Design of Peptide Inhibitors That Bind the bZIP Domain of Epstein–Barr Virus Protein BZLF1

Designing proteins or peptides that bind native protein targets can aid the development of novel reagents and/or therapeutics. Rational design also tests our understanding of the principles underlying protein recognition. This article describes several strategies used to design peptides that bind to the basic region leucine zipper (bZIP) domain of the viral transcription factor BZLF1, which is encoded by the Epstein–Barr virus. BZLF1 regulates the transition of the Epstein–Barr virus from a latent state to a lytic state. It shares some properties in common with the more studied human bZIP transcription factors, but also includes novel structural elements that pose interesting challenges to inhibitor design. In designing peptides that bind to BZLF1 by forming a coiled-coil structure, we considered both affinity for BZLF1 and undesired self-association, which can weaken the effectiveness of an inhibitor. Several designed peptides exhibited different degrees of target-binding affinity and self-association. Rationally engineered molecules were more potent inhibitors of DNA binding than a control peptide corresponding to the native BZLF1 dimerization region itself. The most potent inhibitors included both positive and negative design elements and exploited interaction with the coiled-coil and basic DNA-binding regions of BZLF1.David H. Koch Institute for Integrative Cancer Research at MIT (Graduate Fellowship)National Institutes of Health (U.S.) (Award GM067681)National Science Foundation (U.S.) (Award 0821391

Why rankings of biomedical image analysis competitions should be interpreted with care

International challenges have become the standard for validation of biomedical image analysis methods. Given their scientific impact, it is surprising that a critical analysis of common practices related to the organization of challenges has not yet been performed. In this paper, we present a comprehensive analysis of biomedical image analysis challenges conducted up to now. We demonstrate the importance of challenges and show that the lack of quality control has critical consequences. First, reproducibility and interpretation of the results is often hampered as only a fraction of relevant information is typically provided. Second, the rank of an algorithm is generally not robust to a number of variables such as the test data used for validation, the ranking scheme applied and the observers that make the reference annotations. To overcome these problems, we recommend best practice guidelines and define open research questions to be addressed in the future

Conservation of Nematocida microsporidia gene expression and host response in Caenorhabditis nematodes.

Microsporidia are obligate intracellular parasites that are known to infect most types of animals. Many species of microsporidia can infect multiple related hosts, but it is not known if microsporidia express different genes depending upon which host species is infected or if the host response to infection is specific to each microsporidia species. To address these questions, we took advantage of two species of Nematocida microsporidia, N. parisii and N. ausubeli, that infect two species of Caenorhabditis nematodes, C. elegans and C. briggsae. We performed RNA-seq at several time points for each host infected with either microsporidia species. We observed that Nematocida transcription was largely independent of its host. We also observed that the host transcriptional response was similar when infected with either microsporidia species. Finally, we analyzed if the host response to microsporidia infection was conserved across host species. We observed that although many of the genes upregulated in response to infection are not direct orthologs, the same expanded gene families are upregulated in both Caenorhabditis hosts. Together our results describe the transcriptional interactions of Nematocida infection in Caenorhabditis hosts and demonstrate that these responses are evolutionarily conserved

An intestinally secreted host factor promotes microsporidia invasion of C. elegans.

Microsporidia are ubiquitous obligate intracellular pathogens of animals. These parasites often infect hosts through an oral route, but little is known about the function of host intestinal proteins that facilitate microsporidia invasion. To identify such factors necessary for infection by Nematocida parisii, a natural microsporidian pathogen of Caenorhabditis elegans, we performed a forward genetic screen to identify mutant animals that have a Fitness Advantage with Nematocida (Fawn). We isolated four fawn mutants that are resistant to Nematocida infection and contain mutations in T14E8.4, which we renamed aaim-1 (Antibacterial and Aids invasion by Microsporidia). Expression of AAIM-1 in the intestine of aaim-1 animals restores N. parisii infectivity and this rescue of infectivity is dependent upon AAIM-1 secretion. N. parisii spores in aaim-1 animals are improperly oriented in the intestinal lumen, leading to reduced levels of parasite invasion. Conversely, aaim-1 mutants display both increased colonization and susceptibility to the bacterial pathogen Pseudomonas aeruginosa and overexpression ofaaim-1 reduces P. aeruginosa colonization. Competitive fitness assays show that aaim-1 mutants are favored in the presence of N. parisii but disadvantaged on P. aeruginosa compared to wild-type animals. Together, this work demonstrates how microsporidia exploits a secreted protein to promote host invasion. Our results also suggest evolutionary trade-offs may exist to optimizing host defense against multiple classes of pathogens

High-throughput phenotyping of infection by diverse microsporidia species reveals a wild C. elegans strain with opposing resistance and susceptibility traits.

Animals are under constant selective pressure from a myriad of diverse pathogens. Microsporidia are ubiquitous animal parasites, but the influence they exert on shaping animal genomes is mostly unknown. Using multiplexed competition assays, we measured the impact of four different species of microsporidia on 22 wild isolates of Caenorhabditis elegans. This resulted in the identification and confirmation of 13 strains with significantly altered population fitness profiles under infection conditions. One of these identified strains, JU1400, is sensitive to an epidermal-infecting species by lacking tolerance to infection. JU1400 is also resistant to an intestinal-infecting species and can specifically recognize and destroy this pathogen. Genetic mapping of JU1400 demonstrates that these two opposing phenotypes are caused by separate loci. Transcriptional analysis reveals the JU1400 sensitivity to epidermal microsporidia infection results in a response pattern that shares similarity to toxin-induced responses. In contrast, we do not observe JU1400 intestinal resistance being regulated at the transcriptional level. The transcriptional response to these four microsporidia species is conserved, with C. elegans strain-specific differences in potential immune genes. Together, our results show that phenotypic differences to microsporidia infection amongst C. elegans are common and that animals can evolve species-specific genetic interactions

List of large gene family proteins.

Microsporidia are a large phylum of intracellular parasites that can infect most types of animals. Species in the Nematocida genus can infect nematodes including Caenorhabditis elegans, which has become an important model to study mechanisms of microsporidia infection. To understand the genomic properties and evolution of nematode-infecting microsporidia, we sequenced the genomes of nine species of microsporidia, including two genera, Enteropsectra and Pancytospora, without any previously sequenced genomes. Core cellular processes, including metabolic pathways, are mostly conserved across genera of nematode-infecting microsporidia. Each species encodes unique proteins belonging to large gene families that are likely used to interact with host cells. Most strikingly, we observed one such family, NemLGF1, is present in both Nematocida and Pancytospora species, but not any other microsporidia. To understand how Nematocida phenotypic traits evolved, we measured the host range, tissue specificity, spore size, and polar tube length of several species in the genus. Our phylogenetic analysis shows that Nematocida is composed of two groups of species with distinct traits and that species with longer polar tubes infect multiple tissues. Together, our work details both genomic and trait evolution between related microsporidia species and provides a useful resource for further understanding microsporidia evolution and infection mechanisms.</div