Asexual expansion of Toxoplasma gondii merozoites is distinct from tachyzoites and entails expression of non-overlapping gene families to attach, invade, and replicate within feline enterocytes

Abstract

Background: The apicomplexan parasite Toxoplasma gondii is cosmopolitan in nature, largely as a result of its highly flexible life cycle. Felids are its only definitive hosts and a wide range of mammals and birds serve as intermediate hosts. The latent bradyzoite stage is orally infectious in all warm-blooded vertebrates and establishes chronic, transmissible infections. When bradyzoites are ingested by felids, they transform into merozoites in enterocytes and expand asexually as part of their coccidian life cycle. In all other intermediate hosts, however, bradyzoites differentiate exclusively to tachyzoites, and disseminate extraintestinally to many cell types. Both merozoites and tachyzoites undergo rapid asexual population expansion, yet possess different effector fates with respect to the cells and tissues they develop in and the subsequent stages they differentiate into.

Results: To determine whether merozoites utilize distinct suites of genes to attach, invade, and replicate within feline enterocytes, we performed comparative transcriptional profiling on purified tachyzoites and merozoites. We used high-throughput RNA-Seq to compare the merozoite and tachyzoite transcriptomes. 8323 genes were annotated with sequence reads across the two asexually replicating stages of the parasite life cycle. Metabolism was similar between the two replicating stages. However, significant stage-specific expression differences were measured, with 312 transcripts exclusive to merozoites versus 453 exclusive to tachyzoites. Genes coding for 177 predicted secreted proteins and 64 membrane- associated proteins were annotated as merozoite-specific. The vast majority of known dense-granule (GRA), microneme (MIC), and rhoptry (ROP) genes were not expressed in merozoites. In contrast, a large set of surface proteins (SRS) was expressed exclusively in merozoites.

Conclusions: The distinct expression profiles of merozoites and tachyzoites reveal significant additional complexity within the T. gondii life cycle, demonstrating that merozoites are distinct asexual dividing stages which are uniquely adapted to their niche and biological purpose.

Figure 1

Experimental infection, parasite isolation, and RNA preparation. A) Immunofluorescence assay using sheep immune serum against T. gondii revealed numerous infected enterocytes. Nuclei are counterstained with DAPI. Shown in the top panel is a 20x magnification of a section of the small intestine (bar = 100 μM) where villi are visible. The bottom panel at 100x magnification shows a schizont containing several merozoites (scale bar = 5 μm). B) Enterocytes containing CZ-strain merozoite stages were stripped away selectively, leaving the villus structure and the cells of the lamina propria intact. Histology section (Hematoxilin & Eosin stained) showing stripped villi at day 5 post infection. C) Microscopic examination of parasites in the detergent washed preparation showed only merozoite stages. D) Quality control of total RNA extracted from parasite preparations separated on an Agilent RNA 6000 Pico Chip. The bands generated by host 28S/18S ribosomal RNA (arrowheads) and parasite 26S/18S ribosomal RNA (arrows) as well as a size marker are indicated. The samples analyzed were: raw, unprocessed material from scraped intestinal lining; Tween 80, material that was syringe-passaged and washed twice with PBS/0.05% Tween 80; Percoll, highly enriched parasite fraction after detergent treatment and Percoll gradient centrifugation; Tachy, RNA prepared from tachyzoites grown in cell culture with human foreskin fibroblasts as host cells.

Figure 2

Global comparative transcriptome analysis. A) Scatterplot depicting expression levels as mapped read (DESeq values) of 8323 identified genes in CZ tachyzoites (Tz) and merozoites (Mz). The threshold for stage-regulated expression was set at ≥8-fold difference. B) Pie charts show parsing of 453 tachyzoite and 312 merozoite stage-regulated genes into functional categories.

Figure 3

Differential expression of SRS s and genes coding for secretory organelle (microneme, rhoptry, dense granule) proteins. Bar graphs indicate –fold difference in mRNA levels (DESeq mapped reads); red (tachyzoite), green (merozoite).

Figure 4

Chromosomal distribution and relative expression levels of SRS, GRA, and T. gondii protein A and D families. The chromosomal position and distribution of SRS (◊), GRA (⎔) and T. gondii protein A and D families (Δ) that are differentially expressed between merozoites and tachyzoites is displayed. Tandemly repeated genes are shown as clusters. Uncoloured genes were not expressed in either tachyzoite or merozoite >stage (RPKM <10). Black coloured genes were not differentially expressed between tachyzoites and merozoites. The shade of red (induced in tachyzoites relative to merozoites) or green (induced in merozoites relative to tachyzoites) indicated the fold increase in expression relative to the other life cycle stage. The chromosomal position of SRS pseudogenes is not displayed. The majority of GRA genes were upregulated in tachyzoites. Only GRA11 gene expression was specifically induced in merozoites. The majority of SRSs were upregulated in merozoites. 52 SRS genes were upregulated in merozoites whereas only 14 were upregulated in tachyzoites. Genes in each cluster tended to be coordinately regulated according to life cycle stage, with only 4 exceptions: One gene in each of the SRS16, SRS36, T. gondii Family A (Chromosome XII) and T. gondii Family D (Chromosome XI) clusters was upregulated in tachyzoites, relative to merozoites.

Figure 5

Differential expression of three MIC escorter/adhesin complexes. Expression values are indicated as [RPKM], asterisks: genes coding for MIC escorter proteins.

Figure 6

Genes coding for components the tachyzoite moving junction invasion machinery are expressed at significantly lower levels in merozoites. Cartoon depicting the RON and AMA protein assembly at the moving junction complex (left); bar graph showing expression (RPKM values) of components and paralogs in tachyzoites and merozoites. Mt, microtubules; PM, host cell plasma membrane.

Figure 7

Identification of cis regulatory elements in the predicted promoters of merozoite- and tachyzoite-specific genes. (A) Enriched six-base motifs were identified within the predicted promoters of merozoite- and tachyzoite-specific genes. Listed for both stages are the five most significantly enriched motifs, their occurrence, expected occurrence, and occurrence significance. Asterisks indicate motifs overlapping with the putative cis regulatory elements for promoters of merozoite- and tachyzoite-specific genes, shown in (B) and (C), respectively. (D) Percentage of promoters from merozoite- or tachyzoite-specific genes that contain either the GAAGAAA or GAGACGC putative cis elements. (E) Distribution of the GAGACGC cis element (blue) within the promoters of the fifteen most tachyzoite-specific genes. The predicted promoters correspond to the 500 bp genomic region directly upstream of the transcriptional start site, indicated by an arrow.