Dual RNA-seq reveals no plastic transcriptional response of the coccidian parasite Eimeria falciformis to host immune defenses

Abstract

Background: Parasites can either respond to differences in immune defenses that exist between individual hosts plastically or, alternatively, follow a genetically canalized ("hard wired") program of infection. Assuming that large-scale functional plasticity would be discernible in the parasite transcriptome we have performed a dual RNA-seq study of the lifecycle of Eimeria falciformis using infected mice with different immune status as models for coccidian infections.

Results: We compared parasite and host transcriptomes (dual transcriptome) between naïve and challenge infected mice, as well as between immune competent and immune deficient ones. Mice with different immune competence show transcriptional differences as well as differences in parasite reproduction (oocyst shedding). Broad gene categories represented by differently abundant host genes indicate enrichments for immune reaction and tissue repair functions. More specifically, TGF-beta, EGF, TNF and IL-1 and IL-6 are examples of functional annotations represented differently depending on host immune status. Much in contrast, parasite transcriptomes were neither different between Coccidia isolated from immune competent and immune deficient mice, nor between those harvested from naïve and challenge infected mice. Instead, parasite transcriptomes have distinct profiles early and late in infection, characterized largely by biosynthesis or motility associated functional gene groups, respectively. Extracellular sporozoite and oocyst stages showed distinct transcriptional profiles and sporozoite transcriptomes were found enriched for species specific genes and likely pathogenicity factors.

Conclusion: We propose that the niche and host-specific parasite E. falciformis uses a genetically canalized program of infection. This program is likely fixed in an evolutionary process rather than employing phenotypic plasticity to interact with its host. This in turn might limit the potential of the parasite to adapt to new host species or niches, forcing it to coevolve with its host.

Keywords: Apicomplexa; Coccidia; Dual RNA-seq; Dual transcriptomics; Parasite lifecycle; Phenotypic plasticity; Transcriptional plasticity.

Fig. 1

Oocyst output and changes in intensity of E. falciformis infection in mouse. Oocyst counts in naïve and challenge infection are shown for three different mouse strains. For infection of naïve NMRI 150 oocysts were used, for challenge infection 1500 oocysts. For C57BL/6 and Rag1 ^−/− mice 10 oocysts were used in each infection. a Overall output of shed oocysts and (b) shedding kinetics are depicted. c RT-qPCR data of E. falciformis 18S in NMRI mice displays an increase in parasite mRNA over the course of infection. Significantly less parasite 18S transcripts (normalized against host transcripts of house-keeping genes) were detected in challenge infected mice. Formulas and prediction lines are given for linear models. d The percentage of parasite mRNA detected by RNA-seq increases during infection (shown for NMRI). More mRNA is detected in naïve mice compared to challenge infected mice. Sporozoites and oocysts contained ~100% parasite material

Fig. 2

Differentially abundant mouse mRNAs and clustering thereof. a Venn diagram visualizes the overlap between genes showing differential abundance (FDR < 0.01; edgeR glm likelihood-ratio tests) between i) uninfected controls and different time-points post infection and ii) between different time-points and the sum of all genes reacting to infection. Controls from challenge infection were used. b Hierarchical clustering of differentially abundant mRNAs performed on Euclidean distances using complete linkage. Cluster cut-offs (dendrogram resolution) were set to identify gene-sets with profiles interpretable in relation to the parasite lifecycle and between mice of different immune competence. Clusters are represented with color on the left-hand side of rows and additional numbering is used to refer to clusters (right)

Fig. 3

Correlations of E. falciformis mRNA abundance with ortholgues from other Coccidia. E.falciformis mRNA abundance was compared to that of orthologous genes of E. tenella [45, 46] and T. gondii [47]. Correlation coefficients (Spearman’s ρ) were clustered using complete linkage. T. gondii and Eimeria spp. “late infection” samples cluster together. E. falciformis early infection samples cluster with E. tenella merozoites. E. falciformis sporozoites cluster with E. falciformis early infection, whereas unsporulated oocysts cluster with E. tenella unsporulated oocysts

Fig. 4

Differentially abundant E. falciformis mRNAs and clustering thereof. a Venn diagram visualizes the overlap between genes showing differential abundance (FDR < 0.01; edgeR glm likelihood-ratio tests) between intracellular stages at 3 dpi, 5 dpi and 7 dpi. b Hierarchical clustering of abundance profiles for differentially abundant mRNAs performed on Euclidean distances using complete linkage. Cluster cut-offs (dendrogram resolution) were set to identify gene-sets with profiles interpretable in relation to the parasite lifecycle. Clusters are represented with color on the left-hand side of rows and additional numbering is used to refer to clusters (right)