Dual Transcriptomics To Determine Gamma Interferon-Independent Host Response to Intestinal Cryptosporidium parvum Infection

Abstract

Animals with a chronic infection of the parasite Toxoplasma gondii are protected against lethal secondary infection with other pathogens. Our group previously determined that soluble T. gondii antigens (STAg) can mimic this protection and be used as a treatment against several lethal pathogens. Because treatments are limited for the parasite Cryptosporidium parvum, we tested STAg as a C. parvum therapeutic. We determined that STAg treatment reduced C. parvum Iowa II oocyst shedding in gamma interferon knockout (IFN-γ-KO) mice. Murine intestinal sections were then sequenced to define the IFN-γ-independent transcriptomic response to C. parvum infection. Gene Ontology and transcript abundance comparisons showed host immune response and metabolism changes. Transcripts for type I interferon-responsive genes were more abundant in C. parvum-infected mice treated with STAg. Comparisons between phosphate-buffered saline (PBS) and STAg treatments showed no significant differences in C. parvum gene expression. C. parvum transcript abundance was highest in the ileum and mucin-like glycoproteins and the GDP-fucose transporter were among the most abundant. These results will assist the field in determining both host- and parasite-directed future therapeutic targets.

Keywords: Cryptosporidium parvum; RNA sequencing; STAg; Toxoplasma gondii; cecum; ileum.

FIG 1

Infection time course of C. parvum infected mice treated with STAg or PBS. (A) Oocyst shedding per dpi in experiment 1 over 14 dpi. *, P = 0.020. Oocyst shedding was calculated by qPCR, as described in Materials and Methods. (B) Oocyst shedding per dpi in experiment 2 over 14 dpi. **, P = 0.0058; *, P = 0.0457. Oocyst shedding was calculated by expression of Nluc, as described in Materials and Methods. Each circle represents a mouse. Open circles, uninfected animals; filled circles, infected animals. Statistic differences were determined using R with analysis of variance (ANOVA). The mean for each group is represented by a line. (C) Bioluminescent imaging of C. parvum in IFN-γ-KO mice throughout the intestinal tract: the intestinal tracts of mice infected for 9 days with Nluc-expressing C. parvum Iowa II strain at 1 × 10⁵ showed a higher signal in the ileum and cecum than the duodenum, jejunum, and colon. The radiance scale is shown.

FIG 2

Mouse experiment design and analysis for RNA sequencing. (A) For the RNA-seq analysis, 12 mice (6 infected and 6 noninfected) were treated with PBS or STAg. The treatment was administered 4 h before infection (2.5 × 10⁵ Nluc-expressing C. parvum Iowa II strain oocysts by oral gavage) and 1, 3, and 5 dpi. Fecal samples were collected every other day, and the infection was quantified by Nluc expression. IFN-γ-KO mice were euthanized on 9 dpi, and ileum and cecum samples were collected. (B) Oocyst shedding per dpi in experiment 3 over 9 dpi. Oocyst shedding was calculated by expression of Nluc, as described in Materials and Methods. Significance was determined using R with ANOVA, with P = 0.049. The mean for each group is represented with a line. (C) Relative quantification of C. parvum by qPCR using 18S rRNA primers P4 normalized to mouse GAPDH in ileum samples. Values are means from two independent experiments. Fold change was calculated relative to the reference PBS-infected group at day 6. Significance was determined using R with ANOVA. **, P = 0.0079 (infected STAg treated, day 6 versus day 9). Each circle represents a mouse. (D) RNA sequencing analysis flowchart listing the programs and packages used for reading and analysis.

FIG 3

PCA of ileum and cecum samples. PCA plot from normalized values calculated by DESeq2. Mapping of the cecum and ileum to the Mus musculus genome. Percentages in filled circles correspond to percentages of reads uniquely mapped to the C. parvum Iowa II genome up to one significant figure; for more detailed mapped percentages, see Table S1.

FIG 4

Host differentially expressed genes when comparing treatment (A) and infection (B). The color scale presents higher numbers of DEGs as darker shades of gray.

FIG 5

Host gene ontology (GO) of biological process in STAg- and PBS-treated mice. DEGs in comparisons 5 to 8 were analyzed for GO enrichment of biological process, using the Database for Annotation, Visualization, and Integrated Discovery (DAVID, v6.8). Only GO terms populated by 3 or more DEGs were included in these visualizations (P < 0.05). GO:0006919 stands for “activation of cysteine-type endopeptidase activity involved in apoptotic process.”

FIG 6

Heat map of differentially expressed genes in the ilea of mice treated with STAg (comparison 5). The top 25 more (A) and less (B) abundant transcripts (ranked by log₂ fold change). Each column represents a biological replicate.

FIG 7

Heat map of differentially expressed genes in the ileum of infected mice treated with STAg or PBS. Thirteen genes (ranked by log₂ fold change) were found to be differentially expressed in the ileum between infected mice treated with PBS and those with STAg. Each column represents a biological replicate.