pdm_utils: a SEA-PHAGES MySQL phage database management toolkit.

SUMMARY: Bacteriophages (phages) are incredibly abundant and genetically diverse. The volume of phage genomics data is rapidly increasing, driven in part by the SEA-PHAGES program, which isolates, sequences and manually annotates hundreds of phage genomes each year. With an ever-expanding genomics dataset, there are many opportunities for generating new biological insights through comparative genomic and bioinformatic analyses. As a result, there is a growing need to be able to store, update, explore and analyze phage genomics data. The package pdm_utils provides a collection of tools for MySQL phage database management designed to meet specific needs in the SEA-PHAGES program and phage genomics generally. AVAILABILITY AND IMPLEMENTATION: https://pypi.org/project/pdm-utils/.

Mapping the immunogenic landscape of near-native HIV-1 envelope trimers in non-human primates.

The induction of broad and potent immunity by vaccines is the key focus of research efforts aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins have shown promise as vaccine candidates as they can induce potent autologous neutralizing responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and have characteristics akin to several human-derived broadly neutralizing antibodies.

Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor-binding head domains.

Seasonal influenza virus infections can cause significant morbidity and mortality, but the threat from the emergence of a new pandemic influenza strain might have potentially even more devastating consequences. As such, there is intense interest in isolating and characterizing potent neutralizing antibodies that target the hemagglutinin (HA) viral surface glycoprotein. Here, we use cryo-electron microscopy (cryoEM) to decipher the mechanism of action of a potent HA head-directed monoclonal antibody (mAb) bound to an influenza H7 HA. The epitope of the antibody is not solvent accessible in the compact, prefusion conformation that typifies all HA structures to date. Instead, the antibody binds between HA head protomers to an epitope that must be partly or transiently exposed in the prefusion conformation. The "breathing" of the HA protomers is implied by the exposure of this epitope, which is consistent with metastability of class I fusion proteins. This structure likely therefore represents an early structural intermediate in the viral fusion process. Understanding the extent of transient exposure of conserved neutralizing epitopes also may lead to new opportunities to combat influenza that have not been appreciated previously.

Time-course, negative-stain electron microscopy-based analysis for investigating protein-protein interactions at the single-molecule level.

Several biophysical approaches are available to study protein-protein interactions. Most approaches are conducted in bulk solution, and are therefore limited to an average measurement of the ensemble of molecular interactions. Here, we show how single-particle EM can enrich our understanding of protein-protein interactions at the single-molecule level and potentially capture states that are unobservable with ensemble methods because they are below the limit of detection or not conducted on an appropriate time scale. Using the HIV-1 envelope glycoprotein (Env) and its interaction with receptor CD4-binding site neutralizing antibodies as a model system, we both corroborate ensemble kinetics-derived parameters and demonstrate how time-course EM can further dissect stoichiometric states of complexes that are not readily observable with other methods. Visualization of the kinetics and stoichiometry of Env-antibody complexes demonstrated the applicability of our approach to qualitatively and semi-quantitatively differentiate two highly similar neutralizing antibodies. Furthermore, implementation of machine-learning techniques for sorting class averages of these complexes into discrete subclasses of particles helped reduce human bias. Our data provide proof of concept that single-particle EM can be used to generate a "visual" kinetic profile that should be amenable to studying many other protein-protein interactions, is relatively simple and complementary to well-established biophysical approaches. Moreover, our method provides critical insights into broadly neutralizing antibody recognition of Env, which may inform vaccine immunogen design and immunotherapeutic development.

Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages.

BACKGROUND: A remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity. RESULTS: We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or 'clusters', based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages. CONCLUSIONS: Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.

Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repertoires of tRNA isotypes.

UNLABELLED: Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc(2)155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode. IMPORTANCE: The bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis, provides a higher resolution of the phage population and how it has evolved. Three new phages constituting a new cluster M further expand the diversity of the mycobacteriophages and introduce novel features. As such, they provide insights into phage genome architecture, virion structure, and gene regulation at the transcriptional and translational levels.

Snapshot of haloarchaeal tailed virus genomes.

The complete genome sequences of archaeal tailed viruses are currently highly underrepresented in sequence databases. Here, we report the genomic sequences of 10 new tailed viruses infecting different haloarchaeal hosts. Among these, only two viral genomes are closely related to each other and to previously described haloviruses HF1 and HF2. The approximately 760 kb of new genomic sequences in total shows no matches to CRISPR/Cas spacer sequences in haloarchaeal host genomes. Despite their high divergence, we were able to identify virion structural and assembly genes as well as genes coding for DNA and RNA metabolic functions. Interestingly, we identified many genes and genomic features that are shared with tailed bacteriophages, consistent with the hypothesis that haloarchaeal and bacterial tailed viruses share common ancestry, and that a viral lineage containing archaeal viruses, bacteriophages and eukaryotic viruses predates the division of the three major domains of non-viral life. However, as in tailed viruses in general and in haloarchaeal tailed viruses in particular, there are still a considerable number of predicted genes of unknown function.

From structure to sequence: Antibody discovery using cryoEM.

One of the rate-limiting steps in analyzing immune responses to vaccines or infections is the isolation and characterization of monoclonal antibodies. Here, we present a hybrid structural and bioinformatic approach to directly assign the heavy and light chains, identify complementarity-determining regions, and discover sequences from cryoEM density maps of serum-derived polyclonal antibodies bound to an antigen. When combined with next-generation sequencing of immune repertoires, we were able to specifically identify clonal family members, synthesize the monoclonal antibodies, and confirm that they interact with the antigen in a manner equivalent to the corresponding polyclonal antibodies. This structure-based approach for identification of monoclonal antibodies from polyclonal sera opens new avenues for analysis of immune responses and iterative vaccine design.

Structural Definition of a Neutralization-Sensitive Epitope on the MERS-CoV S1-NTD.

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into the human population in 2012 and has caused substantial morbidity and mortality. Potently neutralizing antibodies targeting the receptor-binding domain (RBD) on MERS-CoV spike (S) protein have been characterized, but much less is known about antibodies targeting non-RBD epitopes. Here, we report the structural and functional characterization of G2, a neutralizing antibody targeting the MERS-CoV S1 N-terminal domain (S1-NTD). Structures of G2 alone and in complex with the MERS-CoV S1-NTD define a site of vulnerability comprising two loops, each of which contain a residue mutated in G2-escape variants. Cell-surface binding studies and in vitro competition experiments demonstrate that G2 strongly disrupts the attachment of MERS-CoV S to its receptor, dipeptidyl peptidase-4 (DPP4), with the inhibition requiring the native trimeric S conformation. These results advance our understanding of antibody-mediated neutralization of coronaviruses and should facilitate the development of immunotherapeutics and vaccines against MERS-CoV.

Genome Sequences of 12 Cluster AN Arthrobacter Phages.

Twelve siphoviral phages isolated using Arthrobacter sp. strain ATCC 21022 were sequenced. The phages all have relatively small genomes, ranging from 15,319 to 15,556 bp. All 12 phages are closely related to previously described cluster AN Arthrobacter phages.

Complete Genome Sequences of Arthrobacter Phages Beans, Franzy, Jordan, Piccoletto, Shade, and Timinator.

We report here the genome sequences of six newly isolated bacteriophages infecting Arthrobacter sp. ATCC 21022. All six have myoviral morphologies and have double-stranded DNA genomes with circularly permuted ends. The six phages are closely related with average nucleotide identities of 73.4 to 93.0% across genomes lengths of 49,797 to 51,347 bp.

Genome Sequences of Mycobacteriophages Jane and Sneeze, New Members of Cluster G.

Jane and Sneeze are newly isolated phages of Mycobacterium smegmatis mc2155 from Hillsborough, NJ, and Palo Verde, Costa Rica, respectively. Both are cluster G, subcluster G1 mycobacteriophages. Notable nucleotide differences exist between genomes in the right half, including the presence of mycobacteriophage mobile element 1 (MPME1) in Jane.

Genome Sequences of Newly Isolated Mycobacteriophages Forming Cluster S.

We describe the genomes of two mycobacteriophages, MosMoris and Gattaca, newly isolated on Mycobacterium smegmatis The two phages are very similar to each other, differing in 61 single nucleotide polymorphisms and six small insertion/deletions. Both have extensive nucleotide sequence similarity to mycobacteriophage Marvin and together form cluster S.

Genome Sequences of Streptomyces Phages Amela and Verse.

Amela and Verse are two Streptomyces phages isolated by enrichment on Streptomyces venezuelae (ATCC 10712) from two different soil samples. Amela has a genome length of 49,452, with 75 genes. Verse has a genome length of 49,483, with 75 genes. Both belong to the BD3 subcluster of Actinobacteriophage.

Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity.

The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery.

Genome Sequence of Mycobacteriophage Mindy.

Mycobacteriophage Mindy is a newly isolated phage of Mycobacterium smegmatis, recovered from a soil sample in Pittsburgh, Pennsylvania, USA. Mindy has a genome length of 75,796 bp, encodes 147 predicted proteins and two tRNAs, and is closely related to mycobacteriophages in cluster E.

Genome Sequence of a Newly Isolated Mycobacteriophage, ShedlockHolmes.

Mycobacteriophage ShedlockHolmes is a newly isolated phage infecting Mycobacterium smegmatis mc(2)155. It has a 61,081-bp genome containing 99 predicted protein-coding genes and one tRNA gene. ShedlockHolmes is closely related to mycobacteriophages Pixie, Keshu, and MacnCheese and is a new member of subcluster K3.

Genome Sequence of Mycobacteriophage Phayonce.

Mycobacteriophage Phayonce is a newly isolated phage recovered from a soil sample in Pittsburgh, PA, using Mycobacterium smegmatis mc(2)155 as a host. Phayonce's genome is 49,203 bp long and contains 77 protein-coding genes, 23 of them having predicted functions. Phayonce shares a strong similarity in nucleotide sequence with phages of cluster P.

Genome Sequence of Mycobacteriophage Momo.

Momo is a newly discovered phage of Mycobacterium smegmatis mc(2)155. Momo has a double-stranded DNA genome 154,553 bp in length, with 233 predicted protein-encoding genes, 34 tRNA genes, and one transfer-messenger RNA (tmRNA) gene. Momo has a myoviral morphology and shares extensive nucleotide sequence similarity with subcluster C1 mycobacteriophages.

Genome Sequences of Mycobacteriophages Luchador and Nerujay.

Luchador and Nerujay are two newly isolated mycobacteriophages recovered from soil samples using Mycobacterium smegmatis. Their genomes are 53,387 bp and 53,455 bp long and have 96 and 97 predicted open reading frames, respectively. Nerujay is related to subcluster A1 phages, and Luchador represents a new subcluster, A14.