BACTERIAL

VIRAL PARASITIC

Viral image: Image from Barrows NJ, Campos RK, Powell ST, Prasanth KR, Schott-Lerner G et al. A screen of FDA-approved drugs for inhibitors of Zikavirus infection. Cell Host Microbe 2016;20(2):259–70

Parasitic image: Image from Automated High-Content Assay for Compounds Selectively Toxic to Trypanosoma cruzi in a Myoblastic Cell Line. Alonso-Padilla, J. et al. PLoS Negl Trop Dis. 2015; Jan, 9(1): e0003493.

Bacterial image: Confocal image of Salmonella bacteria labelled with GFP acquired on Opera Phenix with a 63x water objective NA 1.15. Sample courtesy of D. Goulding, Sanger Institute, UK.

VIRAL DISEASEFeatured Publication Notes

Ebola virus (EBOV) is a serious public health concern, causing severe hemorrhagic disease in humans with a mortality rate of more than 78%.The authors used a kinome analysis to investigate the host kinome response over time in human hepatocyte cells infected with EBOV. This showed that transforming growth factor β (TGF-β)-mediated pathways are modulated during EBOV infection and replication. Phenotypic screening showed that EBOV infection is inhibited in the presence of TGF-β and related pathway kinase inhibitors.

High-content screening and analysis of a panel of 774 FDA-approved drugs identified over 20 molecules that could inhibit Zika virus infection in human cells including cervical, placental, neural stem cells and primary amnion cells – all are potential new therapeutics in the fight against Zika.

An in vivo approach involving UV crosslinking, antisense-mediated affinity purification and mass spectrometry identified host proteins that physically associate with dengue virus RNA. Small interfering RNA-mediated gene silencing, combined with high-content screening demonstrated that over half of these proteins are likely to be involved in regulating dengue virus replication.

Ebola Virus Modulates Transforming Growth Factor β Signaling and Cellular Markers of Mesenchyme-Like Transition in HepatocytesJason Kindrachuk and colleagues

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A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus InfectionNicholas Barrows and colleaguesSEE

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Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus ReplicationStacia Phillips and colleagues

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VIRAL DISEASE

BACTERIAL DISEASEFeatured Publication Notes and Case Study

A Dual Microscopy-Based Assay to Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape J.J. Quereda, J. Pizarro-Cerda and colleagues

Listeriosis-causing L. monocytogenes invades mammalian cells and escapes from the vacuole, enabling proliferation in the host cell cytoplasm. A two-step method involving high-content screening was developed to investigate cell invasion and vacuolar rupture in a single experiment, providing a powerful tool which could be used for identifying the factors involved in these processes in bacteria and other pathogens.

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SEE FILE Infection with the intestinal bacterium Clostridium difficile is the most common cause of healthcare-associated diarrhoea that can develop in

patients after hospitalization and treatment with antibiotics. C. difficile is resistant to a wide range of antibiotics and, for this reason, new treatments for severe cases are desperately needed. C. difficile infection is mediated by the production of toxins by the bacterium, and treatment with toxin-binding agents, such as antibodies is a promising approach to reducing or inhibiting the clinical manifestations. This case study describes the workflow used by researchers at AIMM to generate antibodies against C. difficile ToxB.

B Cell Selection and Therapeutic Antibody Characterization Using the Operetta High-Content Imaging System

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A better understanding of the intracellular stage of M. tuberculosis pathogenesis may aid development of new agents in the battle against tuberculosis infection. High-content screening was used to investigate the role of host genes in intracellular replication, identify small molecule inhibitors within the host cell, and examine the role of bacterial genes in intracellular trafficking.

Testing Chemical & Genetic Modulators in Mycobacterium tuberculosis Infected Cells Using Phenotypic AssaysVincent Delorme and colleagues

BACTERIAL DISEASE

PARASITIC DISEASEFeatured Publication Notes

High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria TransmissionDavid M Plouffe and colleaguesThe Saponin-lysis Sexual Stage Assay (SaLSSA) is a high-throughput, cost-effective assay for identifying small molecules with malaria transmission-blocking activity. A total of 13,983 compounds were analyzed, which included some with consistent low nanomolar transmission-blocking activity. The assay provides a tool for the discovery and development of transmission-blocking drugs.

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PARASITIC DISEASE

Infectious diseases caused by bacterial, viral or parasitic pathogens are a major burden to global health. Devastating infections without effective vaccines or anti-microbial treatment still exist, as illustrated by the 2014 West African Ebola virus outbreak that caused more than 11,000 deaths1 in 10 countries. The increased globalization of modern society, with travel and trade that facilitates the spread of emerging and re-emerging infectious diseases, and phenomena such as anti-microbial resistance, underscore the importance of the development of new preventative and therapeutic approaches.

High-content analysis plays a significant role in infectious disease research as recently reviewed by Ang and Pethe (2016), enabling high-throughput functional and phenotypic assays that can be adapted to a wide range of pathogens. Originally developed as a complementary technology to traditional biochemical high-throughput screening (HTS) in drug discovery, today high-content analysis is established in a far broader area of the life science space as an unbiased imaging method to assess cellular function. Applications include genetic siRNA interference or CRISPR screens for identifying host factors involved in host–pathogen interactions, but also compound screens for drug discovery.

Using High-Content Analysis To Study Infectious Disease

1 Retrieved Dec 12, 2016 from https://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/case-counts.html

More meaningful resultsScreening can be performed within the context of a host cell, allowing drugs that target host cell or microbial factors to be discovered. High-content screening allows the analysis of complex processes such as host cell entry, intracellular trafficking or cell-to-cell spread using fixed samples or live cell imaging.

More information from cellular samplesFully automated imaging and unbiased quantitative image analysis exploits the full potential of microscopy, allowing the characterization of pathogens as well as host cell phenotypes. Infection rates can be determined with high sensitivity, since individual infected cells can be identified. The process of infection can be described with up to hundreds of readouts per cell allowing the linkage of infection rates to other readouts such as host cell morphology or host cell signalling.

More questions answered simultaneouslyData sets are more information-rich compared to non-imaging read-outs, allowing a number of conclusions to be drawn from one experiment, such as drug efficacy against a pathogen as well as drug toxicity on host cells.

More samples analyzedThe automation of imaging tasks allows the analysis of significantly more samples compared to conventional microscopy, enabling genome-wide or kinome-wide siRNA screens or screening of large compound libraries.

Confocal image of Salmonella bacteria labelled with GFP acquired on Opera Phenix with a 63x water objective NA 1.15, sample courtesy of D. Goulding, Sanger Institute, UK.

VIRAL DISEASE BACTERIAL DISEASE PARASITIC DISEASE

Publication Notes and Case Studies

Ebola Virus Modulates Transforming Growth Factor β Signaling and Cellular Markers of Mesenchyme-Like Transition in Hepatocytes Jason Kindrachuk and colleagues

A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus InfectionNicholas Barrows and colleagues

Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus ReplicationStacia Phillips and colleagues

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Testing Chemical & Genetic Modulators in Mycobacterium tuberculosis Infected Cells Using Phenotypic AssaysVincent Delorme and colleagues

A Dual Microscopy-Based Assay to Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape J.J. Quereda, J. Pizarro-Cerda and colleagues

B Cell Selection and Therapeutic Antibody Characterization Using the Operetta High-Content Imaging System

High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria TransmissionDavid M Plouffe and colleagues

· Ang ML and Pethe K. Contribution of high-content imaging technologies to the development of anti-infective drugs. Cytometry A. 2016; Aug; 89(8):755-60. · Barrows NJ. et al. A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection. Cell Host & Microbe. 2016; 20(2):259–70.· Caì Y. et al. Simian hemorrhagic fever virus cell entry is dependent on CD163 and uses a clathrin-mediated endocytosis-like pathway. Journal of Virology. 2015; 89(1):844-856.· Chang SY. et al. A natural component from Euphorbia humifusa Willd displays novel, broad-spectrum anti-influenza activity by blocking nuclear export of viral ribonucleoprotein. Biochemical and biophysical research communications. 2016; 471(2):282-9.

· Chen H. et al. BET-Inhibitors Disrupt Rad21-Dependent Conformational Control of KSHV Latency. PLOS Pathogens. 2017;13(1):e1006100.· Cortjens B. et al. Neutrophil Extracellular Traps Cause Airway Obstruction During Respiratory Syncytial Virus Disease. The Journal of Pathology. 2016; 238(3):401-411.· Ekins S. et al. Machine Learning Models Identify Molecules Active Against the Ebola Virus in vitro. F1000 Research. 2016; 4:1091.· Kim H-Y. et al. Benzothiazepinecarboxamides: Novel Hepatitis C Virus Inhibitors that Interfere with Viral Entry and the Generation of Infectious Virions. Antiviral Research. 2016; 129:39-46.

· Kindrachuk J. et al. Ebola Virus Modulates Transforming Growth Factor β Signaling and Cellular Markers of Mesenchyme-Like Transition in Hepatocytes. Journal of Virology. 2014; 88(17): 9877-9892.

· Ko, M. et al. Screening of FDA-approved drugs using a MERS-CoV clinical isolate from South Korea identifies potential therapeutic options for COVID-19. bioRxiv preprint. 2019· Langsjoen R. et al. Host oxidative folding pathways offer novel anti-chikungunya virus drug targets with broad spectrum potential. Antiviral Research. 2017;143:246-251.· Li M. et al. Quantitative and epitope-specific antigenicity analysis of the human papillomavirus 6 capsid protein in aqueous solution or when adsorbed on particulate adjuvants. Vaccine. 2016;34(37):4422-4428.

· McMullan LK. et al. Characterisation of Infectious Ebola Virus from the Ongoing Outbreak to Guide Response Activities in the Democratic Republic of the Congo: A Phylogenetic and in Vitro Analysis. The Lancet Infectious Diseases. 2019; 19 (9): 1023–32.

· McMullan LK. et al. The Lipid Moiety of Brincidofovir is Required for in vitro Antiviral Activity Against Ebola Virus. Antiviral Research. 2016; 125:71-78.· Park JH. et al. Identification of Novel Membrane-associated Prostaglandin E Synthase-1 (mPGES-1) Inhibitors with Anti-influenza Activities in vitro. Biochemical and Biophysical Research Communications. 2016; 469(4):848-855.

· Phillips SL. et al. Identification of Proteins Bound to Dengue Viral RNA in vivo Reveals New Host Proteins Important for Virus Replication. mBio. 2016; 7(1):e01865-15.· Postnikova EN. et al. Scalable, Semi-Automated Fluorescence Reduction Neutralization Assay for Qualitative Assessment of Ebola Virus-Neutralizing Antibodies in Human Clinical Samples. PLOS ONE. 2019; 14 (8): e0221407.

· Radoshitzky SR. et al. siRNA Screen Identifies Trafficking Host Factors that Modulate Alphavirus Infection. PLoS Pathogens. 2016; 12:e1005466.· Wang C. et al. The Serine/Threonine Kinase AP2-Associated Kinase 1 Plays an Important Role in Rabies Virus Entry. Viruses. 2019; 12 (1): 45. · Wang G. et al. The G Protein-Coupled Receptor FFAR2 Promotes Internalization during Influenza A Virus Entry. Journal of Virology. 2019; 94 (2).

Selected Publications Featuring High-Content Technologies in the Study of Viral Disease

VIRAL DISEASE

· Abdelmohsen U, Cheng C, Reimer A, Kozjak-Pavlovic V, Ibrahim A, Rudel T. et al. Antichlamydial Sterol from the Red Sea Sponge Callyspongia aff. implexa. Planta Medica. 2015;81(05):382-387.

· Ang ML and Pethe K. Contribution of high-content imaging technologies to the development of anti-infective drugs. Cytometry A. 2016; Aug; 89(8):755-60. · Blättner S, Das S, Paprotka K, Eilers U, Krischke M, Kretschmer D. et al. Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epi-thelial Cells and Phagocytes. PLoS Pathogens. 2016;12(9):e1005857.

· Christophe T. et al. High Content Screening Identifies Decaprenyl-phosphoribose 2’ epimerase as a Target for Intracellular Antimycobacterial Inhibitors. PLoS Pathogens. 2009; 5(10):e1000645.

· Clare RH. et al. Development and Validation of a Whole-Cell Screen: A Route to Macrofilaricidal Drugs against Onchocerciasis and Lymphatic Filariasis. J Biomol Screen. 2015; 20(1):64-9. 

· Delorme V, Song O-R, Baulard A and Brodin P. Testing Chemical and Genetic Modulators in Mycobacterium tuberculosis Infected Cells Using Phenotypic Assays. Methods in Molecular Biology. 2015; 1285:387-411.

· Mellouk N. et al. Shigella Subverts the Host Recycling Compartment to Rupture its Vacuole. Cell Host & Microbe. 2014; 16(4):517–30.  · Knight M, Braverman J, Asfaha K, Gronert K, Stanley S. Lipid droplet formation in Mycobacterium tuberculosis infected macrophages requires IFN-γ/HIF-1α signaling and supports host defense. PLOS Pathogens. 2018;14(1):e1006874.

· Pooja S, Pushpanathan M, Gunasekaran P, Rajendhran J. Endocytosis-Mediated Invasion and Pathogenicity of Streptococcus agalactiae in Rat Cardiomyocyte (H9C2). PLoS ONE. 2015;10(10):e0139733.  

· Quereda JJ. et al. A Dual Microscopy-based Assay to Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape. Applied and Environmental Microbiology. 2016; 82(1):211–217.  

· Queval CJ. et al. STAT3 Represses Nitric Oxide Synthesis in Human Macrophages upon Mycobacterium tuberculosis Infection. Scientific Reports. 2016; 6:29297.  · Ranjani J, Pushpanathan M, Mahesh A, Niraimathi M, Gunasekaran P, Rajendhran J. Pseudomonas aeruginosa PAO1 Induces Distinct Cell Death Mechanisms in H9C2 cells and its Differentiated Form. Journal of Basic Microbiology. 2015;55(10):1191-1202.  

Selected Publications Featuring High-Content Technologies in the Study of Bacterial Disease

BACTERIAL DISEASE

· Ang ML and Pethe K. Contribution of high-content imaging technologies to the development of anti-infective drugs. Cytometry A. 2016; Aug; 89(8):755-60. · Alonso-Padilla J. et al. Automated High-Content Assay for Compounds Selectively Toxic to Trypanosoma cruzi in a Myoblastic Cell Line. PLoS Neglected Tropical Diseases. 2015; 9(1):e0003493.

· Clare R, Cook D, Johnston K, Ford L, Ward S, Taylor M. Development and Validation of a High-Throughput Anti-Wolbachia Whole-Cell Screen A Route to Macrofilaricidal Drugs against Onchocerciasis and Lymphatic Filariasis. Journal of Biomolecular Screening. 2015;20(1):64-69.

· De Rycker M. et al. Identification of Trypanocidal Activity for Known Clinical Compounds Using a New Trypanosoma cruzi Hit-Discovery Screening Cascade. PLoS Neglected Tropical Diseases. 2016; 10(4):e0004584.

· Duez J. et al. Splenic Retention of Plasmodium falciparum Gametocytes to Block the Transmission of Malaria. Antimicrobial Agents and Chemotherapy. 2015; 59(7):4206-4214.· Fletcher S, Lucantoni L, Sykes M, Jones A, Holleran J, Saliba K et al. Biological characterization of chemically diverse compounds targeting the Plasmodium falciparum coenzyme A synthesis pathway. Parasites & Vectors. 2016;9(1).

· Hart C, Munro T, Andrews K, Ryan J, Riches A, Skinner-Adams T. A Novel in vitro Image-Based Assay Identifies New Drug Leads for Giardiasis. International Journal for Parasitology: Drugs and Drug Resistance. 2017;7(1):83-89.

· Khare S. et al. Anti-Trypanosomal Treatment with Benznidazole is Superior to Posaconazole Regimens in Mouse Models of Chagas Disease. Antimicrobial Agents and Chemotherapy. 2015; 59(10):6385-6394.

· Khare S. et al. Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease. PLoS Pathogen. 2015; 11(7):e1005058.· Lucantoni L. et al. A Simple and Predictive Phenotypic High Content Imaging Assay for Plasmodium falciparum Mature Gametocytes to Identify Malaria Transmission Blocking Compounds. Scientific Reports. 2015; 5:16414. 

· Plouffe DM. et al. High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria Transmission. Cell Host and Microbe. 2016; 19(1):114–126. · Roth A, Maher SP, Conway AJ, Ubalee R, Chaumeau V, Andolina C et al. A comprehensive model for assessment of liver stage therapies targeting Plasmodium vivax and Plasmodium falciparum Nature Communications 2018; 9:(1837)

· Sisquella X, Nebl T, Thompson J, Whitehead L, Malpede B, Salinas N. et al. Plasmodium Falciparum Ligand Binding to Erythrocytes Induce Alterations in Deformability Essential for Invasion. eLife. 2017; 6:e21083.

· Sykes ML and Avery VM. Development and Application of a Sensitive, Phenotypic, High-throughput Image-based Assay to Identify Compound Activity Against Trypanosoma cruzi Amastigotes. International Journal for Parasitology. Drugs and Drug Resistance. 2015; 5(3):215–28.

· Tegazzini D. et al. A Replicative in vitro Assay for Drug Discovery Against Leishmania donovani. Antimicrobial Agents and Chemotherapy. 2016; 60(6):3524-3532.

Selected Publications Featuring High-Content Technologies in the Study of Parasitic Disease

PARASITIC DISEASE

Confocal Imaging

Spinning disk confocality is a fast and gentle technique to reject light from out of focus planes, allowing live cell imaging with minimal photobleaching and phototoxicity. This option gives full flexibility to optimize image quality, depending on the size and morphology of intracellular and extracellular pathogen samples.

Water Immersion Objectives

Proprietary automated water-immersion objectives with very high numerical aperture deliver and capture more photons allowing imaging of weakly stained samples and provide a higher resolution in XYZ than conventional objectives. This enables good image quality from tiny objects like pathogens inside cells.

Synchrony Optics

The proprietary Synchrony OpticsTM technology reduces spectral crosstalk during simultaneous imaging on multicamera Opera Phenix Plus systems. This increases speed up to 4-fold, allowing genome-wide siRNA screens or large compound screens to be done in reasonable time without compromising on sensitivity.

sCMOS cameras

The large format sCMOS cameras – one in the Operetta CLS and up to four in the Opera Phenix Plus – deliver low noise, wide dynamic range and high resolution – perfect for capturing large numbers of cells, e.g. to detect infection rates with high sensitivity.

High-Content Analysis And Screening Technologies To Study Infectious Disease

PerkinElmer has more than a decade of experience in developing confocal High-Content Screening systems. The Opera Phenix® Plus and Operetta CLS™ systems have been fully designed in-house, which allowed control of all design aspects and technologies. Besides combining market leading automated microscopy and image analysis performance, special attention was paid to the requirements of live cell experiments from light management and environmental control, to the ease of cleaning and minimizing of risk of internal contamination of the system.

High-Content Analysis And Screening Technologies To Study Infectious Disease

Intelligent Image Acquisition

Intelligent acquisition technology – PreciScan™ – employs a pre-scan at low magnification to identify objects of interest and a re-scan of these objects at higher magnification, limiting the acquisition to just part of the wells or particular wells of interest. This saves time and hard drive space when analyzing rare events like low multiplicities of infection (MOI).

Harmony Software Building Blocks

PerkinElmer’s Harmony® software is known for its easy-to-use building blocks, which allow step-by-step image analysis, making it easy for even novice users to generate results, without prior image analysis knowledge. The Operetta CLS and the Opera Phenix Plus are therefore ideally suited for multi-user facilities as new scientists can quickly become familiar with the system and software.

Machine Learning

Machine learning techniques, either supervised or unsupervised, enable the user to distinguish phenotypes using classifiers based on feature combinations rather than on a single parameter or allow image segmentation using a learn-by-example approach. While other systems may require an image analysis expert to create an algorithm, machine learning makes it easy to do it on your own.

PerkinElmer has more than a decade of experience in developing confocal High-Content Screening systems. The Opera Phenix® Plus and Operetta CLS™ systems have been fully designed in-house, which allowed control of all design aspects and technologies. Besides combining market leading automated microscopy and image analysis performance, special attention was paid to the requirements of live cell experiments from light management and environmental control, to the ease of cleaning and minimizing of risk of internal contamination of the system.

Opera Phenix Plus High-Content Screening System

The Opera Phenix® Plus high-content screening system is the premier confocal solution for today’s most demanding applications, such as spheroid imaging or high resolution phenotyping.

Operetta CLS High-Content Analysis System

Uncover deep biological understanding from cell health to bacterial invasion assays with the flexible Operetta CLSTM high-content analysis system.

Harmony High-Content Imaging & Analysis Software

Harmony® provides a complete solution with all the tools you need to set up and automate your experiments, aquire images and analyze data and then store, retrieve and present the results.

Columbus High Volume Data Management & Analysis SystemThe ColumbusTM system is a unique, universal high-volume image data storage and analysis system that brings access to images from a wide range of sources including all major high-content screening instruments via the Internet.

High-Content Profiler

High-Content ProfilerTM enables true multiparametric hit selection from high-content screens, from quality control to normalization to machine learning, in one single workflow powered by TIBCO Spotfire®.

Integrated Laboratory Automation Solutions for Cell-based Assays

PerkinElmer integrated workstations can automate your entire high-content screening workflow from treatment to readout.

Solutions For High-Content Analysis And Screening

Microplates

We offer microplates designed specifically for high-content screening. For example, our validated CellCarrierTM Ultra plates feature an ultra-low bottom with superior flatness and clarity for optimal image quality day in and day out.

MuviCyte Live-cell imaging system

You may also be interested in

The MuviCyteTM system is designed to run long-term live-cell imaging assays. It operates inside your cell-culture incubator, enabling you to maintain your cells under optimal conditions.

www.cellularimaging.com

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A Dual Microscopy-Based Assay to Assess Listeria monocytogenes Cellular Entry and Vacuolar EscapeJ. J. Quereda, J. Pizarro-Cerda and colleagues.Institut Pasteur, Unité des Interactions Bactéries-Cellules/INSERM U604/INRA USC2020 and Unité Dynamique des Interactions Hôte-Pathogène and Imagopole, Paris, France.

FEATUREDPUBLICATION

Quereda JJ et al. A Dual Microscopy-Based Assay To Assess Listeria monocytogenes Cellular Entry and Vacuolar Escape. Appl Environ Microbiol 2016;82(1):211–7.

Listeriosis-causing L. monocytogenes invades mammalian cells and escapes from the vacuole, enabling proliferation in the host cell cytoplasm. A two-step method involving high-content screening was developed to investigate cell invasion and vacuolar rupture in a single experiment, providing a powerful tool which could be used for identifying the factors involved in these processes in bacteria and other intracellular pathogens.The Gram-positive bacterium, Listeria monocytogenes, causes listeriosis in humans, characterised by gastroenteritis in immunocompromised individuals, miscarriage in pregnant women, and newborn meningitis. L. monocytogenes invades mammalian cells and escapes from vacuoles enabling proliferation in the host cell cytoplasm.

Tracking of L. monocytogenes vacuolar escape using a FRET-based high-content assay.Cellular infection with L. innocuaβlact/InlB (A) or L. monocytogenes EGDe PrfA*βlact (B). Images were obtained with an Opera QEHS confocal microscope and a 10x objective, using the following merged channels: intact CCF4 probe peaks at 535 nm (green); cleaved CCF4 probe peaks at 450 nm (blue). L. monocytogenes can escape its vacuole and cleave the CCF4 probe in the cytoplasm, while nonpathogenic L. innocua cannot. Lower panels show Draq5 staining of images A and B.

A novel two-step approach was used to investigate the factors affecting L. monocytogenes invasion and/or escape from the vacuolar compartment. L. monocytogenes and L. innocuareporter strains expressing a β-lactamase covalently attached to the bacterial cell wall were incubated with HeLa cells containing the Försterresonance energy transfer (FRET)probe CCF4 in their cytoplasm. The CCF4 probe was cleaved by the bacterial surface β-lactamase in cells inoculated with L. monocytogenes, but not those inoculated with non-pathogenicL. innocua, thereby demonstrating bacterial access to the host cytoplasm. As a second step, differential immunofluorescence staining was performed to distinguish extracellular versus total bacterial populations in samples. In this assay, extracellular bacteria are first labelled with an anti-Listeria serum and a secondary Alexa Fluor 647 antibody. Cells are then permeabilised and the total Listeria population is labelled with the same primary serum and an Alexa Fluor 488

secondary antibody. Cells were imaged using PerkinElmer’s Opera® QEHS High-Content Screening System. Images were managed and analysed using PerkinElmer’s Columbus database, using building block routines for feature extraction, and automated scoring of the fluorescence signal for each individual cell.

These assays enable high-content screening of host and pathogen factors that differentially alter cell invasion and vacuolar rupture. This approach has broad applicability in the study of other bacteria and pathogens which demonstrate internalisation in host cells.

Reproduced and amended with permission from American Society for Microbiology.

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Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus ReplicationStacia Phillips and colleaguesDepartment of Molecular Genetics and Microbiology, Center for RNA Biology, Duke University, Durham, North Carolina, USA; Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA.

FEATUREDPUBLICATION

An in vivo approach involving UV crosslinking, antisense-mediated affinity purification and mass spectrometry identified host proteins that physically associate with dengue virus RNA. Small interfering RNA-mediated gene silencing with high-content screening demonstrated that over half of these proteins are likely to be involved in regulating dengue virus replication.

Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. There is currently no effective vaccine to prevent infection, and no antivirals for treating dengue virus-associated disease. The development of strategies for the prevention/treatment of dengue requires a detailed understanding of the molecular mechanisms involved in virus replication in both human and insect hosts. Host RNA-binding proteins mediating various aspects of virus replication through their physical interactions with viral RNA have traditionally been identified using in vitro strategies.

Phillips SL et al. Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication mBio 2016;7(1):e01865-15.

High-content imaging of dengue virus infected cells

(A) Representative images used for quantification of virus replication in cells silenced for candidate RNA binding proteins (blue: nuclei, green: viral envelope protein).

(B) Quantification of nuclei per condition by high-content image analysis.(C) Percentage of infected cells measured by high-content imaging of cells expressing viral envelope.

Reproduced and amended with permission from American Society for Microbiology.

Schematic of affinity purification technique

In this study, in vivo UV crosslinking followed by antisense-mediated affinity purification was used to identify proteins associated with dengue virus RNA in infected Huh7 cells.Proteins specifically purified with viral RNA were identified using mass spectrometry. Using stringent selection criteria, twelve host proteins that bind dengue virus RNA in vivo were identified.Seven of these interactions were subsequently validated using RNA immunoprecipitation.

Small interfering RNA-mediated gene silencing was used to test whether the host proteins play a role in mediating efficient dengue virus replication. siRNA-transfected cells were infected with dengue virus and virus replication was analysed by expression of viral envelope protein using PerkinElmer’s Opera Phenix™ High-Content Screening System. Quantification was performed using data collected from 16 fields per well in 96-well format. Nuclei were counted in the Hoechst channel, and infected cells were identified using cytoplasmic signal in the Alexa 488 channel. It was shown that over half of these host proteins are likely to be involved in regulating virus replication.

This in vivo method potentially has broad utility in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches.

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Testing Chemical and Genetic Modulators in Mycobacterium tuberculosis Infected Cells Using Phenotypic AssaysVincent Delorme and colleaguesInstitut Pasteur de Lille, University of Lille Inserm U1019, CNRS UMR8204, 1 rue du Professeur Calmette, BP245, Lille Cedex, 59019, France.

FEATUREDPUBLICATION

Delorme V et al. Testing chemical and genetic modulators in Mycobacterium tuberculosis infected cells using phenotypic assays. Methods Mol Biol. 2015;1285:387-411.

Overall scheme of the various assays used to characterise M. tuberculosis pathogenicity.

A better understanding of the intracellular stage of M. tuberculosis pathogenesis may aid development of new agents in the battle against tuberculosis infection. High-content screening was used to investigate the role of host genes in intracellular replication, identify small molecule inhibitors interfering with replication within the host cell, and examine the role of bacterial genes in intracellular trafficking.

Mycobacterium tuberculosis is able to colonise host cells, with this intracellular stage contributing to tuberculosis pathogenesis and persistent infection. There is a limited understanding of how the tubercle bacillus colonises thehost cell, and the factors associated with intracellular persistence.

The distribution of M. tuberculosis expressing fluorescent proteins can be easily quantified using confocal microscopy. The authors describe three high-content screening methods to investigate quantitatively and

qualitatively the impact of various genetic or chemical modulators onM. tuberculosis-infected host cells: (1) Genome-wide small interfering RNA

(siRNA) library screening was used to investigate which host genes are involved in M. tuberculosisintracellular replication;

(2) Compound libraries were screened to explore whether small molecule compounds can inhibit M. tuberculosisintracellular replication;

(3) M. tuberculosis transposon mutant libraries were screened to investigate which genes are involved in intracellular trafficking. The workflowof each of these approaches is shown in the figure below.

Intracellular M. tuberculosis wasquantified by detecting enhanced GFP-expressing bacteria inside fluorescently labelled phagocytic or non-phagocytic cells. Imaging was performed using PerkinElmer’s Opera® High-Content Screening System recording several fields per well of a 96- or 384-well microplate. Images were analysed using Acapella Studio software, which enabled the development of specific image-analysis routines for the different assays.

These findings contribute to our understanding of M. tuberculosis host cell colonisation, and the factors associated with intracellular persistence.

Copyright1. JoVE shall retain copyright to any figures published by JoVE covered by this agreement.2. Resale of the figure by the client is strictly prohibited.

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A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection

Nicholas Barrows and colleaguesDepartment of Biochemistry and Molecular Biology, University of Texas Medical Branch, Texas, USA; Department of Molecular Genetics and Microbiology, Duke University, Durham, USA.

FEATUREDPUBLICATION

High-content screening and analysis of a panel of 774 FDA-approved drugs identified over 20 molecules that could inhibit Zika virus infection in human cells including cervical, placental, neural stem cells and primary amnion cells – all are potential new therapeutics in the fight against Zika.

Screen for inhibitors of Zika virus infection among FDA-approved drugs

Reprinted from Barrows NJ, Campos RK, Powell ST, Prasanth KR, Schott-Lerner G et al. A screen of FDA-approved drugs for inhibitors of Zika virus infection. Cell Host Microbe 2016;20(2):259–70. with permission from Elsevier.

Although most Zika virus infections are mild and resolve rapidly, cases of severe acute infection in patients with underlying conditions have been reported. There is also compelling evidence linking Zikawith neurological sequelae including Guillain-Barré Syndrome, meningo-encephalitis and myelitis, and a strong causal relationship between Zika virus infection and birth defects, including microcephaly. Despite the significant threat to human health, there are currently no approved vaccines or specific medical therapies to prevent or treat Zikavirus infection.

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Library of774 FDA-approved drugs

PandemicZika virus

HuH-7hepatoma

Validation

Primaryhuman amnionepithelial cells

HeLa andJEG3

HuH-7hepatoma

Neuralstem cells

Imaging usingOpera PhenixHigh-Content

Screening System

Screen for inhibitors of Zika virus infection among FDA-approved drugs. C) Screen reproducibility. Points coloured red were selected for validation studies.D) Infection rate versus cell count.E) Representative high-content images showing viral antigen (red) and host cell nuclei (blue) for selected drugs and negative (DMSO) and positive (NITD008) controls.

High-content screening was used to test a library of 774 FDA-approved drugs for their ability to block infection of human HuH-7 cells by a pandemic Zika virus strain newly isolated from Aedes aegypti mosquitoes in Mexico.

Automated imaging was performed using PerkinElmer’s Opera Phenix™ High-Content Screening System with simultaneous multicolour confocal image acquisition at 10x magnification. Immunofluorescence images were analysed using Harmony® High-Content Imaging and Analysis Software to quantify cell number and the percentage of infected cells.

45 drugs were identified that significantly inhibited Zikavirus infection – crucially, these included drugs approved for use during pregnancy.

A total of 30 compounds were validated in cervical, placental, human neural stem cells and primary amnion cells, with 24 of these confirmed to be inhibitors of Zikavirus infection.

These newly identified drugs are potential candidates for testing in clinical trials of patients infected with Zika virus.

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Ebola Virus Modulates Transforming Growth Factor β Signaling andCellular Markers of Mesenchyme-Like Transition in HepatocytesJason Kindrachuk and colleaguesEmerging Viral Pathogens Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Maryland, USA; Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Maryland, USA

FEATUREDPUBLICATION

Ebola virus (EBOV) is a serious public health concern, causing severe haemorrhagic disease in humans with a mortality rate of more than 78%. Despite the seriousness of this disease, there is relatively little known about the host response during infection. Here the authors use a kinome analysis to investigate the host kinome response over time in human hepatocyte cells infected with EBOV. This kinome analysis shows that transforming growth factor β (TGF-β)-mediated pathways are modulated during EBOV infection and replication. Phenotypic screening shows that EBOV infection is inhibited in the presence of TGF-β and related pathway kinase inhibitors. Additionally, imaging of infected cells shows the phenotypic hallmarks of endothelial-to-mesenchyme transition (EMT), which may contribute to EBOV pathogenesis.

EBOV infection of hepatocytes shows the phenotype of epithelial-to-mesenchyme transition

Kindrachuk J et al. Ebola Virus Modulates Transforming Growth Factor β Signaling and Cellular Markers of Mesenchyme-Like Transition in Hepatocytes. J Virol 2014;88(17):9877–92.

Effect of kinase inhibitors on EBOV replication in hepatocytes as assessed by phenotypic screening

HUH7 cells were treated with kinase inhibitors 1 h prior to infection (pre-infection) or 1 h after infection (post-infection) with EBOV-GFP. Imageswere analysed in Harmony and the percentage inhibition of infection wascalculated from the ratio of infected (GFP-positive) cells to the totalnumber of cells.

Compared to mock-infected cells (top) HUH7 cells infected with EBOV-GFP (bottom) show reduced cell-cell contacts, elongation and spindle-like formation.

HUH7 cells were treated with 10 μM of selected kinase inhibitors 1 h prior to infection (pre-infection) or 1 h after infection (post-infection) with a GFP-expressing Ebola virus (EBOV-GFP). After 24 h in the presence of EBOV-GFP, cells were fixed and imaged on the Operetta®

High-Content Imaging System and images analysedusing Harmony® High-Content Image Analysis Software. The percentage of infection was determined from the ratio of infected cells (showing the presence of EBOV-GFP) to total cells as identified by DAPI staining. Results are based on 3 independent experiments.

EBOV-GFP-infected HUH7 cells also showed phenotypic markers typical of EMT 24 h after infection. Infected cells showed reduced cell-cell contacts, were elongated and formed spindle-like structures.

The identification of specific kinase inhibitors of EBOV infection provides a way to better understand Ebola disease pathogenesis and may represent a starting point for potential treatment regimens. Additional phenotypic screening may also be used to identify modulators of the EMT phenotype induced by EBOV infection.

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High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria TransmissionDavid M Plouffe and colleaguesThe Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Drive, San Diego, CA 92121, USA.

FEATUREDPUBLICATION

Plouffe DM et al. High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria Transmission. Cell Host Microbe 2016;19(1):114–26.

The Saponin-lysis Sexual Stage Assay (SaLSSA) is a high-throughput, cost-effective assay for identifying small molecules with malaria transmission-blocking activity. A total of 13,983 compounds were analysed, which included some with consistent low nanomolar transmission-blocking activity. This assay provides a tool for the discovery and development of transmission-blocking drugs.

Malaria is caused by the Plasmodium parasite, which has a complex life cycle involving both a vertebrate host and the anopheline mosquito. Preventing transmission of the parasite is essential for malaria control. Most of the currently available assays to detect transmission-blocking are relatively low throughput and cannot be applied to large chemical libraries.

The Saponin-lysis Sexual Stage Assay (SaLSSA) is a high-throughput, cost-effective, one-step assay for identifying small molecules with malaria transmission-blocking activity. An overview of the SaLSSA gametocyte assay is shown in the figure below. Gametocyte stages I–V were diluted to 0.5–0.75% gametocytemia plus 1.25% haematocrit in serum-free screening medium. Cultures were dispensed into 384- or 1,536-well plates containing compound (final concentration of 1.25 to 12.5 µM), and incubated at 37°C for 72 h under low-oxygen conditions.To detect viability in the non-replicating gametocytes, the dye MitoTracker CMXRos, which fluoresces at 600 nm in parasites with intact mitochondrial membrane potential, was chosen. MitoTracker Red CMXRos and saponinsolution in screening medium was then added to each well, and plates incubated for 60–120 min at 37°C.Saponin causes the red blood cells to lyse in serum-free medium, and simplifies the identification of parasites with automated microscopy. Imaging and analysis of plates was then performed using PerkinElmer’s Operetta® High-Content Screening system and Harmony® software. Parasite viability indices were calculated by dividing the parasite count of each compound-treated well by the average parasite count of the DMSO control wells.

A total of 13,983 known and new compounds were analysed. Over 90% of these drugs did not show activity against late-stage gametocytes, which are metabolically quiescent. However, several compounds with consistent low nanomolar transmission-blocking activity were identified, some of which showed cross-reactivity against asexual blood and liver stages of parasite development. The data clearly emphasise substantial physiological differences between sexual and asexual parasites, and the assay provides a tool for the discovery and development of transmission-blocking drugs.

Simplified schematic flow diagram of the SaLSSA gametocyte assay.

Stamp compounds MTR Red + saponin High-content imaging and analysis

72 h

Add gametocytes