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Cryptosporidium parvum induces an endoplasmic stress response in the intestinal adenocarcinoma HCT-8 cell line.

ABSTRACT: Invasion of human intestinal epithelial cells (HCT-8) by Cryptosporidium parvum resulted in a rapid induction of host cell spermidine/spermine N(1)-acetyltransferase 1 (hSSAT-1) mRNA, causing a 4-fold increase in SSAT-1 enzyme activity after 24 h of infection. In contrast, host cell SSAT-2, spermine oxidase, and acetylpolyamine oxidase (hAPAO) remained unchanged during this period. Intracellular polyamine levels of C. parvum-infected human epithelial cells were determined, and it was found that spermidine remained unchanged and putrescine increased by 2.5-fold after 15 h and then decreased after 24 h, whereas spermine decreased by 3.9-fold after 15 h. Concomitant with these changes, N(1)-acetylspermine and N(1)-acetylspermidine both increased by 115- and 24-fold, respectively. Increased SSAT-1 has previously been shown to be involved in the endoplasmic reticulum (ER) stress response leading to apoptosis. Several stress response proteins were increased in HCT-8 cells infected with C. parvum, including calreticulin, a major calcium-binding chaperone in the ER; GRP78/BiP, a prosurvival ER chaperone; and Nrf2, a transcription factor that binds to antioxidant response elements, thus activating them. However, poly(ADP-ribose) polymerase, a protein involved in DNA repair and programmed cell death, was decreased. Cumulatively, these results suggest that the invasion of HCT-8 cells by C. parvum results in an ER stress response by the host cell that culminates in overexpression of host cell SSAT-1 and elevated N(1)-acetylpolyamines, which can be used by a parasite that lacks ornithine decarboxylase.

SUBMITTER: Morada M

PROVIDER: S-EPMC3798500 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Cryptosporidium parvum induces an endoplasmic stress response in the intestinal adenocarcinoma HCT-8 cell line.

Morada Mary M Pendyala Lakhsmi L Wu Gang G Merali Salim S Yarlett Nigel N

The Journal of biological chemistry 20130828 42

Invasion of human intestinal epithelial cells (HCT-8) by Cryptosporidium parvum resulted in a rapid induction of host cell spermidine/spermine N(1)-acetyltransferase 1 (hSSAT-1) mRNA, causing a 4-fold increase in SSAT-1 enzyme activity after 24 h of infection. In contrast, host cell SSAT-2, spermine oxidase, and acetylpolyamine oxidase (hAPAO) remained unchanged during this period. Intracellular polyamine levels of C. parvum-infected human epithelial cells were determined, and it was found that ...[more]

PMID: 23986438

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Project description:• The origin of biological samples (In vitro infection of HCT-8 cells with Cryptosporidium parvum) Monolayers of the HCT-8 cells (ATCC CCL-244, American Type Culture Collection) were cultured in RPMI 1640 medium containing 10% fetal bovine serum and additional nutritional supplements. Cells in log phase were plated at 2×106 cells per 150 mm tissue culture dish and infected with sterilised Cryptosporidium parvum oocysts (Iowa strain). Purified oocysts were suspended in phosphate buffered saline, sterilised in 33% bleach for 7 min on ice, washed in Hank's buffered saline solution, and added to HCT-8 cultures at a ratio of one oocyst per cell. Following a 2 h excystation period at 37 °C, cells were washed with warm Hank's buffered saline solution, and cells were incubated in fresh supplemented media. Mock-infected cultures were treated identically, with the exception that oocysts were not added to the cultures. Three independent mock- and C. parvum-infected cultures were prepared for analysis. • The origin of mRNA samples At 6, 12, 24, 48, and 72h post-inoculation, cell culture media were removed and cultures were immediately lysed by the addition of 3.5 ml of Trizol® reagent (GIBCO BRL Life Technologies) directly to the culture plate. Total RNA was prepared as described by the manufacturer, and poly(A) mRNA was isolated using oligo-dT cellulose columns (Amersham Pharmacia Biotech). The qualities of total RNA and poly(A) mRNA preparations were assessed by Northern blot. • Protocols for conducting microarray hybridization Poly(A) mRNA was converted into ‘target’ suitable for hybridisation to Affymetrix microarray chips according to protocols provided by Affymetrix Inc. (Santa Clara, CA). Briefly, 2.0 g of poly(A) mRNA was reverse-transcribed to prepare double-stranded cDNA using T7-(dT)24 primer (GENSET Corp., La Jolla, CA) and Superscript II reverse transcriptase (GIBCO). Approximately 1 g of cDNA was used for in vitro transcription in the presence of biotinylated UTP and CTP using the Enzo BioArray High Yield RNA Transcript Labelling Kit (Enzo Diagnostics, Inc). This labelled cRNA was fragmented in 40 mM Tris–acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate at 94 °C for 35 min. The integrity of cRNA and the efficiency of fragmentation were monitored by Northern blot analysis (not shown). Fragmented biotinylated cRNA targets were hybridised to HG-U95Av2 chips (Affymetrix) that contain probe sets for 12,600 human gene/transcripts. Fifteen micrograms of fragmented cRNA and appropriate controls were hybridised to the chips at 45 °C for 16 h with constant rotation at 60 rev./min. The chips were subsequently washed and stained with streptavidin–phycoerythrin conjugate using the GeneChip Fluidics station protocol EukGE_WS2 (Affymetrix). Following washing and staining, microarray chips were scanned twice at 3 m resolution using a Hewlett-Packard confocal scanner and hybridisation intensities for each of the genes/transcripts were collected from scanned images. • Analysis of microarray data Gene expression data were initially analysed with the GeneChip® expression analysis software (Affymetrix Microarray Suite, version 5.0). The fluorescence intensity of the genes/transcripts was measured for each probe array and, to minimise discrepancies due to non-biological variations, normalised by global scaling to 1000. Further data analyses were performed using GeneSpring software (version 6.0; Silicon Genetics, Redwood City, CA). Per gene normalisation was applied in which the expression signal of each gene in C. parvum-infected cells (raw data) was normalised to the median of its measurements in the mock-infected samples (control); and the ratio of expression levels between mock and infected samples was calculated as the mean value of normalised signal vs. control signal among three replicates. To identify genes with significantly altered expression, a series of statistical analyses (filtering) were performed: cut-off values for ratio of expression levels 1.80 and 0.55 were used to filter genes with expression level fold changes greater than ±1.8 in all three independent samples. Genes with fold change variations >1.5 across the three samples were excluded. Furthermore, a ‘statistical group comparison’ using Student's t-test/ANOVA was conducted to compare mean expression levels between mock-infected and infected samples, and the genes with significant differential expression (P<0.05) were selected. Identified differentially expressed genes were then annotated using GeneSpring's ‘Build Simplified Ontology’ constructor which hierarchically groups genes into meaningful biological categories (gene lists) based on the Gene Ontology Consortium Classifications. Various lists of regulated genes were created by cross-referencing annotated gene lists and applying assorted statistical and visualisation methods. Keywords: time-course

Project description:Purpose: Transcriptome profiling of Crytosporidium parvum infected lung and small intestinal organoids was performed to access the response of epithelial cells upon parasitic infection and to do a temporal analysis of the transcriptome of the parasite inside the organoid lumen. We isolated RNA from infected human lung and small intestinal organoids at 24 and 72 hour post infection. Methods: Organoids were grown in expansion or differentiation media and microinjected with equal amounts of Cryptosporidium oocysts. Media-injected organoids were used as a control .Expanding SI organoids were microinjected at 5-6 days after seeding, differentiated SI organoids were injected at 5 days after inducing differentiation. Lung organoids were incubated for 2 weeks after seeding for microinjection. RNA was extracted from 1-2 matrigel drops containing organoids. RNA was converted to cDNA and libraries were prepared using the CelSeq2 method and sequenced. Samples were sequenced on Illumina NextSeq500 by using 75-bp paired-end sequencing. Methods: Paired-end reads from Illumina sequencing were aligned to the human transcriptome genome and C. parvum transcriptome genome (Iowa strain) by BWA. DeSeq (v1.18.0) was used for read normalization and differential expression analysis (p-value adjustment 0.05 by method Benjamini-Hochberg). Gene set enrichment analysis (GSEA) was performed using gene lists for type I interferon response and regulation against normalized RNA-seq reads of injected SI and lung organoids using GSEA software v3.0 beta2. Results: At 24 hr post-infection,GO (gene ontology)-term analysis revealed that a substantial number of genes related to ‘cytoskeleton’ and ‘cell mobility’ were up-regulated in lung organoids. This suggests that infection by the parasites and subsequent formation of the intracellular stages within 24 hrs might affects cytoskeleton structures of host cells. After 72 hrs, many genes associated with the type I interferon pathway increased dramatically in lung and intestinal organoids. Results: After 72 hrs, many genes associated with the type I interferon pathway increased dramatically in lung and intestinal organoids. Multiple C. parvum genes were differentially expressed with a large fold change between 24 and 72 hr post-injection.At 24 hr post-infection, most of the enriched genes represented ribosomal proteins and ribosomal RNA subunits in both intestinal organoids and lung organoids. By contrast, at 72 hr post-infection, multiple oocyst-wall protein genes were up-regulated, confirming that the parasites formed new oocysts within the organoids. Conclusions: RNA sequencing of injected organoids revealed host epithelial responses upon parasite infection in differentiated SI organoids as well as in lung organoids.Upregulation of genes associated with type I interferon immunity in both SI and lung organoids.

Dataset Information

Cryptosporidium parvum induces an endoplasmic stress response in the intestinal adenocarcinoma HCT-8 cell line.

Publications

Cryptosporidium parvum induces an endoplasmic stress response in the intestinal adenocarcinoma HCT-8 cell line.

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