Toxoplasma Gondii: The Model Apicomplexan - Perspectives and Methods
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Numerous environmental cues initiate maturation and sporulation of shed oocysts, culminating in the production of two sporocysts, that each contains 4 sporozoites encased within a highly impermeable wall [ 10 ]. Sporulated oocysts can withstand harsh conditions and persist for extended periods in the environment [ 6 , 7 , 11 ]. Following ingestion, gastric enzymes and bile degrade the oocyst wall and infective sporozoites are released within the small intestine of an intermediate host where they rapidly invade enterocytes.
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Once inside the enterocyte, the non-replicating sporozoites convert to tachyzoites that swiftly replicate and disseminate to other organs and tissues [ 12 ]. Despite their critical role in transmission and initiation of new T. Notwithstanding these difficulties, comparative transcriptomic and proteomic analyses of T.
These studies showed that while all three infective forms of T.
Radke et al. Additionally, Poukchanski et al. Thus, by describing a sporozoite-specific transcriptome and proteome, these studies reaffirmed that although short-lived, sporozoites are biochemically and functionally distinct from tachyzoites and bradyzoites. These prior studies, however, did not examine the impact of sporozoites on the host cells they infect and they looked at the transcriptomes of sporozoites when they have only just completed development and before they have invaded a host cell.
In this report, we used an in vitro model of infection of the intestine to profile for the first time the host and parasite transcriptomes during infection with Toxoplasma sporozoites. We also show that these intracellular sporozoites are in an intermediate transcriptional state between freshly matured sporozoites day 10 and the tachyzoite form.
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Together, these findings broaden our understanding of the very first interactions of Toxoplasma sporozoites with its host and reveal genes that may mediate fundamental processes of this initial encounter. The Washington State University Institutional Animal Care and Use Committee reviewed and approved the animal protocols associated with the current studies. Efforts were made to minimize the numbers of animals used to generate Toxoplasma organisms.
The kittens used in the study remained healthy throughout. After two weeks of confirmed absence of shedding of Toxoplasma oocysts, the kittens were vaccinated and neutered, then adopted out to pre-screened and approved permanent homes. On the day of the experiment, 10 8 sporulated oocysts were washed three times in 1X PBS to remove sulfuric acid. The oocyst pellet was then resuspended in DMEM and transferred to a 1. Louis, MO. Excysted sporozoites were then washed twice in cold DMEM. Tachyzoites were also derived from M4 sporozoites from the same oocyst harvest described above.
Following egress, tachyzoites were passaged and maintained in culture in MA cells until used to infect IECs.
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Two independent experiments were performed with two technical replicates per experiment. The specific conditions for our study, depicted in Fig 1 , are described below. All experiments were performed in biological duplicate i. SAMseq analysis was used to identify differentially regulated genes of both host and parasite origin in various pairwise comparisons.
Note that infections with tachyzoites were done subsequently to the sporozoite infections after determination of the number of sporozoites excysted from oocyst harvest. To gain access to intracellular parasites, samples were washed three times as above and permeabilized with 0. Samples were washed once with 1X PBS for 5 min and then stained with polyclonal mouse antibody against T. At 8 hpi, 1 ml TRIzol reagent Invitrogen was added to each well. RNA extraction for all 24 samples was performed on the same day. Frozen samples were thawed on ice and equilibrated at room temperature.
RNA in the aqueous phase was transferred into a fresh tube and 0.
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Each tube was inverted three times and incubated at room temperature for 10 min. Supernatants were removed and the RNA pellets were air-dried in open tubes for approximately 10 min. All parameters were left at their default values. The number of reads that mapped to the R.
Many genes are so highly conserved across evolution that they have sequences that are almost identical between Toxoplasma and rat. This makes it difficult to know exactly which reads in a given sample from infected cells derive from the Toxoplasma vs. Because of this, we needed to identify and exclude such genes from our analysis.
To do this, we first searched the uninfected and mock-infected RNASeq data for reads mapping to the Toxoplasma genome; because these samples were uninfected, any such reads would indicate spurious matches. We then compared the number of such reads to the number for the same gene in the infected samples where Toxoplasma infection is present.
Genes with less than 5 exon reads mapping to the rat genome or to the parasite genome in all samples were excluded from further analysis. The number of total reads mapped to each genome after the adjustments described above was used to determine the RPKM Reads Per Kilobase of transcript per Million mapped reads , rounded to the nearest one-tenth value, as the relative expression for each rat and Toxoplasma gene in each sample Tables D and E in S1 File. SAMseq [ 27 ] package for the R platform was used to identify genes with significant changes between two samples.
Lastly, we manually curated the lists of differentially regulated host genes obtained at the end of this analysis pipeline to exclude host genes where the read number might be substantially influenced by Toxoplasma reads. This was done by first creating a merged file of the rat and Toxoplasma transcribed genomes. In practice, this resulted in excluding host genes encoding tubulin, actin, ABCB4, and Rack as genes where we could not eliminate the possibility that Toxoplasma mRNA was substantially contributing to the read number.
Metabolic Pathway Enrichment tool available on ToxoDB release 29 was used to determine enrichment in Toxoplasma differentially expressed gene sets. Following ingestion by an intermediate host, sporozoites are excysted and rapidly invade intestinal epithelial cells IECs [ 12 ].
To study the initial stages of oocyst-initiated Toxoplasma infection, we employed an in vitro model using the IEC cell line from rats and the experimental design depicted on Fig 1.
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As a source of parasites, we used sporulated oocysts from feces of kittens experimentally infected with Toxoplasma M4 strain type II [ 13 ]. The 8-hour infection timeframe was chosen to maximize invasion of the sporozoites but minimize conversion of the sporozoites to tachyzoites and replication once inside the cell; previous workers have shown that sporozoite-to-tachyzoite conversion occurs about 12 hours after infection as assessed by expression of tachyzoite-specific surface markers [ 34 ].
We used half the number of tachyzoites compared to sporozoites as preliminary experiments revealed that the infectivity of the sporozoites in these conditions is about half that of tachyzoites. Parallel infections on cover-slips and subsequent invasion assays Fig 2 revealed that the mass infections used for RNA preparation had actual multiplicities of infection for the sporozoites and tachyzoites of 0.
While not exactly the same, these were judged sufficiently close to allow comparison between the two datasets. Lastly, and as a further control for nonspecific effects, mock infections of the IECs were performed using syringed lysates from uninfected MA cells Mock. All infections and controls were performed in quadruplicate.
Representative fluorescent microscopy images of confluent IEC cells on glass coverslips infected with Toxoplasma type II M4 strain sporozoites and tachyzoites for 8 hours. Parasites were stained with either mouse or rabbit anti- Toxoplasma antibody before and after membrane-permeabilization to identify extracellular parasites red and intracellular parasites green. Images were obtained at 40X magnification.
RNA was extracted at 8 hpi for each of the six conditions depicted in Fig 1 and all 24 samples were submitted for RNA sequencing in a single lane using the NextSeq platform.
We independently mapped the sequenced reads to the genomes of Rattus norvegicus and the Toxoplasma type II Me49 strain Tables A-C in S1 File and identified differentially expressed genes for all pairwise comparisons. Details of data processing are outlined in the Materials and Methods. Given that the oocyst wall includes complex polysaccharides, proteins, and acid-fast lipids [ 37 ], the first pairwise analysis we performed was to check for possible pathogen-associated molecular patterns PAMPs in the oocyst-derived preparations of sporozoites.
The results showed no significant differences using the criteria described above Table F in S1 File and so we conclude that, at least in the conditions being used here, there are no major PAMPs detected by IEC cells in sporozoite preparations derived from oocysts. To determine the transcriptional changes that occur in IECs in response to sporozoite infection, we next compared the transcriptome of IEC cells infected with sporozoites to fzSPZ-exposed IECs although we saw no significant differences between the fzSPZ and the uninfected control, it was nevertheless the most appropriate control.
This indicates that infection with sporozoites does not trigger overwhelming transcriptional changes in IECs, at least at the time point used here, 8 hpi, and recognizing that the MOI was only 0. The most striking characteristic of this set of 26 genes Table 1 , however, is that gene set enrichment analysis GSEA from the Broad Institute [ 28 — 30 ] revealed that at least 21 of the 26 genes are involved in the host inflammatory responses Table 2.
Moreover, five of the 26 genes encode inflammatory chemokines, namely Ccl20 , Cxcl1 , Ccl2 , Ccl7 , and Csf1 , which showed an increase in expression relative to the fzSPZ controls ranging from approximately 2- Ccl7 and Csf1 to fold Ccl As the control for these infections we used IEC cells exposed to scraped, syringed lysates from uninfected cells i. The results showed host genes with significantly higher expression in IECs infected with tachyzoites relative to the mock infection, using the same criteria as described above for the SPZ analysis: 25 of these genes are the same as the genes that were higher in the SPZ-infected host cells as listed in Table 1 ; additional genes with the greatest up-regulation in the TZ-infected IECs not listed in Table 1 are shown in Table 3 while the remaining 52 of the affected genes are in Table G in S1 File.
This comparison revealed no host genes whose expression was significantly different between the two samples Table F in S1 File , confirming that the fzSPZ contains no major PAMPs that significantly affect the transcriptome of IECs, at least in the 8 hours of infection used here. The above results enabled us to next compare the host transcriptomic response from TZ-infection with that seen for SPZ-infection.
Specifically, we compared the host genes whose expression was altered by TZ-infection relative to the mock-infected control with the results of SPZ-infection relative to the fzSPZ control. The results showed that 25 of the 26 genes that were higher in the SPZ-infected samples relative to the fzSPZ controls described above are also among the genes significantly altered during TZ-infection Table 1.
Note that Rcan1 , which was only 1. Additionally, 38 of the 80 remaining genes with increased expression in cells infected with tachyzoites relative to Mock control also showed higher expression in the SPZ infection relative to the fzSPZ control, but not to a statistically significant degree. This set of 38 includes Cd69 , Cx3cl1 , and Traf1 , which are listed in Table 3.