More Like this

1. Identification of Cellular Genes Involved in Baculovirus GP64 Trafficking to the Plasma Membrane

   https://doi.org/10.1128/jvi.00215-22  

   Hodgson, Jeffrey J. ; Buchon, Nicolas ; Blissard, Gary W. ( June 2022 , Journal of Virology)

   Sandri-Goldin, Rozanne M. (Ed.)

   ABSTRACT The baculovirus envelope protein GP64 is an essential component of the budded virus and is necessary for efficient virion assembly. Little is known regarding intracellular trafficking of GP64 to the plasma membrane, where it is incorporated into budding virions during egress. To identify host proteins and potential cellular trafficking pathways that are involved in delivery of GP64 to the plasma membrane, we developed and characterized a stable Drosophila cell line that inducibly expresses the AcMNPV GP64 protein and used that cell line in combination with a targeted RNA interference (RNAi) screen of vesicular protein trafficking pathway genes. Of the 37 initial hits from the screen, we validated and examined six host genes that were important for trafficking of GP64 to the cell surface. Validated hits included Rab GTPases Rab1 and Rab4 , Clathrin heavy chain , clathrin adaptor protein genes AP-1-2β and AP-2μ , and Snap29 . Two gene knockdowns ( Rab5 and Exo84 ) caused substantial increases (up to 2.5-fold) of GP64 on the plasma membrane. We found that a small amount of GP64 is released from cells in exosomes and that some portion of cell surface GP64 is endocytosed, suggesting that recycling helps to maintain GP64 atmore »the cell surface. IMPORTANCE While much is known regarding trafficking of viral envelope proteins in mammalian cells, little is known about this process in insect cells. To begin to understand which factors and pathways are needed for trafficking of insect virus envelope proteins, we engineered a Drosophila melanogaster cell line and implemented an RNAi screen to identify cellular proteins that aid transport of the model baculovirus envelope protein (GP64) to the cell surface. For this we developed an experimental system that leverages the large array of tools available for Drosophila and performed a targeted RNAi screen to identify cellular proteins involved in GP64 trafficking to the cell surface. Since viral envelope proteins are often critical for production of infectious progeny virions, these studies lay the foundation for understanding how either pathogenic insect viruses (baculoviruses) or insect-vectored viruses (e.g., flaviviruses, alphaviruses) egress from cells in tissues such as the midgut to enable systemic virus infection.« less
2. High-Throughput Screen for Inhibitors of the Type IV Pilus Assembly ATPase PilB

   https://doi.org/10.1128/mSphere.00129-21  

   Dye, Keane J. ; Vogelaar, Nancy J. ; Sobrado, Pablo ; Yang, Zhaomin ( April 2021 , mSphere)

   Dunman, Paul (Ed.)

   ABSTRACT The bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant. Antivirulence chemotherapeutics are considered a promising alternative to antibiotics because they target the disease process instead of bacterial viability. However, a roadblock to the discovery of anti-T4P compounds is the lack of a high-throughput screen (HTS) that can be implemented relatively easily and economically. Here, we describe the first HTS for the identification of inhibitors specifically against the T4P assembly ATPase PilB in vitro . Chloracidobacterium thermophilum PilB ( Ct PilB) had been demonstrated to have robust ATPase activity and the ability to bind its expected ligands in vitro. We utilized Ct PilB and MANT-ATP, a fluorescent ATP analog, to develop a binding assay and adapted it for an HTS. As a proof of principle, we performed a pilot screen with a small compound library of kinase inhibitors and identified quercetin as a PilB inhibitor in vitro . Using Myxococcus xanthus as a model bacterium, we found quercetin to reduce its T4P-dependent motility and T4P assembly in vivo. These results validated our HTS as effective in identifying PilB inhibitors. This assay may prove valuable inmore »seeking leads for the development of antivirulence chemotherapeutics against PilB, an essential and universal component of all bacterial T4P systems. IMPORTANCE Many bacterial pathogens use their type IV pili (T4P) to facilitate and maintain infection of a human host. Small chemical compounds that inhibit the production or assembly of T4P hold promise in the treatment and prevention of infections, especially in the era of increasing threats from antibiotic-resistant bacteria. However, few chemicals are known to have inhibitory or anti-T4P activity. Their identification has not been easy due to the lack of a method for the screening of compound collections or libraries on a large scale. Here, we report the development of an assay that can be scaled up to screen compound libraries for inhibitors of a critical T4P assembly protein. We further demonstrate that it is feasible to use whole cells to examine potential inhibitors for their activity against T4P assembly in a bacterium.« less
3. Integration of postmortem amygdala expression profiling, GWAS, and functional cell culture assays: neuroticism-associated synaptic vesicle glycoprotein 2A (SV2A) gene is regulated by miR-133a and miR-218

   https://doi.org/10.1038/s41398-020-00966-4  

   Jurkiewicz, Magdalena ; Moser, Dirk ; Koller, Antonius ; Yu, Lei ; Chen, Emily I. ; Bennett, David A. ; Canli, Turhan ( August 2020 , Translational Psychiatry)

   Recent genome-wide studies have begun to identify gene variants, expression profiles, and regulators associated with neuroticism, anxiety disorders, and depression. We conducted a set of experimental cell culture studies of gene regulation by micro RNAs (miRNAs), based on genome-wide transcriptome, proteome, and miRNA expression data from twentypostmortemsamples of lateral amygdala from donors with known neuroticism scores. Using Ingenuity Pathway Analysis and TargetScan, we identified a list of mRNA–protein–miRNA sets whose expression patterns were consistent with miRNA-based translational repression, as a function of trait anxiety. Here, we focused on one gene from that list, which is of particular translational significance in Psychiatry: synaptic vesicle glycoprotein 2A (SV2A) is the binding site of the anticonvulsant drug levetiracetam ((S)-α-Ethyl-2-oxo-1-pyrrolidineacetamide), which has shown promise in anxiety disorder treatments. We confirmed thatSV2Ais associated with neuroticism or anxiety using an original GWAS of a community cohort (N = 1,706), and cross-referencing a published GWAS of multiple cohorts (Ns ranging from 340,569 to 390,278).Postmortemamygdala expression profiling implicated three putative regulatory miRNAs to targetSV2A: miR-133a, miR-138, and miR-218. Moving from association to experimental causal testing in cell culture, we used a luciferase assay to demonstrate that miR-133a and miR-218, but not miR-138, significantly decreased relative luciferase activity from theSV2Adual-luciferasemore »construct. In human neuroblastoma cells, transfection with miR-133a and miR-218 reduced both endogenousSV2AmRNA and protein levels, confirming miRNA targeting of theSV2Agene. This study illustrates the utility of combiningpostmortemgene expression data with GWAS to guide experimental cell culture assays examining gene regulatory mechanisms that may contribute to complex human traits. Identifying specific molecular mechanisms of gene regulation may be useful for future clinical applications in anxiety disorders or other forms of psychopathology.

   « less
4. Quinine copolymer reporters promote efficient intracellular DNA delivery and illuminate a protein-induced unpackaging mechanism

   https://doi.org/10.1073/PNAS.2016860117  

   Van Bruggen, Craig ; Punihaole, David ; Keith, Allison R. ; Schmitz, Andrew J. ; Tolar, Jakub ; Frontiera, Renee R. ; Reineke, Theresa M. ( December 2020 , Proceedings of the National Academy of Sciences)

   Polymeric vehicles that efficiently package and controllably release nucleic acids enable the development of safer and more efficacious strategies in genetic and polynucleotide therapies. Developing delivery platforms that endogenously monitor the molecular interactions, which facilitate binding and release of nucleic acids in cells, would aid in the rational design of more effective vectors for clinical applications. Here, we report the facile synthesis of a copolymer containing quinine and 2-hydroxyethyl acrylate that effectively compacts plasmid DNA (pDNA) through electrostatic binding and intercalation. This polymer system poly(quinine- co -HEA) packages pDNA and shows exceptional cellular internalization, transgene expression, and low cytotoxicity compared to commercial controls for several human cell lines, including HeLa, HEK 293T, K562, and keratinocytes (N/TERTs). Using quinine as an endogenous reporter for pDNA intercalation, Raman imaging revealed that proteins inside cells facilitate the unpackaging of polymer–DNA complexes (polyplexes) and the release of their cargo. Our work showcases the ability of this quinine copolymer reporter to not only facilitate effective gene delivery but also enable diagnostic monitoring of polymer–pDNA binding interactions on the molecular scale via Raman imaging. The use of Raman chemical imaging in the field of gene delivery yields unprecedented insight into the unpackaging behavior of polyplexes inmore »cells and provides a methodology to assess and design more efficient delivery vehicles for gene-based therapies.« less
5. Genome mining of biosynthetic and chemotherapeutic gene clusters in Streptomyces bacteria

   https://doi.org/10.1038/s41598-020-58904-9  

   Belknap, Kaitlyn C. ; Park, Cooper J. ; Barth, Brian M. ; Andam, Cheryl P. ( February 2020 , Scientific Reports)

   Streptomycesbacteria are known for their prolific production of secondary metabolites, many of which have been widely used in human medicine, agriculture and animal health. To guide the effective prioritization of specific biosynthetic gene clusters (BGCs) for drug development and targeting the most prolific producer strains, knowledge about phylogenetic relationships ofStreptomycesspecies, genome-wide diversity and distribution patterns of BGCs is critical. We used genomic and phylogenetic methods to elucidate the diversity of major classes of BGCs in 1,110 publicly availableStreptomycesgenomes. Genome mining ofStreptomycesreveals high diversity of BGCs and variable distribution patterns in theStreptomycesphylogeny, even among very closely related strains. The most common BGCs are non-ribosomal peptide synthetases, type 1 polyketide synthases, terpenes, and lantipeptides. We also found that numerousStreptomycesspecies harbor BGCs known to encode antitumor compounds. We observed that strains that are considered the same species can vary tremendously in the BGCs they carry, suggesting that strain-level genome sequencing can uncover high levels of BGC diversity and potentially useful derivatives of any one compound. These findings suggest that a strain-level strategy for exploring secondary metabolites for clinical use provides an alternative or complementary approach to discovering novel pharmaceutical compounds from microbes.