microbial communities.IMPORTANCEShigella spp. bacteria are causative agents of dysentery and affect more than 164 million people worldwide every year. Despite the need to combat antibiotic-resistant Shigella strains, relatively few Shigella-infecting bacteriophages have been described. By specifically looking for Shigella-infecting phages, this work has identified new isolates that (i) may be useful to combat Shigella infections and (ii) fill gaps in our knowledge of bacteriophage biology. The rare qualities of these new isolates emphasize the importance of isolating phages on "nontraditional" laboratory strains of bacteria to more fully understand both the basic biology and diversity of bacteriophages.

Breaking Symmetry in Viral Icosahedral Capsids as Seen through the Lenses of X-ray Crystallography and Cryo-Electron Microscopy.

The majority of viruses on Earth form capsids built by multiple copies of one or more types of a coat protein arranged with 532 symmetry, generating an icosahedral shell. This highly repetitive structure is ideal to closely pack identical protein subunits and to enclose the nucleic acid genomes. However, the icosahedral capsid is not merely a passive cage but undergoes dynamic events to promote packaging, maturation and the transfer of the viral genome into the host. These essential processes are often mediated by proteinaceous complexes that interrupt the shell's icosahedral symmetry, providing a gateway through the capsid. In this review, we take an inventory of molecular structures observed either internally, or at the 5-fold vertices of icosahedral DNA viruses that infect bacteria, archea and eukaryotes. Taking advantage of the recent revolution in cryo-electron microscopy (cryo-EM) and building upon a wealth of crystallographic structures of individual components, we review the design principles of non-icosahedral structural components that interrupt icosahedral symmetry and discuss how these macromolecules play vital roles in genome packaging, ejection and host receptor-binding.

Genes affecting progression of bacteriophage P22 infection in Salmonella identified by transposon and single gene deletion screens.

Bacteriophages rely on their hosts for replication, and many host genes critically determine either viral progeny production or host success via phage resistance. A random insertion transposon library of 240,000 mutants in Salmonella enterica serovar Typhimurium was used to monitor effects of individual bacterial gene disruptions on bacteriophage P22 lytic infection. These experiments revealed candidate host genes that alter the timing of phage P22 propagation. Using a False Discovery Rate of < 0.1, mutations in 235 host genes either blocked or delayed progression of P22 lytic infection, including many genes for which this role was previously unknown. Mutations in 77 genes reduced the survival time of host DNA after infection, including mutations in genes for enterobacterial common antigen (ECA) synthesis and osmoregulated periplasmic glucan (OPG). We also screened over 2000 Salmonella single gene deletion mutants to identify genes that impacted either plaque formation or culture growth rates. The gene encoding the periplasmic membrane protein YajC was newly found to be essential for P22 infection. Targeted mutagenesis of yajC shows that an essentially full-length protein is required for function, and potassium efflux measurements demonstrated that YajC is critical for phage DNA ejection across the cytoplasmic membrane.