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Introduction: Prolyl-4-hydroxylases ( P4H ) catalyse the irreversible conversion of proline to hydroxyproline, constituting a common posttranslational modification of proteins found in humans, plants, and microbes. Hydroxyproline residues can be further modified in plants to yield glycoproteins containing characteristic O-glycans. It is currently unknown how these plant endogenous modifications impact protein functionality and they cause considerable concerns for the recombinant production of therapeutic proteins in plants. In this study, we carried out host engineering to generate a therapeutic glycoprotein largely devoid of plant-endogenous O-glycans for functional characterization. Methods: Genome editing was used to inactivate two genes coding for enzymes of the P4H10 subfamily in the widely used expression host Nicotiana benthamiana . Using glycoengineering in plants and expression in human HEK293 cells we generated four variants of a potent, SARS-CoV-2 neutralizing antibody, COVA2-15 IgA1. The variants that differed in the number of modified proline residues and O-glycan compositions of their hinge region were assessed regarding their physicochemical properties and functionality. Results: We found that plant endogenous O-glycan formation was strongly reduced on IgA1 when transiently expressed in the P4H10 double mutant N. benthamiana plant line. The IgA1 glycoforms displayed differences in proteolytic stability and minor differences in receptor binding thus highlighting the importance of O-glycosylation in the hinge region of human IgA1. Discussion: This work reports the successful protein O-glycan engineering of an important plant host for recombinant protein expression. While the complete removal of endogenous hydroxyproline residues from the hinge region of plant-produced IgA1 is yet to be achieved, our engineered line is suitable for structure-function studies of O-glycosylated recombinant glycoproteins produced in plants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Uetz, Göritzer, Vergara, Melnik, Grünwald-Gruber, Figl, Deghmane, Groppelli, Reljic, Ma, Stöger and Strasser.)

All aspects of biodiversity research, from taxonomy to conservation, rely on data associated with species names. Effective integration of names across multiple fields is paramount and depends on the coordination and organization of taxonomic data. We assess current efforts and find that even key applications for well-studied taxa still lack commonality in taxonomic information required for integration. We identify essential taxonomic elements from our interoperability assessment to support improved access and integration of taxonomic data. A stronger focus on these elements has the potential to involve taxonomic communities in biodiversity science and overcome broken linkages currently limiting research capacity. We encourage a community effort to democratize taxonomic expertise and language in order to facilitate maximum interoperability and integration., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Species are usually described by morphological terms. In order to simplify and shorten descriptions these are often abbreviated (e.g., SVL for snout-vent-length). However, there has been no systematic attempt to define and standardize such terms or their abbreviations. Here we present an initial list of 594 unique abbreviations from a total list of 1,223 abbreviations collected from >50 reptile species descriptions, resulting in a non-redundant list of 344 abbreviations. Most of these abbreviations describe either meristic characters such as scale counts (46%) or measurements such as SVL (snout-vent-length) (30%). The remainder describe presence/absence states, colors, or formulas such as ratios. We highlight the common problem of synonyms and homonyms, i.e., different terms and abbreviations for the same character or the same term for different characters. We propose to standardize definitions of terms and abbreviations in future species descriptions. In order to future-proof species descriptions for machine-readability such as text-mining, standardization is needed for all species descriptions in biology, not just reptiles.

Over the last 25 years, biology has entered the genomic era and is becoming a science of 'big data'. Most interpretations of genomic analyses rely on accurate functional annotations of the proteins encoded by more than 500 000 genomes sequenced to date. By different estimates, only half the predicted sequenced proteins carry an accurate functional annotation, and this percentage varies drastically between different organismal lineages. Such a large gap in knowledge hampers all aspects of biological enterprise and, thereby, is standing in the way of genomic biology reaching its full potential. A brainstorming meeting to address this issue funded by the National Science Foundation was held during 3-4 February 2022. Bringing together data scientists, biocurators, computational biologists and experimentalists within the same venue allowed for a comprehensive assessment of the current state of functional annotations of protein families. Further, major issues that were obstructing the field were identified and discussed, which ultimately allowed for the proposal of solutions on how to move forward., (© The Author(s) 2022. Published by Oxford University Press.)

The properties of host plants used for molecular farming can be modified by CRISPR/Cas9 genome editing to improve the quality and yield of recombinant proteins. However, it is often necessary to target multiple genes simultaneously, particularly when using host plants with large and complex genomes. This is the case for Nicotiana benthamiana, an allotetraploid relative of tobacco frequently used for transient protein expression. A multiplex genome editing system incorporating the DsRed2 fluorescent marker for the identification and selection of transgenic plants was established. As proof of principle, NbP4H4 was targeted encoding a prolyl-4-hydroxylase involved in protein O-linked glycosylation. Using preselected gRNAs with efficiencies confirmed by transient expression, transgenic plant lines with knockout mutations in all four NbP4H4 genes were obtained. Leaf fluorescence was then used to screen for the absence of the SpCas9 transgene in T1 plants, and transgene-free lines with homozygous or biallelic mutations were identified. The analysis of plant-produced recombinant IgA1 as a reporter protein revealed changes in the number of peptides containing hydroxyproline residues and pentoses in the knockout plants. The selection of efficient gRNAs combined with the DsRed2 marker reduces the effort needed to generate N. benthamiana mutants and simplifies the screening processes to obtain transgene-free progeny., (© 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

The modes of interactions between plants and plant-associated microbiota are manifold, and secondary metabolites often play a central role in plant-microbe interactions. Abiotic and biotic (including both plant pathogens and endophytes) stress can affect the composition and concentration of secondary plant metabolites, and thus have an influence on chemical compounds that make up for the taste and aroma of fruit. While the role of microbiota in growth and health of plants is widely acknowledged, relatively little is known about the possible effect of microorganisms on the quality of fruit of plants they are colonizing. In this work, tomato ( Solanum lycopersicum L.) plants of five different cultivars were grown in soil and in hydroponics to investigate the impact of the cultivation method on the flavor of fruit, and to assess whether variations in their chemical composition are attributable to shifts in bacterial microbiota. Ripe fruit were harvested and used for bacterial community analysis and for the analysis of tomato volatiles, sugars and acids, all contributing to flavor. Fruit grown in soil showed significantly higher sugar content, whereas tomatoes from plants under hydroponic conditions had significantly higher levels of organic acids. In contrast, aroma profiles of fruit were shaped by the tomato cultivars, rather than the cultivation method. In terms of bacterial communities, the cultivation method significantly defined the community composition in all cultivars, with the bacterial communities in hydroponic tomatoes being more variable that those in tomatoes grown in soil. Bacterial indicator species in soil-grown tomatoes correlated with higher concentrations of volatiles described to be perceived as "green" or "pungent." A soil-grown specific reproducibly occurring ASV (amplicon sequence variants) classified as Bacillus detected solely in "Solarino" tomatoes, which were the sweetest among all cultivars, correlated with the amount of aroma-relevant volatiles as well as of fructose and glucose in the fruit. In contrast, indicator bacterial species in hydroponic-derived tomatoes correlated with aroma compounds with "sweet" and "floral" notes and showed negative correlations with glucose concentrations in fruit. Overall, our results point toward a microbiota-related accumulation of flavor and aroma compounds in tomato fruit, which is strongly dependent on the cultivation substrate and approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Escobar Rodríguez, Novak, Buchholz, Uetz, Bragagna, Gumze, Antonielli and Mitter.)

The secretive behavior and life history of snakes makes studying their biology, distribution, and the epidemiology of venomous snakebite challenging. One of the most useful, most versatile, and easiest to collect types of biological data are photographs, particularly those that are connected with geographic location and date-time metadata. Photos verify occurrence records, provide data on phenotypes and ecology, and are often used to illustrate new species descriptions, field guides and identification keys, as well as in training humans and computer vision algorithms to identify snakes. We scoured eleven online and two offline sources of snake photos in an attempt to collect as many photos of as many snake species as possible, and attempt to explain some of the inter-species variation in photograph quantity among global regions and taxonomic groups, and with regard to medical importance, human population density, and range size. We collected a total of 725,565 photos-between 1 and 48,696 photos of 3098 of the world's 3879 snake species (79.9%), leaving 781 "most wanted" species with no photos (20.1% of all currently-described species as of the December 2020 release of The Reptile Database). We provide a list of most wanted species sortable by family, continent, authority, and medical importance, and encourage snake photographers worldwide to submit photos and associated metadata, particularly of "missing" species, to the most permanent and useful online archives: The Reptile Database, iNaturalist, and HerpMapper., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Glycolysis is regulated by numerous mechanisms including allosteric regulation, post-translational modification or protein-protein interactions (PPI). While glycolytic enzymes have been found to interact with hundreds of proteins, the impact of only some of these PPIs on glycolysis is well understood. Here we investigate which of these interactions may affect glycolysis in E. coli and possibly across numerous other bacteria, based on the stoichiometry of interacting protein pairs (from proteomic studies) and their conservation across bacteria. We present a list of 339 protein-protein interactions involving glycolytic enzymes but predict that ~70% of glycolytic interactors are not present in adequate amounts to have a significant impact on glycolysis. Finally, we identify a conserved but uncharacterized subset of interactions that are likely to affect glycolysis and deserve further study.

YhcB, a poorly understood protein conserved across gamma-proteobacteria, contains a domain of unknown function (DUF1043) and an N-terminal transmembrane domain. Here, we used an integrated approach including X-ray crystallography, genetics, and molecular biology to investigate the function and structure of YhcB. The Escherichia coli yhcB KO strain does not grow at 45 °C and is hypersensitive to cell wall-acting antibiotics, even in the stationary phase. The deletion of yhcB leads to filamentation, abnormal FtsZ ring formation, and aberrant septum development. The Z-ring is essential for the positioning of the septa and the initiation of cell division. We found that YhcB interacts with proteins of the divisome (e.g., FtsI, FtsQ) and elongasome (e.g., RodZ, RodA). Seven of these interactions are also conserved in Yersinia pestis and/or Vibrio cholerae. Furthermore, we mapped the amino acid residues likely involved in the interactions of YhcB with FtsI and RodZ. The 2.8 Å crystal structure of the cytosolic domain of Haemophilus ducreyi YhcB shows a unique tetrameric α-helical coiled-coil structure likely to be involved in linking the Z-ring to the septal peptidoglycan-synthesizing complexes. In summary, YhcB is a conserved and conditionally essential protein that plays a role in cell division and consequently affects envelope biogenesis. Based on these findings, we propose to rename YhcB to ZapG (Z-ring-associated protein G). This study will serve as a starting point for future studies on this protein family and on how cells transit from exponential to stationary survival., Competing Interests: Conflict of interest G. T. M. is the founder of Nexomics Biosciences Inc. The other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

No central online repository exists for the collection of animal images; hence it remains unclear how extensively species have been illustrated in the published literature or online. Here we compiled a list of more than 8000 reptile species (out of 11,341) that have photos in one of six popular online repositories, namely iNaturalist (6,349 species), the Reptile Database (5,144), Flickr (4,386), CalPhotos (3,071), Wikimedia (2,952), and Herpmapper (2,571). These sites have compiled over one million reptile photos, with some species represented by tens of thousands of images. Despite the number of images, many species have only one or a few images. This suggests that a considerable fraction of morphological and geographic variation is under documented or difficult to access. We highlight prominent gaps in amphisbaenians, lizards, and snakes, with geographic hotspots for species without images in Central Africa, Pacific Islands, and the Andes Mountains. We present a list of ~3,000 species without photos in any of the six databases and ask the community to fill the gaps by depositing images on one of these sites (preferably with minimal copyright restrictions).

Viruses infect their human hosts by a series of interactions between viral and host proteins, indicating that detailed knowledge of such virus-host interaction interfaces are critical for our understanding of viral infection mechanisms, disease etiology and the development of new drugs. In this review, we primarily survey human host-virus interaction data that are available from public databases following the standardized PSI-MS format. Notably, available host-virus protein interaction information is strongly biased toward a small number of virus families including herpesviridae, papillomaviridae, orthomyxoviridae and retroviridae. While we explore the reliability and relevance of these protein interactions we also survey the current knowledge about viruses functional and topological targets. Furthermore, we assess emerging frontiers of host-virus protein interaction research, focusing on protein interaction interfaces of hosts that are infected by different viruses and viruses that infect multiple hosts. Finally, we cover the current status of research that investigates the relationships of virus-targeted host proteins to other comorbidities as well as the influence of host-virus protein interactions on human metabolism., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We compared the species names in the Reptile Database, a dedicated taxonomy database, with those in the NCBI taxonomy database, which provides the taxonomic backbone for the GenBank sequence database. About 67% of the known ~11,000 reptile species are represented with at least one DNA sequence and a binary species name in GenBank. However, a common problem arises through the submission of preliminary species names (such as "Pelomedusa sp. A CK-2014") to GenBank and thus the NCBI taxonomy. These names cannot be assigned to any accepted species names and thus create a disconnect between DNA sequences and species. While these names of unknown taxonomic meaning sometimes get updated, often they remain in GenBank which now contains sequences from ~1,300 such "putative" reptile species tagged by informal names (~15% of its reptile names). We estimate that NCBI/GenBank probably contain tens of thousands of such "disconnected" entries. We encourage sequence submitters to update informal species names after they have been published, otherwise the disconnect will cause increasing confusion and possibly misleading taxonomic conclusions.

We present information on primary type specimens for 13,282 species and subspecies of reptiles compiled in the Reptile Database, that is, holotypes, neotypes, lectotypes, and syntypes. These represent 99.4% of all 13,361 currently recognized taxa (11,050 species and 2311 subspecies). Type specimens of 653 taxa (4.9%) are either lost or not located, were never designated, or we did not find any information about them. 51 species are based on iconotypes. To map all types to physical collections we have consolidated all synonymous and ambiguous collection acronyms into an unambiguous list of 364 collections holding these primary types. The 10 largest collections possess more than 50% of all (primary) reptile types, the 36 largest collections possess more than 10,000 types and the largest 73 collections possess over 90% of all types. Of the 364 collections, 107 hold type specimens of only 1 species or subspecies. Dozens of types are still in private collections. In order to increase their utility, we recommend that the description of type specimens be supplemented with data from high-resolution images and CT-scans, and clear links to tissue samples and DNA sequence data (when available). We request members of the herpetological community provide us with any missing type information to complete the list.

By August 2017 an estimated 13,047 species and subspecies of extant reptiles have been described by a total of 6,454 papers and books which are listed in a supplementary file. For 1,052 species a total of 2,452 subspecies (excluding nominate subspecies) had been described by 2017, down from 1,295 species and 4,411 subspecies in 2009, due to the elevation of many subspecies to species. Here we summarize the history of these taxon description beginning with Linnaeus in 1758. While it took 80 years to reach the first 1,000 species in 1838, new species and subspecies descriptions since then have been added at a roughly constant rate of 1000 new taxa every 12-17 years. The only exception were the decades during World Wars I and II and the beginning of this millennium when the rate of descriptions increased to now about 7 years for the last 1,000 taxa. The top 101 most productive herpetologists (in terms of "taxon output") have described more than 8,000 species and subspecies, amounting to over 60% of all currently valid taxa. More than 90% of all species were described in either English (68.2%), German (12.7%) or French (9.3%).

Two-hybrid methods remain among the most preferred choices for detecting protein-protein interactions (PPIs) and much of the PPI data in databases have been produced using yeast two-hybrid (Y2H) screens. The Y2H methods are extensively used to detect PPIs because of their scalability and accessibility. Several variants of Y2H methods have been developed and used by different research groups, increasing the accessibility of these methods and their applications in detecting different types of PPIs. However, the availability of variations on the same core methodology emphasizes the need to have a systematic comparison of available Y2H methods in the context of their applicability, coverage and efficiency. In this chapter, we discuss the key parameters of Y2H methods, namely proteins of interest, vectors, libraries, screening strategies, data analysis, and provide a flowchart that should help to decide which Y2H strategy is most appropriate for a protein interaction screen.

Mycobacteriophage are viruses that infect mycobacteria. More than 1,400 mycobacteriophage genomes have been sequenced, coding for over one hundred thousand proteins of unknown functions. Here we investigate mycobacteriophage Giles-host protein-protein interactions (PPIs) using yeast two-hybrid screening (Y2H). A total of 25 reproducible PPIs were found for a selected set of 10 Giles proteins, including a putative virion assembly protein (gp17), the phage integrase (gp29), the endolysin (gp31), the phage repressor (gp47), and six proteins of unknown function (gp34, gp35, gp54, gp56, gp64, and gp65). We note that overexpression of the proteins is toxic to M. smegmatis, although whether this toxicity and the associated changes in cellular morphology are related to the putative interactions revealed in the Y2H screen is unclear.

The family Tectiviridae comprises a group of tailless, icosahedral, membrane-containing bacteriophages that can be divided into two groups by their hosts, either Gram-negative or Gram-positive bacteria. While the first group is composed of PRD1 and nearly identical well-characterized lytic viruses, the second one includes more variable temperate phages, like GIL16 or Bam35, whose hosts are Bacillus cereus and related Gram-positive bacteria. In the genome of Bam35, nearly half of the 32 annotated open reading frames (ORFs) have no homologs in databases (ORFans), being putative proteins of unknown function, which hinders the understanding of their biology. With the aim of increasing knowledge about the viral proteome, we carried out a comprehensive yeast two-hybrid analysis of all the putative proteins encoded by the Bam35 genome. The resulting protein interactome comprised 76 unique interactions among 24 proteins, of which 12 have an unknown function. These results suggest that the P17 protein is the minor capsid protein of Bam35 and P24 is the penton protein, with the latter finding also being supported by iterative threading protein modeling. Moreover, the inner membrane transglycosylase protein P26 could have an additional structural role. We also detected interactions involving nonstructural proteins, such as the DNA-binding protein P1 and the genome terminal protein (P4), which was confirmed by coimmunoprecipitation of recombinant proteins. Altogether, our results provide a functional view of the Bam35 viral proteome, with a focus on the composition and organization of the viral particle. IMPORTANCE Tailless viruses of the family Tectiviridae can infect commensal and pathogenic Gram-positive and Gram-negative bacteria. Moreover, they have been proposed to be at the evolutionary origin of several groups of large eukaryotic DNA viruses and self-replicating plasmids. However, due to their ancient origin and complex diversity, many tectiviral proteins are ORFans of unknown function. Comprehensive protein-protein interaction (PPI) analysis of viral proteins can eventually disclose biological mechanisms and thus provide new insights into protein function unattainable by studying proteins one by one. Here we comprehensively describe intraviral PPIs among tectivirus Bam35 proteins determined using multivector yeast two-hybrid screening, and these PPIs were further supported by the results of coimmunoprecipitation assays and protein structural models. This approach allowed us to propose new functions for known proteins and hypothesize about the biological role of the localization of some viral ORFan proteins within the viral particle that will be helpful for understanding the biology of tectiviruses infecting Gram-positive bacteria., (Copyright © 2017 American Society for Microbiology.)

The human genome contains a large number of protein polymorphisms due to individual genome variation. How many of these polymorphisms lead to altered protein-protein interaction is unknown. We have developed a method to address this question. The intersection of the SKEMPI database (of affinity constants among interacting proteins) and CAPRI 4.0 docking benchmark was docked using HADDOCK, leading to a training set of 166 mutant pairs. A random forest classifier based on the differences in resulting docking scores between the 166 mutant pairs and their wild-types was used, to distinguish between variants that have either completely or partially lost binding ability. Fifty percent of non-binders were correctly predicted with a false discovery rate of only 2 percent. The model was tested on a set of 15 HIV-1 - human, as well as seven human- human glioblastoma-related, mutant protein pairs: 50 percent of combined non-binders were correctly predicted with a false discovery rate of 10 percent. The model was also used to identify 10 protein-protein interactions between human proteins and their HIV-1 partners that are likely to be abolished by rare non-synonymous single-nucleotide polymorphisms (nsSNPs). These nsSNPs may represent novel and potentially therapeutically-valuable targets for anti-viral therapy by disruption of viral binding.

Background: Protein-protein interactions (PPIs) can offer compelling evidence for protein function, especially when viewed in the context of proteome-wide interactomes. Bacteria have been popular subjects of interactome studies: more than six different bacterial species have been the subjects of comprehensive interactome studies while several more have had substantial segments of their proteomes screened for interactions. The protein interactomes of several bacterial species have been completed, including several from prominent human pathogens. The availability of interactome data has brought challenges, as these large data sets are difficult to compare across species, limiting their usefulness for broad studies of microbial genetics and evolution., Results: In this study, we use more than 52,000 unique protein-protein interactions (PPIs) across 349 different bacterial species and strains to determine their conservation across data sets and taxonomic groups. When proteins are collapsed into orthologous groups (OGs) the resulting meta-interactome still includes more than 43,000 interactions, about 14,000 of which involve proteins of unknown function. While conserved interactions provide support for protein function in their respective species data, we found only 429 PPIs (~1% of the available data) conserved in two or more species, rendering any cross-species interactome comparison immediately useful. The meta-interactome serves as a model for predicting interactions, protein functions, and even full interactome sizes for species with limited to no experimentally observed PPI, including Bacillus subtilis and Salmonella enterica which are predicted to have up to 18,000 and 31,000 PPIs, respectively., Conclusions: In the course of this work, we have assembled cross-species interactome comparisons that will allow interactomics researchers to anticipate the structures of yet-unexplored microbial interactomes and to focus on well-conserved yet uncharacterized interactors for further study. Such conserved interactions should provide evidence for important but yet-uncharacterized aspects of bacterial physiology and may provide targets for anti-microbial therapies.

Two-hybrid systems are one of the most popular, preferred, cost effective, and scalable in vivo genetic approaches for screening protein-protein interactions. A number of variants of yeast and bacterial two-hybrid systems exist, rendering them ideal for modern, flexible proteomics-driven studies. For mapping protein interactions at genome scales (that is, constructing an interactome), the yeast two-hybrid system has been extensively tested and is preferred over bacterial two-hybrid systems, given that users have created more resources such as a variety of vectors and other modifications. Each system has its own advantages and limitations and thus needs to be compared directly. For instance, the bacterial two-hybrid method seems a better fit than the yeast two-hybrid system to screen membrane-associated proteins. In this chapter, we provide detailed protocols for yeast and bacterial two-hybrid systems as well as a comparison of outcomes for each approach using our own and published data., (© 2017 Elsevier Inc. All rights reserved.)

Although an abundance of bacteriophages exists, little is known about interactions between their proteins and those of their bacterial hosts. Here, we experimentally determined the phage-host interactomes of the phages Dp-1 and Cp-1 and their underlying protein interaction network in the host Streptococcus pneumoniae. We compared our results to the interaction patterns of E. coli phages lambda and T7. Dp-1 and Cp-1 target highly connected host proteins, occupy central network positions, and reach many protein clusters through the interactions of their targets. In turn, lambda and T7 targets cluster to conserved and essential proteins in E. coli, while such patterns were largely absent in S. pneumoniae. Furthermore, targets in E. coli were mutually strongly intertwined, while targets of Dp-1 and Cp-1 were strongly connected through essential and orthologous proteins in their immediate network vicinity. In both phage-host systems, the impact of phages on their protein targets appears to extend from their network neighbors, since proteins that interact with phage targets were located in central network positions, have a strong topologically disruptive effect and touch complexes with high functional heterogeneity. Such observations suggest that the phages, biological impact is accomplished through a surprisingly limited topological reach of their targets.

The investigation of host-pathogen interaction interfaces and their constituent factors is crucial for our understanding of an organism's pathogenesis. Here, we explored the interactomes of HIV, hepatitis C virus, influenza A virus, human papillomavirus, herpes simplex virus, and vaccinia virus in a human host by analyzing the combined sets of virus targets and human genes that are required for viral infection. We also considered targets and required genes of bacteriophages lambda and T7 infection in Escherichia coli . We found that targeted proteins and their immediate network neighbors significantly pool with proteins required for infection and essential for cell growth, forming large connected components in both the human and E. coli protein interaction networks. The impact of both viruses and phages on their protein targets appears to extend to their network neighbors, as these are enriched with topologically central proteins that have a significant disruptive topological effect and connect different protein complexes. Moreover, viral and phage targets and network neighbors are enriched with transcription factors, methylases, and acetylases in human viruses, while such interactions are much less prominent in bacteriophages. IMPORTANCE While host-virus interaction interfaces have been previously investigated, relatively little is known about the indirect interactions of pathogen and host proteins required for viral infection and host cell function. Therefore, we investigated the topological relationships of human and bacterial viruses and how they interact with their hosts. We focused on those host proteins that are directly targeted by viruses, those that are required for infection, and those that are essential for both human and bacterial cells (here, E. coli ). Generally, we observed that targeted, required, and essential proteins in both hosts interact in a highly intertwined fashion. While there exist highly similar topological patterns, we found that human viruses target transcription factors through methylases and acetylases, proteins that played no such role in bacteriophages.

Hepatitis E virus (HEV) is an emerging virus causing epidemic acute hepatitis in developing countries as well as sporadic cases in industrialized countries. The life cycle of HEV is still poorly understood and the lack of efficient cell culture systems and animal models are the principal limitations for a detailed study of the viral replication cycle. Here we exhaustively examine all possible intraviral protein-protein interactions (PPIs) of HEV by systematic Yeast two-hybrid (Y2H) and LuMPIS screens, providing a basis for studying the function of these proteins in the viral replication cycle. Key PPIs correlate with the already published HEV 3D structure. Furthermore, we report 20 novel PPIs including the homodimerization of the RNA dependent RNA polymerase (RdRp), the self-interaction of the papain like protease, and ORF3 interactions with the papain-like protease and putative replicase components: RdRp, methylase and helicase. Furthermore, we determined the dissociation constant (Kd) of ORF3 interactions with the viral helicase, papain-like protease and methylase, which suggest a regulatory function for ORF3 in orchestrating the formation of the replicase complex. These interactions may represent new targets for antiviral drugs.

Helicobacter pylori (H. pylori) is a ε-proteobacterium that colonizes the stomach of about half of the world's population. Persistent infections have been associated with several gastric diseases. Mainly rod- or spiral shaped but also coccoid H. pylori forms have been isolated from mucus layer biopsies of patients. It is still being debated whether the coccoid form can be transformed back into the spiral form or whether this morphology is a result of bacterial cell death or persistence. We established stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomics of H. pylori and applied it to investigate differences between the spiral and the coccoid morphology. We detected 72% and were able to relatively quantify 47% of the H. pylori proteome. Proteins involved in cell division and transcriptional and translational processes showed a lower abundance in coccoid cells. Additionally, proteins related to host colonization, including CagA, the arginase RocF, and the TNF-α inducing protein were down-regulated. The fact that outer membrane proteins were observed at higher abundances might represent a mechanism for immune evasion but also preserves adherence to host cells. The established protocol for relative protein quantification of H. pylori samples offers new possibilities for research on H. pylori., Biological Significance: Our study shows that SILAC can be employed to study protein abundance changes in H. pylori. We have chosen to establish SILAC for H. pylori because it facilitates fractionation on both, protein and peptide level and thus enables deep proteome coverage. Furthermore, SILAC allows robust and highly accurate protein quantification. The manuscript includes a detailed description of the applied method, suggestions for further improvement as well as a practical application. The investigation of differences between the coccoid and infectious spiral morphology of H. pylori with SILAC revealed the regulation of proteins that are involved in host colonization, motility, cell division as well as transcriptional and translational processes. The data will help molecular biologist to focus on relevant pathways that were found to be regulated in response to morphological changes. Furthermore, the application of SILAC offers new possibilities to study the biology of H. pylori. It enables to monitor protein abundance changes in response to certain stimuli such as oxygen stress or antibiotics. Moreover, SILAC raises the possibility to study co-cultures of host cells and H. pylori on protein level. Additionally, pulsed SILAC experiments enable the quantification of protein turnover., (Copyright © 2015. Published by Elsevier B.V.)

Unlabelled: Mycobacteriophages are viruses that infect mycobacterial hosts and are prevalent in the environment. Nearly 700 mycobacteriophage genomes have been completely sequenced, revealing considerable diversity and genetic novelty. Here, we have determined the protein complement of mycobacteriophage Giles by mass spectrometry and mapped its genome-wide protein interactome to help elucidate the roles of its 77 predicted proteins, 50% of which have no known function. About 22,000 individual yeast two-hybrid (Y2H) tests with four different Y2H vectors, followed by filtering and retest screens, resulted in 324 reproducible protein-protein interactions, including 171 (136 nonredundant) high-confidence interactions. The complete set of high-confidence interactions among Giles proteins reveals new mechanistic details and predicts functions for unknown proteins. The Giles interactome is the first for any mycobacteriophage and one of just five known phage interactomes so far. Our results will help in understanding mycobacteriophage biology and aid in development of new genetic and therapeutic tools to understand Mycobacterium tuberculosis., Importance: Mycobacterium tuberculosis causes over 9 million new cases of tuberculosis each year. Mycobacteriophages, viruses of mycobacterial hosts, hold considerable potential to understand phage diversity, evolution, and mycobacterial biology, aiding in the development of therapeutic tools to control mycobacterial infections. The mycobacteriophage Giles protein-protein interaction network allows us to predict functions for unknown proteins and shed light on major biological processes in phage biology. For example, Giles gp76, a protein of unknown function, is found to associate with phage packaging and maturation. The functions of mycobacteriophage-derived proteins may suggest novel therapeutic approaches for tuberculosis. Our ORFeome clone set of Giles proteins and the interactome data will be useful resources for phage interactomics., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Virtually all processes in living cells are dependent on protein-protein interactions (PPIs). Understanding PPI networks is thus essential for molecular biology and disease research. One powerful genetic system for mapping PPIs both at a small scale and in a high-throughput manner is the yeast two-hybrid (Y2H) screen. In Y2H screening, PPIs are detected through the activation of reporter genes responding to a reconstituted transcription factor. In this introduction, we describe library- and array-based Y2H methods and explain their basic theory. We also include the rationale behind different Y2H approaches and strategies for optimizing results., (© 2015 Cold Spring Harbor Laboratory Press.)

Yeast two-hybrid (Y2H) screens are an efficient system for mapping protein-protein interactions and whole interactomes. The screens can be performed using random libraries or collections of defined open reading frames (ORFs) called ORFeomes. This protocol describes both library and array-based Y2H screening, with an emphasis on array-based assays. Array-based Y2H is commonly used to test a number of "prey" proteins for interactions with a single "bait" (target) protein or pool of proteins. The advantage of this approach is the direct identification of interacting protein pairs without further downstream experiments: The identity of the preys is known and does not require further confirmation. In contrast, constructing and screening a random prey library requires identification of individual prey clones and systematic retesting. Retesting is typically performed in an array format., (© 2015 Cold Spring Harbor Laboratory Press.)

Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 "gold standard" protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 "gold standard" protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial "model" species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies.