Name: high-throughput transcriptome analysis for investigating host-pathogen interactions
Uploaded: 2022-03-05T12:30:00
Description: Watch this Scientific Journal Video about High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions at JoVE.com

HN is funded by FAPESP (grant numbers: #2017/50137-3, 2012/19278-6, 2018/14933-2, 2018/21934-5, and 2013/08216-2) and CNPq (313662/2017-7).
We are particularly thankful to the following grants for fellows: ANAG (FAPESP Process 2019/13880-5), VEM (FAPESP Process 2019/16418-0), IMSC (FAPESP Process 2020/05284-0), APV (FAPESP Process 2019/27146-1) and, RLTO (CNPq Process 134204/2019-0).

The samples used in this protocol were approved by the ethics committees from both the Department of Microbiology of the Institute of Biomedical Sciences at the University of S&#227;o Paulo and the Federal University of Sergipe (Protocols: 54937216.5.0000.5467 and 54835916.2.0000.5546, respectively).
1. Docker desktop installation
NOTE: Steps to prepare the Docker environment are different among the operating systems (OSs). Therefore, Mac users must f...

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq+analysis+of+chikungunya+virus+infection+and+identification+of+granzyme+A+as+a+major+promoter+of+arthritic+inflammation.">RNA-Seq analysis of chikungunya virus infection and identification of granzyme A as a major promoter of arthritic inflammation.</a> PLOS Pathogens. 13 (2), 1006155 (2017).</li><li>Gon&#231;alves, A. N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+impact+of+sample+heterogeneity+on+transcriptome+analysis+of+human+diseases+using+MDP+webtool.">Assessing the impact of sample heterogeneity on transcriptome analysis of human diseases using MDP webtool.</a> Frontiers in Genetics. 10, 971 (2019).</li><li>Russo, P. S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CEMiTool:+a+Bioconductor+package+for+performing+comprehensive+modular+co-expression+analyses.">CEMiTool: a Bioconductor package for performing comprehensive modular co-expression analyses.</a> BMC Bioinformatics. 19 (1), 56 (2018).</li><li>Costa-Silva, J., Domingues, D., Lopes, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq+differential+expression+analysis:+An+extended+review+and+a+software+tool.">RNA-Seq differential expression analysis: An extended review and a software tool.</a> PloS One. 12 (12), 0190152 (2017).</li><li>Seyednasrollah, F., Laiho, A., Elo, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+software+packages+for+detecting+differential+expression+in+RNA-seq+studies.">Comparison of software packages for detecting differential expression in RNA-seq studies.</a> Briefings in Bioinformatics. 16 (1), 59-70 (2015).</li><li>Zhang, B., Horvath, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+framework+for+weighted+gene+co-expression+network+analysis.">A general framework for weighted gene co-expression network analysis.</a> Statistical Applications in Genetics and Molecular Biology. 4, (2005).</li><li>Cheng, C. W., Beech, D. J., Wheatcroft, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+CEMiTool+for+gene+co-expression+analysis+of+RNA-seq+data.">Advantages of CEMiTool for gene co-expression analysis of RNA-seq data.</a> Computers in Biology and Medicine. 125, 103975 (2020).</li><li>Cardozo, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=webCEMiTool:+Co-expression+modular+analysis+made+easy.">webCEMiTool: Co-expression modular analysis made easy.</a> Frontiers in Genetics. 10, 146 (2019).</li><li>de Lima, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+noncoding+RNAs+are+involved+in+multiple+immunological+pathways+in+response+to+vaccination.">Long noncoding RNAs are involved in multiple immunological pathways in response to vaccination.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (34), 17121-17126 (2019).</li><li>Prada-Medina, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+immunology+of+diabetes-tuberculosis+comorbidity+reveals+signatures+of+disease+complications.">Systems immunology of diabetes-tuberculosis comorbidity reveals signatures of disease complications.</a> Scientific Reports. 7 (1), 1999 (2017).</li><li>Chen, E. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichr:+interactive+and+collaborative+HTML5+gene+list+enrichment+analysis+tool.">Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool.</a> BMC Bioinformatics. 14, 128 (2013).</li><li>Kuleshov, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichr:+a+comprehensive+gene+set+enrichment+analysis+web+server+2016+update.">Enrichr: a comprehensive gene set enrichment analysis web server 2016 update.</a> Nucleic Acids Research. 44, 90-97 (2016).</li><li>Raudvere, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=g:Profiler:+a+web+server+for+functional+enrichment+analysis+and+conversions+of+gene+lists+(2019+update).">g:Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update).</a> Nucleic Acids Research. 47, 191-198 (2019).</li><li>Young, M. D., Wakefield, M. J., Smyth, G. 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The preparation of the sequencing libraries is a crucial step toward answering biological questions in the best possible way. The type of transcripts of interest of the study will guide which type of sequencing library will be chosen and drive bioinformatic analyses. For example, from the sequencing of a pathogen and host interaction, according to the type of sequencing, it is possible to identify sequences from both or just from the host transcripts.
Next-generation sequencing equipment, e.g....

Arboviruses, such as dengue, yellow fever, chikungunya, and zika, have been widely associated with several endemic outbreaks and have emerged as one of the main pathogens responsible for infecting humans in the last decades1,2. Individuals infected with the chikungunya virus (CHIKV) often have fever, headache, rash, polyarthralgia, and arthritis3,4,5. Viruses can subvert the gene expression of the cell and influence various host signaling pathways. Recently, blood transcriptome studies utilized RNA-seq to identify the differentially expressed genes (DEGs) associated with acute CHIKV infection in comparison with convalescence6 or healthy controls7. CHIKV-infected children had up-regulated genes that are involved in innate immunity, such as the ones related to cellular sensors for viral RNA, JAK/STAT signaling, and toll-like receptor signaling pathways6. Adults acutely infected with CHIKV also showed induction of genes related to innate immunity, such as the ones related to monocytes and dendritic cell activation, and to antiviral responses7. The signaling pathways enriched with down-regulated genes included the ones related to adaptive immunity, such as T cell activation and differentiation and enrichment in T and B cells7.
Several methods can be used to analyze transcriptome data of host and pathogen genes. Often, RNA-seq library preparation starts with the enrichment of mature poly-A transcripts. This step removes most of the ribosomal RNA (rRNA) and in some of the cases viral/bacterial RNAs. However, when the biological question involves the pathogen transcript detection and RNA are sequenced independent of the previous selection, many other different transcripts could be detected by sequencing. For example, subgenomic mRNAs have been shown to be an important factor to verify the severity of the diseases8. In addition, for certain viruses such as CHIKV and SARS-CoV-2, even poly-A enriched libraries generate viral reads that can be utilized in downstream analyses9,10. When focused on the analysis of the host transcriptome, researchers can investigate the biological perturbation across samples, identify differentially expressed genes and enriched pathways, and generate co-expression modules7,11,12. This protocol highlights transcriptome analyses of CHIKV-infected patients and healthy individuals using different bioinformatic approaches (Figure 1A). Data from a previously published study7 consisting of 20 healthy and 39 CHIKV acutely infected individuals were used to generate the representative results.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CEMiTool</td>
			<td>Computational Systems Biology Laboratory</td>
			<td>1.12.2</td>
			<td>Discovery and the analysis of co-expression gene modules in a fully automatic manner, while providing a user-friendly HTML report with high-quality graphs.</td>
		</tr>
		<tr>
			<td>EdgeR</td>
			<td>Bioconductor (Maintainer: Yunshun Chen [yuchen at wehi.edu.au])</td>
			<td>3.30.3</td>
			<td>Differential expression analysis of RNA-seq expression profiles with biological replication</td>
		</tr>
		<tr>
			<td>EnhancedVolcano</td>
			<td>Bioconductor (Maintainer: Kevin Blighe [kevin at clinicalbioinformatics.co.uk])</td>
			<td>1.6.0</td>
			<td>Publication-ready volcano plots with enhanced colouring and labeling</td>
		</tr>
		<tr>
			<td>FastQC</td>
			<td>Babraham Bioinformatics</td>
			<td>0.11.9</td>
			<td>Aims to provide a simple way to do some quality control checks on raw sequence data coming from high throughput sequencing</td>
		</tr>
		<tr>
			<td>FeatureCounts</td>
			<td>Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research</td>
			<td>2.0.0</td>
			<td>Assign mapped sequencing reads to specified genomic features</td>
		</tr>
		<tr>
			<td>MDP</td>
			<td>Computational Systems Biology Laboratory</td>
			<td>1.8.0</td>
			<td>Molecular Degree of Perturbation calculates scores for transcriptome data samples based on their perturbation from controls</td>
		</tr>
		<tr>
			<td>R</td>
			<td>R Core Group</td>
			<td>4.0.3</td>
			<td>Programming language and free software environment for statistical computing and graphics</td>
		</tr>
		<tr>
			<td>STAR</td>
			<td>Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research</td>
			<td>2.7.6a</td>
			<td>Aligner designed to specifically address many of the challenges of RNA-seq data mapping using a strategy to account for spliced alignments</td>
		</tr>
		<tr>
			<td>Bowtie2</td>
			<td>Johns Hopkins University</td>
			<td>2.4.2</td>
			<td>Ultrafast and memory-efficient tool for aligning sequencing reads to long reference sequences</td>
		</tr>
		<tr>
			<td>Trimmomatic</td>
			<td>THE USADEL LAB</td>
			<td>0.39</td>
			<td>Trimming adapter sequence tasks for Illumina paired-end and single-ended data</td>
		</tr>
		<tr>
			<td>Get Docker</td>
			<td>Docker</td>
			<td>20.10.2</td>
			<td>Create a bioinformatic environment reproducible and predictable (https://docs.docker.com/get-docker/)</td>
		</tr>
		<tr>
			<td>WSL2-Kernel</td>
			<td>Windows</td>
			<td>NA</td>
			<td>https://docs.microsoft.com/en-us/windows/wsl/wsl2-kernel</td>
		</tr>
		<tr>
			<td>Get Docker Linux</td>
			<td>Docker</td>
			<td>NA</td>
			<td>https://docs.docker.com/engine/install/ubuntu/</td>
		</tr>
		<tr>
			<td>Docker Linux Repository</td>
			<td>Docker</td>
			<td>NA</td>
			<td>https://docs.docker.com/engine/install/ubuntu/#install-using-the-repository</td>
		</tr>
		<tr>
			<td>MDP Website</td>
			<td>Computational Systems Biology Laboratory</td>
			<td>NA</td>
			<td>https://mdp.sysbio.tools</td>
		</tr>
		<tr>
			<td>Enrichr Website</td>
			<td>MaayanLab</td>
			<td>NA</td>
			<td>https://maayanlab.cloud/Enrichr/</td>
		</tr>
		<tr>
			<td>webCEMiTool</td>
			<td>Computational Systems Biology Laboratory</td>
			<td>NA</td>
			<td>https://cemitool.sysbio.tools/</td>
		</tr>
		<tr>
			<td>gProfiler</td>
			<td>Bioinformatics, Algorithmics and Data Mining Group</td>
			<td>NA</td>
			<td>https://biit.cs.ut.ee/gprofiler/gost</td>
		</tr>
		<tr>
			<td>goseq</td>
			<td>Bioconductor (Maintainer: Matthew Young [my4 at sanger.ac.uk])</td>
			<td>NA</td>
			<td>http://bioconductor.org/packages/release/bioc/html/goseq.html</td>
		</tr>
		<tr>
			<td>SRA NCBI study</td>
			<td>NCBI</td>
			<td>NA</td>
			<td>https://www.ncbi.nlm.nih.gov/bioproject/PRJNA507472/</td>
		</tr>
	</tbody>
</table>

high-throughput transcriptome analysis for investigating host-pathogen interactions

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of the disease.&#160;To study such processes, researchers can use high-throughput sequencing techniques (RNA-seq) that measure the dynamic changes of the host transcriptome at different stages of infection, clinical outcomes, or disease severity.This investigation can lead to a better understanding of the diseases, as well as uncovering potential drug targets and treatments. The protocol presented here describes a complete pipeline to analyze RNA-sequencing data from raw reads to functional analysis. The pipeline is divided into five steps: (1) quality control of the data; (2) mapping and annotation of genes; (3) statistical analysis to identify differentially expressed genes and co-expressed genes; (4) determination of the molecular degree of the perturbation of samples; and (5) functional analysis. Step 1 removes technical artifacts that may impact the quality of downstream analyses. In step 2, genes are mapped and annotated according to standard library protocols.&#160;The statistical analysis in step 3 identifies genes that are differentially expressed or co-expressed in infected samples, in comparison with non-infected ones. Sample variability and the presence of potential biological outliers are verified using the molecular degree of perturbation approach in step 4. Finally, the functional analysis in step 5 reveals the pathways associated with the disease phenotype. The presented pipeline aims to support researchers through the RNA-seq data analysis from host-pathogen interaction studies and drive future&#160;in vitro or in vivo experiments, that are essential to understand the molecular mechanism of infections.

The computing environment for transcriptome analyses was created and configured on the Docker platform. This approach allows beginner Linux users to use Linux terminal systems without a priori management knowledge. The Docker platform uses the resources of the host OS to create a service container that includes specific users&#39; tools (Figure 1B). A container based on the Linux OS Ubuntu 20.04 distribution was created and it was fully configured for transcriptomic analyses, which is access...

Watch this Scientific Journal Video about High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions at JoVE.com

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

The protocol presented here describes a complete pipeline to analyze RNA-sequencing transcriptome data from raw reads to functional analysis, including quality control and preprocessing steps to advanced statistical analytical approaches.

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of ...

Welcome to the protocol of high-throughput transcriptome analysis for investigating host-pathogen interactions. This protocol is divided in the following steps. Quality control to filter low-quality reads and also to remove adapter sequences Sequencing and annotations, where are you have to map the reads into a reference genomes and annotate the reads into the genes.Statistical and co-expression analysis, which defines the differentially expressed genes and also finds the co-expression modules. Molecular degree of perturbation analysis to find potential outlier samples. And finally, the functional analysis to determine the biological functions of differentially expressed genes.All the tools utilizing these pipelines were pre-installed in a Linux system and encapsulated into a Docker container. The samples utilizing these protocols derived from a paper published by our group in PLOS Pathogen. The samples comprise 20 healthy people and 39 patients infected with Chikungunya virus.The blood samples were collected, and RNA sequencing was performed. To install Docker in Windows system, you have to follow these steps. Go to the official webpage of Docker, and click in Get Started.Find the installer for Docker Desktop for windows. Download the file. Install locally in your machine.Make sure that these two options are marked. After installing the program, downloads the Docker image for this protocol. Go to the Windows terminal.Execute the commands to downloads the image. After downloading the image, you can see the file in the Docker desktop, and from this image, we can initiate the container. After you click in the round button, you have to expand the original parameters and options to define the name of the container and to associate a folder in your local computer with the folder inside Docker.After this, you click in Run to initiate the container. You can then access the terminal, which is in the Linux system inside the Docker. Type the bash commands, and then you can execute all the commands of this protocol.First, we have to execute the source to make all the tools of this protocol available. You should access the directory scripts. To perform a transcriptomic analysis, you have to download first the reference genome.For this, you have to execute the following commands. After the genome is download, you have to download the annotation of the genes. To do this, you have to type the following commands.Next, you have to configure the fastq-dump. This is allow you to downloads the sequencing files of the examples. After typing the following commands, you have to use the Tab button to go to the Tools option and to mark the options currents directory.Use the Tab buttons to save, and then ok. And then exit the tool fastq-dump. Now we can initiate the downloads of the reads by typing the following commands.The quality control consists and evaluates graphically the probability of errors in the sequencing reads. In this step, you have also to remove the technical sequences such as adapters. To generate the quality control graphs, you have to run the FastQC program.To remove the adapter sequences and the low-quality sequences, you have to type the following commands. With the good-quality reads, we have now to map the reads into the reference genome. After the mapping, we are gonna have to annotate the genes according to the human genes and then count the number of reads that match each human gene.The first step is to index the reference genome by typing the following command. And then we type this commands to map the reads into the human genome. Next, you should run the scripts that annotate the reads.After mapping and annotation, you can perform the differential expression analysis which it consists in finding the genes whose expression is higher or lower in one group compared to another. To identify the differentially expressed genes, or DEGs, you have to run following commands. After this, you can transfer the data results from the Docker to your local computer.For this, go to the terminal and type the following commands to save all the results to a local folder. To perform the remaining analysis, you also have to copy all the files of the directory data to a directory in your local computer. In your local computer, you will be able to see the directories where you saved the data from Docker.As you can see, you can access all the libraries. You can also open the HTML file containing the quality control reports. You can also access a directory containing the differentially expressed genes.And inside this directory, you will find the volcano plots where you can see the genes that are up-or downregulated in the one group versus another, in this case, patients infected with Chikungunya virus versus healthy controls. All the remaining steps of this protocol are gonna be executed in web tools using your browser. Let's first start with CEMiTool.Go to the browser and type the following address. CEMiTool identifies co-expression modules from expression data sets provided by the users. In the main page, you can go to the menu and click in the button Run.This will open a new page where you can upload the expression file. This file is in the directory data of your local computer. You will see that there three expression files, and the one that we are gonna use for the CEMiTool is a normalization call tmm.Then you have to select the phenodata file, the same thing for the file containing the protein-protein interactions, and finally, upload the file containing the gene sets or pathways. The gene sets file enables CEMiTool to perform enrichment analysis for each one of the co-expression module. Next, you should to expand the parameter section and click in Apply VST.After that, you can just click Run CEMiTool. After you run CEMiTool, you will see that 12 co-expression modules were identified. By clicking here, you can download all the results of these analysis.Another tool that we are gonna utilize in this protocol is MDP, or Molecular Degree of Perturbation. Just type in your browser mdp.sysbio.tools. MDP calculates the molecular distance of each sample compared to a reference group of samples, in this case, the healthy controls, in order to find not only potential outliers but also how perturbed are each samples compared to this group.In the Run page, you can just upload the expression file by clicking the button and selecting the file. Then you have to upload the phenodata file. Then you have to define which column contain the information about the group or the class and then which class or group correspond to the control group.After this, you can just run MDP. The bar graph shows for each one of the samples as a bar the score of molecular degree of perturbation, and the colors represent the different groups. And the box plot is another way of visualizing the same results where you see on each dots the is a different samples separate by two groups.To perform the functional analysis, we are gonna use the Enrichr tool. For this, you have to select the list of genes that were differentially expressed, either up-or downregulated, and use it as a input gene list in Enrichr tool. You will see that there are different tabs.All the results can also be downloaded to your local computer. The computer environment for transcriptome analysis has been placed on the Docker platform. This approach allows users with no prior experience with Linux system to utilize a terminal.In this container, there is a predefined folder structure for dataset and scripts which are necessary for all the analysis. In the pipeline, users will utilize blood transcriptome data from 20 healthy individuals and 39 patients acutely infected with Chikungunya virus. The sequencing platform returns a set of FASTQ files containing the DNA sequence, i.e.the reads, and the associated quality for each nucleotide base. The Phred quality scale indicates the probability of an incorrect reading for each base. Tools identify and remove low-quality reads from samples and to increase the probability of mapping reads.In this step, the mapping module, the high-quality reads recovered are used as inputs to align them against the human reference genome. CEMiTool identifies and analyze co-expression modules. Genes within the same module are co-expressed, which means that they exhibit similar patterns of expression across the samples of the data sets.The network analysis provides information about the most connected genes, i.e. the hubs. The names of those genes are shown in the network.The size of the nodes is proportional to its degree of connectivity. The results obtained from the DEG analysis were summarized in the volcano plots. The analysis of the molecular degree of perturbation permits the identification of perturbed samples from healthy and infected individuals.MDP suggests which samples are potential biological outliers. Removing those samples will impact the downstream results. A functional enrichment analysis using AURA can be performed with Enrichr tool.These steps helps to interpret the results by revealing common functional roles of several genes that were differentially expressed. The biological process shown in the bar graphs are the top 10 enriched gene sets based on their p-value ranking. In conclusion, these protocols covers all steps of RNA-Seq analysis.The pipeline was developed and encapsulated into the non-commercial system named Docker. On an image and made available for the scientific community. Due to the container system, all scripts and tools are under the same specific version to guarantee reproducibility.Furthermore, parts of the bioinformatics analysis was performed via free user-friendly web tools.

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Research

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JoVE Core

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Immunology and Infection

Unit 1

Control Charts

SCIENTISTS IN ACTION

Use of an Optical Trap for Study of Host-Pathogen Interactions for Dynamic Live Cell Imaging

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. Historically, intercellular contact events such as phagocytosis3 have been imaged by mixing two cell types, and then continuously scanning the field-of-view to find serendipitous intercellular contacts at the appropriate stage of interaction. The stochastic nature of these events renders this process tedious, and it is difficult to observe early or fleeting events in cell-cell contact by this approach. This method requires finding cell pairs that are on the verge of contact, and observing them until they consummate their contact, or do not. To address these limitations, we use optical trapping as a non-invasive, non-destructive, but fast and effective method to position cells in culture.
Optical traps, or optical tweezers, are increasingly utilized in biological research to capture and physically manipulate cells and other micron-sized particles in three dimensions4. Radiation pressure was first observed and applied to optical tweezer systems in 19705, 6, and was first used to control biological specimens in 19877. Since then, optical tweezers have matured into a technology to probe a variety of biological phenomena8-13.
We describe a method14 that advances live cell imaging by integrating an optical trap with spinning disk confocal microscopy with temperature and humidity control to provide exquisite spatial and temporal control of pathogenic organisms in a physiological environment to facilitate interactions with host cells, as determined by the operator. Live, pathogenic organisms like Candida albicans and Aspergillus fumigatus, which can cause potentially lethal, invasive infections in immunocompromised individuals15, 16 (e.g. AIDS, chemotherapy, and organ transplantation patients), were optically trapped using non-destructive laser intensities and moved adjacent to macrophages, which can phagocytose the pathogen. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability in immunology, primary T-cells were also trapped and manipulated to form synapses with anti-CD3 coated microspheres in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine spatial control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.

A method is described to individually select, manipulate, and image live pathogens using an optical trap coupled to a spinning disk microscope. The optical trap provides spatial and temporal control of organisms and places them adjacent to host cells. Fluorescence microscopy captures dynamic intercellular interactions with minimal perturbation to cells.

Dynamic live cell imaging allows direct visualization of real-time interactions between cells of the immune system1, 2; however, the lack of spatial and ...

Use of the EpiAirway Model for Characterizing Long-term Host-pathogen Interactions

Nontypeable Haemophilus influenzae (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory mucosa 1; 2. To study the mechanisms by which these organisms survive on and inside respiratory tissues, a model in which successful long-term co-culture of bacteria and human cells can be performed is required. We use primary human respiratory epithelial tissues raised to the air-liquid interface, the EpiAirway model (MatTek, Ashland, MA). These are non-immortalized, well-differentiated, 3-dimensional tissues that contain tight junctions, ciliated and nonciliated cells, goblet cells that produce mucin, and retain the ability to produce cytokines in response to infection. 
This biologically relevant in vitro model of the human upper airway can be used in a number of ways; the overall goal of this method is to perform long-term co-culture of EpiAirway tissues with NTHi and quantitate cell-associated and internalized bacteria over time. As well, mucin production and the cytokine profile of the infected co-cultures can be determined. This approach improves upon existing methods in that many current protocols use submerged monolayer or Transwell cultures of human cells, which are not capable of supporting bacterial infections over extended periods3. For example, if an organism can replicate in the overlying media, this can result in unacceptable levels of cytotoxicity and loss of host cells, arresting the experiment. The EpiAirway model allows characterization of long-term host-pathogen interactions. Further, since the source for the EpiAirway is normal human tracheo-bronchial cells rather than an immortalized line, each is an excellent representation of actual human upper respiratory tract tissue, both in structure and in function4.
For this method, the EpiAirway tissues are weaned off of anti-microbial and anti-fungal compounds for 2 days prior to delivery, and all procedures are performed under antibiotic-free conditions. This necessitates special considerations, since both bacteria and primary human tissues are used in the same biosafety cabinet, and are co-cultured for extended periods.

This method allows characterization of extended bacterial co-culture with EpiAirways, primary human respiratory epithelial tissue grown at the air-liquid interface, a biologically relevant in vitro model. The approach can be used with any microbe that is amenable to long-term co-culture.

Nontypeable Haemophilus influenzae  (NTHi) are human-adapted Gram-negative bacteria that can cause recurrent and chronic infections of the respiratory ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation. The study of biofilms is important in many biological fields, including environmental science, food production, and medicine. However, few studies have focused on such bacterial biofilms, partially due to the difficulty of investigating them. Most of the methods for qualitative and quantitative biofilm analyses described in the literature are only suitable for biofilms forming on abiotic surfaces or on homogeneous and thin biotic surfaces, such as a monolayer of epithelial cells.
While laser scanning confocal microscopy (LSCM) is often used to analyze in situ and in vivo biofilms, this technology becomes very challenging when applied to bacterial biofilms on fungal hyphae, due to the thickness and the three dimensions of the hyphal networks. To overcome this shortcoming, we developed a protocol combining microscopy with a method to limit the accumulation of hyphal layers in fungal colonies. Using this method, we were able to investigate the development of bacterial biofilms on fungal hyphae at multiple scales using both LSCM and scanning electron microscopy (SEM). This report describes the protocol, including microorganism cultures, bacterial biofilm formation conditions, biofilm staining, and LSCM and SEM visualizations.

This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Bacterial biofilms frequently form on fungal surfaces and can be involved in numerous bacterial-fungal interaction processes, such as metabolic ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...

High Throughput Co-culture Assays for the Investigation of Microbial Interactions

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many approaches used for the study of microbial interactions require specialized equipment and are expensive and time intensive. This paper presents a protocol for co-culture interaction assays that are inexpensive, scalable to large sample numbers, and easily adaptable to numerous experimental designs. Microorganisms are cultured together, with each well representing one pairwise combination of microorganisms. A test organism is cultured on one side of each well and first incubated in monoculture. Subsequently, target organisms are simultaneously inoculated onto the opposite side of each well using a 3D-printed inoculation stamp. After co-culture, the completed assays are scored for visual phenotypes, such as growth or inhibition. These assays can be used to confirm phenotypes or identify patterns among isolates of interest. Using this simple and effective method, users can analyze combinations of microorganisms rapidly and efficiently. This co-culture approach is applicable to antibiotic discovery as well as culture-based microbiome research and has already been successfully applied to both applications.

The co-culture interaction assays presented in this protocol are inexpensive, high throughput, and simple. These assays can be used to observe microbial interactions in co-culture, identify interaction patterns, and characterize the inhibitory potential of a microbial strain of interest against human and environmental pathogens.

The study of interactions between microorganisms has led to numerous discoveries, from novel antimicrobials to insights in microbial ecology. Many ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...