The transcriptome from asexual to sexual in vitro development of Cystoisospora suis (Apicomplexa: Coccidia)

Abstract

The apicomplexan parasite Cystoisospora suis is an enteropathogen of suckling piglets with woldwide distribution. As with all coccidian parasites, its lifecycle is characterized by asexual multiplication followed by sexual development with two morphologically distinct cell types that presumably fuse to form a zygote from which the oocyst arises. However, knowledge of the sexual development of C. suis is still limited. To complement previous in vitro studies, we analysed transcriptional profiles at three different time points of development (corresponding to asexual, immature and mature sexual stages) in vitro via RNASeq. Overall, transcription of genes encoding proteins with important roles in gametes biology, oocyst wall biosynthesis, DNA replication and axonema formation as well as proteins with important roles in merozoite biology was identified. A homologue of an oocyst wall tyrosine rich protein of Toxoplasma gondii was expressed in macrogametes and oocysts of C. suis. We evaluated inhibition of sexual development in a host-free culture for C. suis by antiserum specific to this protein to evaluate whether it could be exploited as a candidate for control strategies against C. suis. Based on these data, targets can be defined for future strategies to interrupt parasite transmission during sexual development.

Figure 1

Identification of upregulated and downregulated genes in sexual stages using differential expression analysis. (a) Summary of the differential expression analysis of early sexual stages compared to merozoites (T1-T2), late sexual stages (T1-T3) and late sexual stages compared to early sexual stages (T2-T3) showing the number of genes up or down-regulated of all predicted C. suis. (b) Venn diagrams showing the overlap between the genes that were up- and down-regulated in early and late sexual stages compared with asexual stages. A total of 443 upregulated and 689 downregulated genes were identified in this overlapping region. (c) Summary of total number of genes (937 upregulated and 1188 downregulated) in early and late sexual stages.

Figure 2

Verification of the gene expression profiles by qRT-PCR. Six genes were selected randomly for validation of the RNA-seq data. According to the RNA-seq results, the expression levels of CSUI_008252, CSUI_003709, CSUI_000190 were upregulated at T2 and T3, and the expression levels of CSUI_005927, CSUI_003422 and CSUI_006265 were downregulated at T2 and T3. Glyceraldehyde-3-phosphate and actin were used for normalization. Values represent the mean ± standard deviation (SD). Asterisks represent significant difference (*P < 0.05, **P < 0.01***, P < 0.001, ****P < 0.0001).

Figure 3

Gene ontology (GO) analysis of differently expressed genes. Differentially expressed genes (DEGs) are classified into three main categories: biological process (BP), molecular function (MF) and cellular component (CC). The identified functions for each corresponding numbers GO category are shown in supplemental file 2.

Figure 4

Biological functions of proteins coded by downregulated (a) and upregulated (b) genes. The biological functions of proteins coded by each of the 1188 down- and 937 upregulated transcripts was assigned manually based on ToxoDB®, Blast2Go® or previously published annotations. The pie charts represent the relative proportion of these different biological functions within up- and down regulated genes.

Figure 5

Localization of CSUI_001473 antigens in differents C. suis stages. (a) Merozoite from day 6 of in vitro culture. (b) Microgamont from day 9 of culture. (c) Macrogamont from day 9 of culture. (d) Unsporulated oocyst from day 14. (e) Sporulated oocyst ex vivo (isolated from the feces of experimentally infected piglets). (f) Sporozoite released from in vitro excysted oocysts. DIC, differential interference contrast microscopy; DAPI staining appears in blue; green indicates binding of anti-rCSUI-001473 antibodies, and turquoise indicates merged results. Scale bar = 10 μm.

Figure 6

Serum Inhibition assay. (a) Inhibition rates for asexual, early (mostly gamonts) and late (mostly gametes) sexual stages of C. suis in a host-cell free culture 0–4 days after transfer of merozoites. (b to d) Total numbers of counted stages by day of cultivation in host-cell free culture. n.s.: not singificant, *: P ≤ 0.05, **: P ≤ 0.01. Values represent the mean ± standard deviation (SD) from three independent experiments. n.s.: not singificant, *: P ≤ 0.05, **: P ≤ 0.01.