Targeted Transcriptomic Analysis of C57BL/6 and BALB/c Mice During Progressive Chronic Toxoplasma gondii Infection Reveals Changes in Host and Parasite Gene Expression Relating to Neuropathology and Resolution

Toxoplasma gondii is a resilient parasite that infects a multitude of warm-blooded hosts and results in a lifelong chronic infection requiring continuous responses by the host. Chronic infection is characterized by a balanced immune response and neuropathology that are driven by changes in gene expression. Previous research pertaining to these processes has been conducted in various mouse models, and much knowledge of infection-induced gene expression changes has been acquired through the use of high throughput sequencing techniques in different mouse strains and post-mortem human studies. However, lack of infection time course data poses a prominent missing link in the understanding of chronic infection, and there is still much that is unknown regarding changes in genes specifically relating to neuropathology and resulting repair mechanisms as infection progresses throughout the different stages of chronicity. In this paper, we present a targeted approach to gene expression analysis during T. gondii infection through the use of NanoString nCounter gene expression assays. Wild type C57BL/6 and BALB/c background mice were infected, and transcriptional changes in the brain were evaluated at 14, 28, and 56 days post infection. Results demonstrate a dramatic shift in both previously demonstrated and novel gene expression relating to neuropathology and resolution in C57BL/6 mice. In addition, comparison between BALB/c and C57BL/6 mice demonstrate initial differences in gene expression that evolve over the course of infection and indicate decreased neuropathology and enhanced repair in BALB/c mice. In conclusion, these studies provide a targeted approach to gene expression analysis in the brain during infection and provide elaboration on previously identified transcriptional changes and also offer insights into further understanding the complexities of chronic T. gondii infection.

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Laser capture microdissection protocol for gene expression analysis in the brain

Histochemistry and Cell Biology ◽

10.1007/s00418-017-1585-1 ◽

2017 ◽

Vol 148 (3) ◽

pp. 299-311 ◽

Cited By ~ 6

Author(s):

P. Garrido-Gil ◽

P. Fernandez-Rodríguez ◽

J. Rodríguez-Pallares ◽

Jose L. Labandeira-Garcia

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Laser Capture Microdissection ◽

Gene Expression Analysis ◽

Laser Capture ◽

The Brain

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Time Course Global Gene Expression Analysis of an In VivoCandidaBiofilm

The Journal of Infectious Diseases ◽

10.1086/599838 ◽

2009 ◽

Vol 200 (2) ◽

pp. 307-313 ◽

Cited By ~ 110

Author(s):

Jeniel E. Nett ◽

Alexander J. Lepak ◽

Karen Marchillo ◽

David R. Andes

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Time Course ◽

Gene Expression Analysis ◽

Global Gene Expression ◽

Global Gene Expression Analysis

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Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

PLoS ONE ◽

10.1371/journal.pone.0238157 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0238157

Author(s):

Liz M. Florez ◽

Reiny W. A. Scheper ◽

Brent M. Fisher ◽

Paul W. Sutherland ◽

Matthew D. Templeton ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Reference Genes ◽

Time Course ◽

Gene Expression Analysis ◽

Virulence Genes ◽

Functional Characterization ◽

Post Inoculation ◽

In Planta ◽

Regulation Of Expression

European canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Reverse transcription quantitative real-time PCR (RT-qPCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate RT-qPCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

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Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

10.1101/2020.08.12.247601 ◽

2020 ◽

Author(s):

Liz M. Florez ◽

Reiny W. A. Scheper ◽

Brent M. Fisher ◽

Paul W. Sutherland ◽

Matthew D. Templeton ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Reference Genes ◽

Time Course ◽

Gene Expression Analysis ◽

Virulence Genes ◽

Functional Characterization ◽

Post Inoculation ◽

In Planta ◽

Qrt Pcr

AbstractEuropean canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Real-time quantitative reverse transcription PCR (qRT-PCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate qRT-PCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

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Proteomic and transcriptomic analyses of early and late-chronic Toxoplasma gondii infection shows novel and stage specific transcripts

BMC Genomics ◽

10.1186/s12864-019-6213-0 ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 4

Author(s):

Andrew L. Garfoot ◽

Gary M. Wilson ◽

Joshua J. Coon ◽

Laura J. Knoll

Keyword(s):

Toxoplasma Gondii ◽

Chronic Infection ◽

Time Course ◽

Protozoan Pathogen ◽

Toxoplasma Gondii Infection ◽

High Level ◽

Protein Rich ◽

The Brain ◽

Unique Ability

Abstract Background The protozoan pathogen Toxoplasma gondii has the unique ability to develop a chronic infection in the brain of its host by transitioning from the fast growing tachyzoite morphology to latent bradyzoite morphology. A hallmark of the bradyzoite is the development of neuronal cysts that are resilient against host immune response and current therapeutics. The bradyzoite parasites within the cyst have a carbohydrate and protein-rich wall and a slow-replication cycle, allowing them to remain hidden from the host. The intracellular, encysted lifestyle of T. gondii has made them recalcitrant to molecular analysis in vivo. Results Here, we detail the results from transcriptional and proteomic analyses of bradyzoite-enriched fractions isolated from mouse brains infected with T. gondii over a time course of 21 to 150 days. The enrichment procedure afforded consistent identification of over 2000 parasitic peptides from the mixed-organism sample, representing 366 T. gondii proteins at 28, 90, and 120 day timepoints. Deep sequencing of transcripts expressed during these three timepoints revealed that a subpopulation of genes that are transcriptionally expressed at a high level. Approximately one-third of these transcripts are more enriched during bradyzoite conditions compared to tachyzoites and approximately half are expressed at similar levels during each phase. The T. gondii transcript which increased the most over the course of chronic infection, sporoAMA1, shows stage specific isoform expression of the gene. Conclusions We have expanded the transcriptional profile of in vivo bradyzoites to 120 days post-infection and provided the first in vivo proteomic profile of T. gondii bradyzoites. The RNA sequencing depth of in vivo bradyzoite T. gondii was over 250-fold greater than previous reports and allowed us to identify low level transcripts and a novel bradyzoite-specific isoform of sporoAMA1.

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gEAR: gene Expression Analysis Resource portal for community-driven, multi-omic data exploration

10.1101/2020.08.28.272039 ◽

2020 ◽

Cited By ~ 1

Author(s):

Joshua Orvis ◽

Brian Gottfried ◽

Jayaram Kancherla ◽

Ricky S. Adkins ◽

Yang Song ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Gene Expression Analysis ◽

De Novo ◽

Marker Gene ◽

Rna Seq ◽

Data Types ◽

Multiple Species ◽

User Friendly ◽

The Brain

ABSTRACTThe gEAR portal (gene Expression Analysis Resource, umgear.org) is an open access community-driven tool for multi-omic and multi-species data visualization, analysis and sharing. The gEAR supports visualization of multiple RNA-seq data types (bulk, sorted, single cell/nucleus) and epigenomics data, from multiple species, time points and tissues in a single-page, user-friendly browsable format. An integrated scRNA-seq workbench provides access to raw data of scRNA-seq datasets for de novo analysis, as well as marker-gene and cluster comparisons of pre-assigned clusters. Users can upload, view, analyze and privately share their own data in the context of previously published datasets. Short, permanent URLs can be generated for dissemination of individual or collections of datasets in published manuscripts. While the gEAR is currently curated for auditory research with over 90 high-value datasets organized in thematic profiles, the gEAR also supports the BRAIN initiative (via nemoanalytics.org) and is easily adaptable for other research domains.

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Gene expression tomography

Physiological Genomics ◽

10.1152/physiolgenomics.00090.2001 ◽

2002 ◽

Vol 8 (2) ◽

pp. 159-167 ◽

Cited By ~ 14

Author(s):

VANESSA M. BROWN ◽

ALEX OSSADTCHI ◽

ARSHAD H. KHAN ◽

SANJIV S. GAMBHIR ◽

SIMON R. CHERRY ◽

...

Keyword(s):

Gene Expression ◽

Image Reconstruction ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Expression Patterns ◽

Brain Slices ◽

Axial Rotation ◽

Gene Expression Patterns ◽

Rnase Protection ◽

The Brain

Gene expression tomography, or GET, is a new method to increase the speed of three-dimensional (3-D) gene expression analysis in the brain. The name is evocative of the method’s dual foundations in high-throughput gene expression analysis and computerized tomographic image reconstruction, familiar from techniques such as positron emission tomography (PET) and X-ray computerized tomography (CT). In GET, brain slices are taken using a cryostat in conjunction with axial rotation about independent axes to create a series of “views” of the brain. Gene expression information obtained from the axially rotated views can then be used to recreate 3-D gene expression patterns. GET was used to successfully reconstruct images of tyrosine hydroxylase gene expression in the mouse brain, using both RNase protection and real-time quantitative reverse transcription PCR (QRT-PCR). A Monte-Carlo analysis confirmed the good quality of the GET image reconstruction. By speeding acquisition of gene expression patterns, GET may help improve our understanding of the genomics of the brain in both health and disease.

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