Toxoplasma gondii merozoite gene expression analysis with comparison to the life cycle discloses a unique expression state during enteric development
© Behnke et al.; licensee BioMed Central Ltd. 2014
Received: 31 December 2013
Accepted: 2 May 2014
Published: 8 May 2014
Considerable work has been carried out to understand the biology of tachyzoites and bradyzoites of Toxoplasma gondii in large part due to in vitro culture methods for these stages. However, culturing methods for stages that normally develop in the gut of the definitive felid host, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite’s life cycle. Here, we begin to unravel the molecular aspects of enteric stages by providing new data on merozoite stage gene expression.
To profile gene expression differences in enteric stages we harvested merozoites from the intestine of infected cats and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by comparing it to previously published data for the oocyst, tachyzoite, and bradyzoite stages. Principal component analysis highlighted the unique profile of merozoites, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen one (SAG1) and many dense granule proteins do not label merozoites: our microarray data confirms that these genes were not expressed at this stage. Also, the expression for many rhoptry and microneme proteins was drastically reduced while the expression for many surface antigens was increased at the merozoite stage. Gene Ontology and KEGG analysis revealed that genes involved in transcription/translation and many metabolic pathways were upregulated at the merozoite stage, highlighting unique growth requirements of this stage. To functionally test these predictions, we demonstrated that an upstream promoter region of a merozoite specific gene was sufficient to control expression in merozoites in vivo.
Merozoites are the first developmental stage in the coccidian cycle that takes place within the gut of the definitive host. The data presented here describe the global gene expression profile of the merozoite stage and the creation of transgenic parasite strains that show stage-specific expression of reporter genes in the cat intestine. These data and reagents will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate enteric development.
KeywordsToxoplasma gondii Merozoite Enteric stages Definitive host Life cycle Parasite Gene expression
Intracellular parasites represent a significant portion of human disease burden throughout the world. The apicomplexan parasite Toxoplasma gondii is one of the most successful intracellular parasites and it is estimated up to a third of the human population has been infected . This high infection rate results in approximately 1.5 million new infections in the U.S. per year. Fortunately, most infections do not result in debilitating symptoms as individuals with healthy immune systems are able to control the growth of the parasite, yet they are generally not able to eliminate chronic infection. Toxoplasmosis has been an indicator disease for patients suffering from complications of AIDS since the advent of the HIV viral epidemic . Unborn babies can become infected in mothers who convert during pregnancy, resulting in 400–4,000 new congenital toxoplasma infections in the U.S. per year . Also, the parasite can thrive in immune privileged areas of the eye, resulting in approximately 5,000 symptomatic ocular toxoplasmosis cases in the U.S. every year . Major routes of human infection are via either the ingestion of undercooked infected meat or the accidental ingestion of oocysts shed into the environment, for example, by gardening or cleaning cat litter. Recent estimates by the CDC indicate that of known etiological agents, toxoplasmosis is the fourth leading cause of hospitalization and the second leading cause of death by foodborne illness in the U.S today . The ability of Toxoplasma to infect such a large number of individuals, approximately 30 million in the U.S., results in meaningful disease burden in those individuals where the parasite circumvents normal modes of control .
Much work has been carried out to understand the biology of the asexual stages, tachyzoites and bradyzoites, which occur in the intermediate host. One reason for this is the relative ease in culturing these stages in the laboratory, both in tissue culture and the mouse model. Tachyzoites grow well in various cell lines, such as human foreskin fibroblasts (HFFs), and bradyzoites can be induced in vitro [12, 13] via cellular stresses [14, 15] or in vivo. These culturing methods have allowed researchers to develop an extensive molecular toolbox for Toxoplasma, resulting in an increased understanding of its biology including host cell invasion and egress , how the parasite modulates the intracellular environment , and the identification and characterization of virulence factors that allow the parasite to evade host innate immune defenses . Culturing methods for stages beyond the bradyzoite, the merozoite and sexual stages, have not been developed, hindering the ability to study a large portion of the parasites life cycle and restricting such work to a few laboratories with the resources to house cats.
There are several reports describing various aspects of the enteric stages of T. gondii within felid intestinal cells [19–22]. These studies used electron microscopy to observe ultrastructural features or performed immunohistochemistry (IHC) to determine the labeling of parasites with various antibodies, useful in characterizing the expression of a limited number of proteins in enteric stages. Another report demonstrated the feasibility of isolating merozoite stage parasites from the intestinal mucosa of infected cat intestine  and showed these parasites illicit unique antigenic responses . Based on this report, we undertook isolation of merozoites for genome wide expression profiling study in order to identify merozoite-specific genes.
Isolation of merozoite stage parasites and mRNA hybridization
To provide insight into gene expression of the enteric stage, we harvested intestines of three cats (cat numbers: c48, c50, and c52) infected with the type II parasite, TgNmBr1 . The harvesting process resulted in the removal of the majority of host cells and debris, leaving crescent shaped merozoite parasites. To determine that the purified parasites were indeed T. gondii, a portion of the sample was fixed and stained with sera from mice immunized with type II Me49 parasites (Figure 1B). Indeed, the crescent shaped purified parasites were labeled with the Toxoplasma specific antibody. Purified parasites from all three felid intestines were processed for mRNA isolation and hybridized to the Affymetrix Toxoplasma GeneChip  (array sample names: Mc48, Mc50, and Mc52). In order to place the newly acquired merozoite gene expression in context of the life cycle, the data were analyzed in combination with a recently published dataset by Fritz HM et al. 2012  covering day 0, 4, 10 oocyst (array sample names: OD0, OD4, OD10), day 2 tachyzoite (TD2), and day 4, 8, 21 bradyzoite (BD4, BD8, BD21) development (Figure 1C). Although the parasites used in the present study (TgNmBr1 strain) and the Fritz HM et al. study  (M4 strain) are both clonal type II strains, they are of different origins; the former is from a guinea fowl from Brazil, and the latter is from a sheep in United Kingdom. To control for any strain specific differences in identifying merozoite specific genes, we also hybridized mRNA from TgNmBr1 tachyzoites (TNm). Plotting of RMA normalized values for all the samples used in the analysis demonstrated that the medians of the distributions converge and that the ranges of extreme values across all samples were similar, lending weight that the samples from the current study and the Fritz HM et al.  study are comparable (Figure 1C).
Life cycle gene expression analysis
To determine the major trends across the samples we conducted principal component analysis (PCA) that highlighted the uniqueness of the merozoite samples (Figure 2B), clustering them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. The distinct grouping of the merozoite samples was not the result of strain specific differences as the tachyzoite TgNmBr1 strain sample (TNm), the same strain used to generate the merozoite parasites, grouped with the tachyzoite M4 strain Day 2 sample (TD2) Interestingly, the PCA analysis revealed that compared to the merozoite and oocyst stages, the tachyzoite and bradyzoite stages were quite similar and they all group together. Although studies have shown there are gene expression differences between these two stages [28–30], the PCA of global expression profiles indicates that bradyzoites resemble dormant tachyzoites when taken in context of the whole life cycle. Lastly, the circular progression of samples, from the OD0 to OD4/OD10 cluster, to the tachyzoite/bradyzoite cluster, and finally to the merozoite cluster, follows the progression of the life cycle (Figures 1A and 2B).
We identified life cycle regulated genes by performing an analysis of variance test (ANOVA p-value cutoff .05) on genes with expression above background levels in at least one sample. This analysis resulted in the identification of 5,969 genes, a large proportion (73.4%) of the genes in the Toxoplasma genome. There were 1,571 non-expressed genes (19.3%) for which expression was at or below background in all samples, and 591 non-regulated genes (7.3%) that were not significantly differentially expressed between the samples. Although the life cycle regulated gene set (5,969) captures most of the T.gondii genome, a fifth of the genes represented on the array did not have detectable expression. Some of these non-expressed genes may represent stage specific genes for stages which we do not have expression data, such as the micro- and macrogametes.
We also looked at the expression of parasite-specific gene families in the merozoite stage. As seen in Figure 2A, many of the annotated GRA genes (50%) are not expressed at the merozoite stage (Figure 3C). Genes in two other parasite specific gene families, the rhoptry (ROP) (61%) and microneme (MIC) (60%), similarly lacked expression in the merozoite stage. For example, none of the annotated rhoptry neck (RON) or apical membrane (AMA) genes, important for parasite invasion in intermediate stages, were expressed at the merozoite stage. One RON paralog, RON4L1 , had slight expression in the merozoite, but the expression was downregualted as compared to the tachyzoite/bradyzoite stages. Likewise, genes that have been shown to be important for host immune evasion, such as ROP18, ROP5, ROP16, GRA15, and GRA24 [18, 32], were not expressed. Although essentially half of the genes in these gene families were not expressed as merozoites, others had low constitutive expression, and a few were upregulated. Those that exhibited upregulated expression include: GRA family (DG32 antigen, DG32 protein, GRA12 and NTPase I), ROP family (ROP21, ROP32, ROP33, ROP36, TGME49_281790 (kinase), and TGME49_249470 (rhoptry kinase)) and MIC family (TGMe49_200270 (PAN/Apple domain), TGME49_275800 (SRP72), TGME49_286150 (PAN/Apple domain), and TGME49_254430 (microneme)), but even most of these were low abundance expressed genes. See Additional file 2 for specific genes and expression values. The downregulated expression state upon entering the enteric stages for many members of these gene families is unexpected as many have been shown to play a role in host-parasite interactions in intermediate hosts, for example invasion  or host immune modulation/resistance , and suggests they are not needed during enteric development. Converse to the GRA, ROP, and MIC families, many members of the parasite surface antigen gene family (SRS) were upregulated (44.8%) upon differentiating into the first of the enteric stages as a merozoite (Figure 3C). Although developmentally regulated expression of various SRS genes has been shown for other stages, in comparison, quite a large number were upregulated at the merozoite stage. Members of the SRS gene family are involved in parasite adhesion, invasion, and virulence, possible roles they may have in allowing the parasite to develop effectively in the cat intestine .
Timing and control of life cycle gene expression
By calculating the maximum and minimum values of the first derivate (f’(x)) of the spline curve, one can determine the inflection points in the graph which correspond to the points of maximum rate of synthesis and degradation. For example, GRA1 (Figure 4A) had an upwardly trending inflection point just after the OD0 sample corresponding to the maximum rate of synthesis just before the peak of expression between OD4 and OD10, and a downward trending inflection point between the BD8 and Mc52 samples indicating the maximum rate of degradation just before the gene is no longer expressed in the merozoite. There was a wave of synthesis throughout the life cycle starting with entry into the merozoite stage, Mc52, steadily tapering through the oocyst stages (Figure 4C). This progression largely mirrored the maximum expression distribution where maximum rates of synthesis (Figure 4C) precede maximum points of expression (Figure 4B) by half a stage. The distribution seen for maximum rate of synthesis was shifted one stage down the life cycle when genes were ordered by maximum rate of degradation (Figure 4C), reflecting the peak of expression that occurred between synthesis and degradation. Lastly, we determined the time, or number of stages, to maximum or minimum expression by subtracting the maximum or minimum expression value (Figure 4B) for each regulated gene from the corresponding rate value (Figure 4C). For the upregulation of gene expression, most genes reached peak expression very rapidly, within one stage (Figure 4D), indicating that once the cell commits to the rapid synthesis of a gene the expression peaks quickly in relation to the life cycle. This was also a result of the stage-specific expression for many genes. When genes were downregulated, many genes reached their minimum of expression rapidly, but there was also a group of genes that had a delayed minimum after the greatest rate of degradation, approximately four stages later (Figure 4D). This delayed grouping indicates that the minimum of expression for a large number of genes was just before the upregulation of that gene, as there are only 6 stages represented in this analysis.
Promoter control of merozoite specific expression
Here we describe the global gene expression of the merozoite stage of Toxoplasma gondii and analyzed this stage in context of the life cycle in combination with previously published array data from other life cycle stages . We confirm that the expression patterns observed for the merozoite samples matched data on protein expression for enteric stage parasites . We also substantially extend these prior studies by showing that many parasite specific gene families such as GRAs, ROPs and MICs were downregulated at the merozoite stage. Functions for many members of these families have been described for the intermediate stages of the parasite, and the regulation at the merozoite stage suggests that these genes are not needed during intraepithelial development in the definitive host. Interesting, genes known to be critical for moving junction formation and thus parasite invasion during the intermediate stages are not expressed in the merozoite. Although not much is known about how merozoites invade, the shared morphological characteristics of tachyzoites and merozoites suggests an active invasion process, which based on the expression data is not reliant on known RON/AMA interactions. Speciation of apicomplexan parasites may have occurred via evolutionary divergence of definitive hosts [44, 45]. Many members of the parasite-specific gene families are the result of gene duplication and expansion events, and those few members that are upregulated at the merozoite stage, the first stage the parasite differentiates into within the definitive host, may represent the ancestral copies from which the expanded intermediate stage expressed genes arose.
The merozoite occupies a unique place in the overall gene expression continuum of the T. gondii life cycle, clustering between tachyzoite/bradyzoite and oocyst samples as shown by PCA. It will be of interest to isolate other enteric forms of Toxoplasma, such as the micro- and macrogamete for global gene expression studies to determine if the missing forms will follow this progression, thus being placed between the merozoite and oocysts clusters. Additionally, a large number of genes were regulated when the parasites enter coccidian development. Many of these genes related to cell growth/maintenance (Translation and Transcription GO categories) and metabolic processes (Glycolysis and TCA Cycle GO categories) were upregulated at the merozoite stage. Different than the intermediate stages, the merozoite divides by processes similar to schizogony termed endopolygeny, where multiple daughters are generated before the plasma membrane resolves the individual parasites . It is possible that the merozoite specific upregulation of cell growth and metabolism related genes is a consequence or even a requirement for the endopolygeny form of division. On the other hand, the elevated expression of growth and metabolism related genes is reminiscent of the Warburg effect, where, for example, cancer cells within hypoxic tumor microenvironments increase expression of metabolism related genes, such as glycolysis . The merozoite preferably grows in the microaerobic environment of the small intestine and it may be the hypoxic nature of the small intestine  that partially explains the unique expression pattern of growth and metabolic related genes in the merozoite.
We also demonstrate that promoter control of gene expression is conserved at the merozoite stage. Quite a number of conserved motifs were found in co-regulated genes, whether when ordering genes by the maximum or minimum of expression. Identifying conserved over-represented motifs by ordering genes in this manner may indicate regulatory control for a particular motif. For example, the large number of motifs overrepresented throughout the life cycle when genes were ordered by the minimum of expression indicates that these motifs correspond to points in the life cycle when expression is at the lowest and thus may be associated with repressor mechanisms. Indeed, recently the AP2 transcription factor, AP2IX-9, was shown to operate as a repressor by inhibiting bradyzoite-specific gene expression , a role many of the 67 annotated AP2 genes, or other cis element binding proteins, in Toxoplasma may share. In addition to identifying conserved motifs associated with gene expression patterns, we confirm that a merozoite specific promoter is sufficient to control the stage specific expression of a reporter gene.
The microarray data of Toxoplasma merozoites provide a global gene expression dataset for this stage. Not only will this information be valuable in understanding the life cycle of Toxoplasma, it can be used to develop reagents and tools to further characterize the developmental biology of the sexual stages of coccidian parasites. For example, the transgenic parasites developed in this study, which express drug selectable markers only at the merozoite stage, will be used in forward selection strategies to screen for tissue culture conditions that are favorable to merozoite growth. As the merozoite is the first stage the parasite differentiates into within the gut of the definitive host, it is the first hurdle to better understand the sexual stages of the parasite. If we can determine the correct conditions that coax the parasite into the merozoite stage in vitro, those conditions may be sufficient to allow the parasite to complete sexual development.
Epithelial gut stages are a common life cycle feature of apicomplexan parasites, and most often the sexual stages develop in the gut, whether the host be vertebrate or invertebrate. This shared tropism is the result of the passive route of ingestion with which the parasite can gain access to and exit from the host. The triggers and specific processes that control parasite differentiation within gut environments are relatively unknown for apicomplexan parasites. In large part, this is because there are no tissue culture systems for gut stages. For example, it is necessary to use mosquitoes to perform crosses in Plasmodium  and to use chickens to grow Eimeria tenella . It is not for lack of trying that efforts haven’t been successful in the past, but with the advent of -omic technologies these efforts can be re-explored. Using genomic based technologies the unique molecular characteristics of gut stages can be acquired and assessed for possible drivers of differentiation. This strategy has recently led to a major advance in understanding the tsetse fly specific developmental stages of Trypanosome brucei. Analysis of transcriptional data allowed researchers to show for the first time that the expression of just one RNA-binding protein in procyclics induced them in vitro into long and short form epimastigotes and eventually infectious metacyclics . This approach has the potential to work in other systems, such as Toxoplasma that has many tools already developed for study, and may reveal unique and/or shared aspects of apicomplexan gut stage development.
Merozoites are the first developmental stage in the coccidian cycle that takes place within the gut of the definitive host. The data presented here describe the global gene expression profile of the merozoite stage and the creation of transgenic parasite strains that show stage-specific expression of reporter genes in the cat intestine. These data and reagents will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate enteric development.
Laboratory mice were used for maintaining chronic infections of the parasite T. gondii. Mice were housed according to instructions in the “Guide to Care and Use of Laboratory Animals” under supervision of a fully trained, veterinary staff in the Washington University Animal Care Facility. Protocols were approved by the Institutional Care Committee and are covered by animal welfare assurance number A-3381-01.
Members of the cat family are the only known host for the sexual stages of T. gondii. Protocols were conducted in the laboratory of Dr. J. P. Dubey at the USDA in Beltsville MD. Dr. Dubey’s laboratory is approved for these procedures by USDA, ARS, Beltsville Agricultural Research Center Animal Care Committee (BAACUC) and are covered by animal welfare assurance number A4400-01.
Parasite lines and tissue culture
The type II TgNmBr1 strain was provided by J.P. Dubey . The type II Me49-Fudr strain was used to make transgenic parasites . For tissue culture, parasites were grown in human foreskin fibroblasts (HFFs) in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (HyClone) at 37°C with 5% CO2. For the plaque assays, parasites were seeded to confluent HFF monlayers at either 100 or 2000 per well and allowed to grow for 9 days (+/-pyrimethamine (Sigma), 10 μM), at which time the monolayers were washed with PBS, fixed with 100% ethanol and stained with Crystal Violet (.05%).
The TgNmBr1 parasite was grown in tissue culture as tachyzoites for mRNA isolation. Two day infected HFF monolayers were scrapped, needle passed, and parasites filtered through a 3 μm polycarbinate membrane (GE). Filtered parasites were spun at 1,410 rpm and parasite pellets were frozen at -80°C until processed for mRNA.
Sexual stage parasite production and harvest
To produce intraepithelial intestinal sexual stage parasites, the TgNmBr1 strain was intraperitoneally (i.p.) injected into mice that were allowed to develop chronic infections characterized by tissue cysts containing bradyzoites. After 30 days, brains from the infected mice were harvested and fed to three separate cats (c48, c50, and c52). Production of oocysts was monitored, and once oocysts were detected (5–6 days post feeding), the small intestine was harvested and placed in 1X PBS with antibiotics and kept at 4°C. To harvest sexual stage parasites, intestines were flayed open, lightly scrapped to remove the mucus and first layer of intestinal cells, and the scrapped material was suspended in PBS. Samples were passed through progressively smaller needles (16G – 23G) to break host cells and debris, and centrifuged in an Eppendorf 5810R centrifuge at 400 rpm to remove large material. The supernatant was filtered through 3 μm membranes, centrifuged at 1,410 rpm to pellet parasites, and parasite pellets were frozen at -80°C until processed for RNA. A portion of each sample was resuspended in PBS and observed for purity under the microscope. The harvesting process resulted in removing the majority of host cells and debris, leaving crescent shaped felid intestinal derived parasites.
To test enteric stage expression of the MSF promoter, the transgenic Me49-Fudr-MSFp-DHFR-Ty parasite was i.p. injected into CD-1 mice to generate in vivo bradyzoite cysts. After 30 days, mice were sacrificed and cysts were isolated from brain samples and kept on ice until fed to a cat. The cat was monitored for oocyst shedding, and once oocysts were observed, the intestine was harvested, fixed in 10% formalin, and processed for IHC.
RNA isolation and microarray hybridization
The Qiagen RNeasy kit (Qiagen) was used to harvest RNA as described previously . Briefly, frozen parasite pellets were resuspended in RLT buffer with β-mercaptoethanol and processed on the RNeasy column. Samples were treated with DNase and resuspended in deionized water and frozen at -80°C. RNA quality was determined on an Agilent Bioanalyzer (Agilent Technologies). The RNA quality was good, showing lack of degradation and little host contamination. Two samples for the c52 RNA, one each for the c48 and c50 RNA, and two for tachyzoite TgNmBr1 RNA were labeled using the Ambion MessageAmp Premier RNA Amplification Kit (Life Technologies) using 500 ng total RNA. Using standard hybridization protocols, 5.5 μg of labeled cRNA was hybridized to the Affymetrix Toxoplasma GeneChip  and imaged with an Affymetrix GeneChip Scanner (Affymetrix). The microarray CEL files created for this study have been deposited at NCBI GEO submission GSE51780.
Microarray data processing and analysis
To analyze the merozoite gene expression in context of the life cycle we obtained a previously published set of arrays from NCBI GEO GSE32427 , comprising the following sample types; day 0, 4, 10 oocyst (OD0, OD4, OD10), day 2 tachyzoite (TD2), and day 4, 8, 21 bradyzoite (BD4, BD8, BD21). Arrays from GSE32427 and those hybridized for this study (TNm, Mc48, Mc50, Mc52), GSE51780, were combined and analyzed as a set. Microarray data were loaded into R using the “affy” library and processed using Robust Multi-array Average (RMA) and quantile normalization . RMA normalization converged the distributions and medians across all samples. RMA normalized data were used in subsequent analyses. The log2 RMA normalized intensity values can be acquired from NCBI GEO submission GSE51780. Annotations and Gene Ontology (GO) assignments were obtained from ToxoDB.org .
R normalized values were imported into GeneSpring (Agilent Technologies). Duplicate samples were grouped by type and average values were calculated. To identify those 8,131 Toxoplasma probesets, or genes, represented on the microarray with significant gene expression differences across sample types we used the following criteria: 1. genes with raw expression values of 30 or lower in all samples were flagged as not expressed and removed from analysis. This removed 1571 genes. 2. An analysis of variance (ANOVA) test was run on the remaining 6560 genes using a p-value cutoff of .05 and multiple testing correction: Benjamini and Hochberg False Discovery Rate. There were 43 genes that had insufficient data. This procedure identified 5969 genes significantly different in at least one sample across the experiment, of which 5% (298) may have been called by chance.
Heatmaps were generated using gene tree clustering with the standard correlation similarity measure.
PCA: Principle component analysis (PCA) was carried out on the 5,969 gene set by sample type, or condition, to determine the strongest expression themes in the data. This method calculates the standard correlation between each condition vector and each eigenvector, or principal component vector. PCA identified three principal components (PCA1: 43% variance, PCA2: 32.2% variance, PCA3: 10% variance), together these comprise 85% of the variance across sample types.
Spline curves: To accurately describe expression profiles across the life cycle for the 5,969 significantly differentially expressed genes we used the spline function in R. The TNm, BD4, BD21, Mc48, and Mc50 samples were not included in spline analysis as they are correlative to either the TD2, BD8, Mc52 samples. The excluded samples essentially represent replicate sample types for many genes that would hinder the correct determination of maximum and minimum spline values, confounding the ordering or grouping of genes in context of the life cycle. A spline curve was generated for each of the non-redundant 5969 genes using a “n” number of points 20*length (dataset) and the “natural” spline method. The global maximum and minimum, or extrema, spline values for each gene were determined, which correspond to points of maximum and minimum expression across the life cycle for a particular gene. The spline function was also used to determine the extrema of the first derivative, f’(x), using “splinefun” and “deriv = 1”. The extrema of the first derivative are the inflection points along the spline curve that correspond to the maximum rate of synthesis or degradation for a particular gene. The differences between maximum, or minimum, expression and the respective rate values were calculated to determine the time to maximum or minimum expression.
FIRE: To identify putative cis elements in the 5’ regions of co-regulated genes across the life cycle we used the Finding Informative Regulatory Elements (FIRE) program . Sequence 2,000-0 bp 5′ of each gene was obtained from ToxoDB.org  and combined with the continuous dataset of ordered genes according to the maximum or minimum of expression. A minimum cutoff z-score of 8.5 was used and motifs were sorted by phase of expression. No significant motifs were found for 100 permutations of randomly ordered genes. Also, no significant motifs were found for correctly ordered maximum of expression genes and only one significant motif was found for minimum of expression genes when compared to sequence 4,000-2,000 bp 5′ of each gene, and no significant motifs were found when correctly ordered genes were compared to sequence 6,000-4,000 bp 5′ of each gene.
KEGG: Normalized ratio expression values were used to map genes and expression into the Kyoto Encyclopedia of Genes and Genomes (KEGG) . The Toxoplasma gondii genome is represented at KEGG, entry T01093, and life cycle regulated genes with KEGG assignments were mapped onto the PathwayMap using the KegArray program, pathways 2 fold downregulated (blue), non-regulated (yellow), and 2 fold upregulated (red). Images for the Metabolic Pathways map (tgo01100) were obtained for each sample and a GIF file was created using the convert program.
Immunofluorescence, immunohistochemistry and microscopy
For IFA: Extracellular merozoite parasites were adhered to poly-L-lysine treated coverslips. Intracellular parasites were imaged on coverslips with infected HFF monolayers. All coverslips were fixed with 4% formaldehyde with .01% TritonX-100 in 1X PBS. Coverslips were blocked with 5% Fetal-bovine/Normal goat serum (FBS/NGS) in PBS. Coverslips were mounted in Prolong Gold containing DAPI (Life Technologies).
For IHC: Felid intestines were fixed in 10% formalin and stored in 70% EtOH. Samples were mounted in paraffin tissue blocks and thin sectioned.
For IFA and IHC: Antibodies were suspended in 1X PBS with 1% FBS. Primary antibodies used were all diluted 1:1,000 and include; mouse α-Me49, rabbit α-RH, mouse α-Ty, conjugated α-SAG1-594, and rabbit α-BAG5 (BAG5 is an alternate name for BAG1). Alexa Fluor 488 α-mouse or Alexa Fluor 595 α-rabbit (Life Technologies) were used for the secondary at 1:1000. Images were obtained on a Zeiss Axioskop 2 MOT Plus microscope using a AxioCam MRm camera (Carl Zeiss, Inc.).
Plasmid construction and creation of transgenic parasites
To construct the merozoite promoter expression plasmids we used the pDEST-GRA1p as a template. The pDEST-GRA1p plasmid has a Destination Gateway cloning site flanked by an upstream TgGRA1 promoter (GRA1p) and a downstream TgDHFR 3′ UTR, and contains a bleomycin cassette for selection in Toxoplasma. The GRA1p (611 bp) was excised from pDEST-GRA1p using the Hind-III and Bgl-II restriction enzymes and replaced with either the MSF (998 bp upstream of the start codon) or the SRS22B (672 bp upstream) promoters that were amplified from type II Me49 lysate using the iProof polymerase (Bio-Rad Laboratories) with standard PCR amplification techniques, resulting in pDEST-MSFp and pDEST-SRS22Bp plasmids. Primers used to amplify the merozoite promoters: Megap Hind-III for (1) (5′-CTAGTAAGCTTTCTCCCCTGGGAAAAGACAGG-3′), Megap Bgl-II rev (5′-CTAGTAGATCTGCCGTTTTGGTGCGTCCAAG-3′), SRS22Bp Hind-III for (1) (5′-CTAGTAAGCTTCTGTGCGTCCTCCACCTTC-3′), SRS22Bp Bgl-II rev (5′-CTAGTAGATCTCTTGAATTAACTGAGACCAGGGCCAC-3′). To create a Gateway cloning fragment for the pDEST plasmids, the pyrimethamine resistant DHFR gene was cloned into the pDONR221 plasmid with a C-terminal Ty tag using the standard BP reaction protocol, resulting in pDONR-DHFR-Ty. Primers used to clone pDONR-DHFR-Ty: DHFR-Ty CDS attB1 for (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCAGAAACCGGTGTGTCTGG-3′), DHFR-Ty CDS attB2 rev (5′- GGGGACCACTTTGTACAAGAAAGCTGGGTCATCGAGCGGGTCCTGGTTCGTGTGGACCTCGACAGCCATCTCCATCTGGA -3′). The pDONR-DHFR-Ty plasmid was then cloned into the three pDEST plasmids using the LR reaction to create, pDEST-GRA1p-DHFR-Ty, pDEST-MSFp-DHFR-Ty, and pDEST-SRS22Bp-DHFR-Ty. The three plasmids were individually electroporated into Me49-Fudr, parasites were selected with phloemycin using standard protocols , cloned, and screened for plasmid integration using specific primers (See Figure 6A). Primers used to screen clones (for (1), see above): GRA1p for (G1) (5′- AAACCCTCGAAGGCTGCTAGTACT 3′), GRA1p rev (G2) (5′- TCTTGCTTGATTTCTTCAAAGAACAACAGCAAG-3′), DHFR-Ty in 5′ of CDS rev (2) (5′- CCCGTCTTTGCAAATTTCCTGG-3′), DHFR 3′ UTR rev (3) (5′- CCGCGGTGTCACTGTAGCC -3′). This resulted in three transgenic strains; Me49-GRA1p-DHFR-Ty (GRA1 strain), Me49-MSFp-DHFR-Ty (MSF strain) and Me49-SRS22Bp-DHFR-Ty (SRS22B strain).
Availability of supporting data
Microarray CEL files and log2 normalized intensity values are available at NCBI GEO, http://www.ncbi.nlm.nih.gov/geo/, under Accession number GSE51780.
Analysis of Variance
Principal component analysis
Finding Informative Regulatory Elements
Megakaryocyte Stimulating Factor.
We would like to thank Joanna Gress of the Functional Genomics Core at Montana State University for her expertise in amplifying, labeling, and hybridizing the mRNA samples to the Toxoplasma gondii Affymetrix microarray and to the Advanced Imaging and Tissue Analysis Core (AITAC) at Washington University School of Medicine for processing the histological samples for immunohistochemistry. We are also grateful to Jennifer Barks for help assistance with parasite culture and to the EuPathDB Project Team for the creation and maintenance of ToxoDB.org. Partially supported by a grant from the NIH (AI059176).
- Montoya JG, Liesenfeld O: Toxoplasmosis. Lancet. 2004, 363 (9425): 1965-1976. 10.1016/S0140-6736(04)16412-X.PubMedView ArticleGoogle Scholar
- Jones JL, Roberts JM: Toxoplasmosis hospitalizations in the United States, 2008, and trends, 1993–2008. Clin Infect Dis. 2012, 54 (7): e58-e61. 10.1093/cid/cir990.PubMedView ArticleGoogle Scholar
- Jones JL, Dargelas V, Roberts J, Press C, Remington JS, Montoya JG: Risk factors for Toxoplasma gondii infection in the United States. Clin Infect Dis. 2009, 49 (6): 878-884. 10.1086/605433.PubMedView ArticleGoogle Scholar
- Jones JL, Holland GN: Annual burden of ocular toxoplasmosis in the US. Am J Trop Med Hyg. 2010, 82 (3): 464-465. 10.4269/ajtmh.2010.09-0664.PubMed CentralPubMedView ArticleGoogle Scholar
- Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, Roy SL, Jones JL, Griffin PM: Foodborne illness acquired in the United States–major pathogens. Emerg Infect Dis. 2011, 17 (1): 7-15. 10.3201/eid1701.P11101.PubMed CentralPubMedView ArticleGoogle Scholar
- Jones JL, Kruszon-Moran D, Sanders-Lewis K, Wilson M: Toxoplasma gondii infection in the United States, 1999 2004, decline from the prior decade. Am J Trop Med Hyg. 2007, 77 (3): 405-410.PubMedGoogle Scholar
- Dubey JP: History of the discovery of the life cycle of Toxoplasma gondii. Int J Parasitol. 2009, 39 (8): 877-882. 10.1016/j.ijpara.2009.01.005.PubMedView ArticleGoogle Scholar
- Hill DE, Chirukandoth S, Dubey JP: Biology and epidemiology of Toxoplasma gondii in man and animals. Anim Health Res Rev. 2005, 6 (1): 41-61. 10.1079/AHR2005100.PubMedView ArticleGoogle Scholar
- Su C, Evans D, Cole RH, Kissinger JC, Ajioka JW, Sibley LD: Recent expansion of Toxoplasma through enhanced oral transmission. Science. 2003, 299: 414-416. 10.1126/science.1078035.PubMedView ArticleGoogle Scholar
- Dubey JP: Toxoplasma, Hammondia, Besniotia, Sarcocystis, and Other Tissue Cyst-forming Coccidia of Man and Animals. Parasitic Protozoa. Edited by: Kreier JP. 1977, New York: Academic Press, 101-237.Google Scholar
- Dubey JP: The history of Toxoplasma gondii–the first 100 years. J Eukaryot Microbiol. 2008, 55 (6): 467-475. 10.1111/j.1550-7408.2008.00345.x.PubMedView ArticleGoogle Scholar
- Hogan MJ, Yoneda C, Feeney L, Zweigart P, Lewis A: Morphology and culture of Toxoplasma. Trans Am Ophthalmol Soc. 1960, 58: 167-187.PubMed CentralPubMedGoogle Scholar
- Matsubayashi H, Akao S: Morphological studies on the development of the Toxoplasma cyst. Am J Trop Med Hyg. 1963, 12: 321-333.PubMedGoogle Scholar
- Weiss LM, Laplace D, Takvorian PM, Tanowitz HB, Cali A, Wittner M: A cell culture system for study of the development of Toxoplasma gondii bradyzoites. J Eukaryot Microbiol. 1995, 42 (2): 150-157. 10.1111/j.1550-7408.1995.tb01556.x.PubMedView ArticleGoogle Scholar
- Soete M, Camus D, Dubremetz JF: Experimental induction of bradyzoite-specific antigen expression and cyst formation by the RH strain of Toxoplasma gondii in vitro. Exp Parasitol. 1994, 78 (4): 361-370. 10.1006/expr.1994.1039.PubMedView ArticleGoogle Scholar
- Sibley LD: Invasion and intracellular survival by protozoan parasites. Immunol Rev. 2011, 240 (1): 72-91. 10.1111/j.1600-065X.2010.00990.x.PubMed CentralPubMedView ArticleGoogle Scholar
- Boyle JP, Radke JR: A history of studies that examine the interactions of Toxoplasma with its host cell: Emphasis on in vitro models. Int J Parasitol. 2009, 39 (8): 903-914. 10.1016/j.ijpara.2009.01.008.PubMedView ArticleGoogle Scholar
- Hunter CA, Sibley LD: Modulation of innate immunity by Toxoplasma gondii virulence effectors. Nat Rev Microbiol. 2012, 10 (11): 766-778. 10.1038/nrmicro2858.PubMed CentralPubMedView ArticleGoogle Scholar
- Dubey JP, Frenkel JK: Cyst-induced toxoplasmosis in cats. J Protozool. 1972, 19 (1): 155-177. 10.1111/j.1550-7408.1972.tb03431.x.PubMedView ArticleGoogle Scholar
- Ferguson DJ: Use of molecular and ultrastructural markers to evaluate stage conversion of Toxoplasma gondii in both the intermediate and definitive host. Int J Parasitol. 2004, 34 (3): 347-360. 10.1016/j.ijpara.2003.11.024.PubMedView ArticleGoogle Scholar
- Ferguson DJ, Hutchison WM, Dunachie JF, Siim JC: Ultrastructural study of early stages of asexual multiplication and microgametogony of Toxoplasma gondii in the small intestine of the cat. Acta Pathol Microbiol Scand B: Microbiol Immunol. 1974, 82 (2): 167-181.Google Scholar
- Speer CA, Dubey JP: Ultrastructural differentiation of Toxoplasma gondii schizonts (types B to E) and gamonts in the intestines of cats fed bradyzoites. Int J Parasitol. 2005, 35 (2): 193-206. 10.1016/j.ijpara.2004.11.005.PubMedView ArticleGoogle Scholar
- Omata Y, Taka A, Terada K, Koyama T, Kanda M, Saito A, Dubey JP: Isolation of coccidian enteroepithelial stages of Toxoplasma gondii from the intestinal mucosa of cats by Percoll density-gradient centrifugation. Parasitol Res. 1997, 83 (6): 574-577. 10.1007/s004360050300.PubMedView ArticleGoogle Scholar
- Taka A, Omata Y, Ohsawa T, Koyama T, Kanda M, Saito A, Toyoda Y: Antibody reactivity in mice and cats to feline enteroepithelial stages of Toxoplasma gondii. Vet Parasitol. 1999, 83 (1): 73-78. 10.1016/S0304-4017(99)00023-0.PubMedView ArticleGoogle Scholar
- Dubey JP, Passos LM, Rajendran C, Ferreira LR, Gennari SM, Su C: Isolation of viable Toxoplasma gondii from feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctolagus cuniculus) from Brazil. J Parasitol. 2011, 97 (5): 842-845. 10.1645/GE-2728.1.PubMedView ArticleGoogle Scholar
- Bahl A, Davis PH, Behnke M, Dzierszinski F, Jagalur M, Chen F, Shanmugam D, White MW, Kulp D, Roos DS: A novel multifunctional oligonucleotide microarray for Toxoplasma gondii. BMC Genomics. 2010, 11: 603-10.1186/1471-2164-11-603.PubMed CentralPubMedView ArticleGoogle Scholar
- Fritz HM, Buchholz KR, Chen X, Durbin-Johnson B, Rocke DM, Conrad PA, Boothroyd JC: Transcriptomic analysis of toxoplasma development reveals many novel functions and structures specific to sporozoites and oocysts. PLoS One. 2012, 7 (2): e29998-10.1371/journal.pone.0029998.PubMed CentralPubMedView ArticleGoogle Scholar
- Behnke MS, Radke JB, Smith AT, Sullivan WJ, White MW: The transcription of bradyzoite genes in Toxoplasma gondii is controlled by autonomous promoter elements. Mol Microbiol. 2008, 68 (6): 1502-1518. 10.1111/j.1365-2958.2008.06249.x.PubMed CentralPubMedView ArticleGoogle Scholar
- Cleary MD, Singh U, Blader IJ, Brewer JL, Boothroyd JC: Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene expression. Eukaryot Cell. 2002, 1 (3): 329-340. 10.1128/EC.1.3.329-340.2002.PubMed CentralPubMedView ArticleGoogle Scholar
- Radke JR, Behnke MS, Mackey AJ, Radke JB, Roos DS, White MW: The transcriptome of Toxoplasma gondii. BMC Biol. 2005, 3: 26-10.1186/1741-7007-3-26.PubMed CentralPubMedView ArticleGoogle Scholar
- Samuelson J, Bushkin GG, Chatterjee A, Robbins PW: Strategies to discover the structural components of cyst and oocyst walls. Eukaryot Cell. 2013, 12 (12): 1578-1587. 10.1128/EC.00213-13.PubMed CentralPubMedView ArticleGoogle Scholar
- Braun L, Brenier-Pinchart MP, Yogavel M, Curt-Varesano A, Curt-Bertini RL, Hussain T, Kieffer-Jaquinod S, Coute Y, Pelloux H, Tardieux I, Sharma A, Belrhali H, Bougdour A, Hakimi MA: A Toxoplasma dense granule protein, GRA24, modulates the early immune response to infection by promoting a direct and sustained host p38 MAPK activation. J Exp Med. 2013, 210 (10): 2071-2086. 10.1084/jem.20130103.PubMed CentralPubMedView ArticleGoogle Scholar
- Carruthers VB, Tomley FM: Microneme proteins in apicomplexans. Subcell Biochem. 2008, 47: 33-45. 10.1007/978-0-387-78267-6_2.PubMed CentralPubMedView ArticleGoogle Scholar
- Wasmuth JD, Pszenny V, Haile S, Jansen EM, Gast AT, Sher A, Boyle JP, Boulanger MJ, Parkinson J, Grigg ME: Integrated bioinformatic and targeted deletion analyses of the SRS gene superfamily identify SRS29C as a negative regulator of Toxoplasma virulence. MBio. 2012, 3 (6): e00321-e00312.PubMed CentralPubMedView ArticleGoogle Scholar
- Behnke MS, Wootton JC, Lehmann MM, Radke JB, Lucas O, Nawas J, Sibley LD, White MW: Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii. PLoS One. 2010, 5 (8): e12354-10.1371/journal.pone.0012354.PubMed CentralPubMedView ArticleGoogle Scholar
- Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL: The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol. 2003, 1 (1): E5-PubMed CentralPubMedView ArticleGoogle Scholar
- Radke JR, Gubbels MJ, Jerome ME, Radke JB, Striepen B, White MW: Identification of a sporozoite-specific member of the Toxoplasma SAG superfamily via genetic complementation. Mol Microbiol. 2004, 52 (1): 93-105. 10.1111/j.1365-2958.2003.03967.x.PubMedView ArticleGoogle Scholar
- Bohne W, Wirsing A, Gross U: Bradyzoite-specific gene expression in Toxoplasma gondii requires minimal genomic elements. Mol Biochem Parasitol. 1997, 85 (1): 89-98. 10.1016/S0166-6851(96)02814-9.PubMedView ArticleGoogle Scholar
- Nakaar V, Bermudes D, Peck KR, Joiner KA: Upstream elements required for expression of nucleoside triphosphate hydrolase genes of Toxoplasma gondii. Mol Biochem Parasitol. 1998, 92 (2): 229-239. 10.1016/S0166-6851(97)00220-X.PubMedView ArticleGoogle Scholar
- Soldati D, Boothroyd JC: Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii. Science. 1993, 260 (5106): 349-352. 10.1126/science.8469986.PubMedView ArticleGoogle Scholar
- Elemento O, Slonim N, Tavazoie S: A universal framework for regulatory element discovery across all genomes and data types. Mol Cell. 2007, 28 (2): 337-350. 10.1016/j.molcel.2007.09.027.PubMed CentralPubMedView ArticleGoogle Scholar
- Van Poppel NF, Welagen J, Vermeulen AN, Schaap D: The complete set of Toxoplasma gondii ribosomal protein genes contains two conserved promoter elements. Parasitology. 2006, 133 (Pt 1): 19-31.PubMedView ArticleGoogle Scholar
- Walker R, Gissot M, Huot L, Alayi TD, Hot D, Marot G, Schaeffer-Reiss C, Van Dorsselaer A, Kim K, Tomavo S: Toxoplasma transcription factor TgAP2XI-5 regulates the expression of genes involved in parasite virulence and host invasion. J Biol Chem. 2013, 288 (43): 31127-31138. 10.1074/jbc.M113.486589.PubMed CentralPubMedView ArticleGoogle Scholar
- Goodswen SJ, Kennedy PJ, Ellis JT: A review of the infection, genetics, and evolution of Neospora caninum: from the past to the present. Infect Genet Evol. 2013, 13: 133-150.PubMedView ArticleGoogle Scholar
- David Sibley L: Recent origins among ancient parasites. Vet Parasitol. 2003, 115 (2): 185-198. 10.1016/S0304-4017(03)00206-1.PubMedView ArticleGoogle Scholar
- Piekarski G, Pelster B, Witte HM: Endopolygeny in Toxoplasma gondii. Z Parasitenkd. 1971, 36 (2): 122-130.PubMedGoogle Scholar
- Cairns RA, Harris IS, Mak TW: Regulation of cancer cell metabolism. Nat Rev Cancer. 2011, 11 (2): 85-95. 10.1038/nrc2981.PubMedView ArticleGoogle Scholar
- Colgan SP, Taylor CT: Hypoxia: an alarm signal during intestinal inflammation. Nat Rev Gastroenterol Hepatol. 2010, 7 (5): 281-287. 10.1038/nrgastro.2010.39.PubMed CentralPubMedView ArticleGoogle Scholar
- Radke JB, Lucas O, De Silva EK, Ma Y, Sullivan WJ, Weiss LM, Llinas M, White MW: ApiAP2 transcription factor restricts development of the Toxoplasma tissue cyst. Proc Natl Acad Sci U S A. 2013, 110 (17): 6871-6876. 10.1073/pnas.1300059110.PubMed CentralPubMedView ArticleGoogle Scholar
- Baker DA: Malaria gametocytogenesis. Mol Biochem Parasitol. 2010, 172 (2): 57-65. 10.1016/j.molbiopara.2010.03.019.PubMed CentralPubMedView ArticleGoogle Scholar
- Muller J, Hemphill A: In vitro culture systems for the study of apicomplexan parasites in farm animals. Int J Parasitol. 2013, 43 (2): 115-124. 10.1016/j.ijpara.2012.08.004.PubMedView ArticleGoogle Scholar
- Kolev NG, Ramey-Butler K, Cross GA, Ullu E, Tschudi C: Developmental progression to infectivity in Trypanosoma brucei triggered by an RNA-binding protein. Science. 2012, 338 (6112): 1352-1353. 10.1126/science.1229641.PubMed CentralPubMedView ArticleGoogle Scholar
- Behnke MS, Khan A, Wootton JC, Dubey JP, Tang K, Sibley LD: Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases. Proc Natl Acad Sci U S A. 2011, 108 (23): 9631-9636. 10.1073/pnas.1015338108.PubMed CentralPubMedView ArticleGoogle Scholar
- Gautier L, Cope L, Bolstad BM, Irizarry RA: affy–analysis of Affymetrix GeneChip data at the probe level. Bioinformatics. 2004, 20 (3): 307-315. 10.1093/bioinformatics/btg405.PubMedView ArticleGoogle Scholar
- Gajria B, Bahl A, Brestelli J, Dommer J, Fischer S, Gao X, Heiges M, Iodice J, Kissinger JC, Mackey AJ, Pinney DF, Roos DS, Stoeckert CJ, Wang H, Brunk BP: ToxoDB: an integrated Toxoplasma gondii database resource. Nucleic Acids Res. 2008, 36 (Database issue): D553-D556.PubMed CentralPubMedGoogle Scholar
- Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M: KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res. 2012, 40 (Database issue): D109-D114.PubMed CentralPubMedView ArticleGoogle Scholar
- Messina M, Niesman I, Mercier C, Sibley LD: Stable DNA transformation of Toxoplasma gondii using phleomycin selection. Gene. 1995, 165 (2): 213-217. 10.1016/0378-1119(95)00548-K.PubMedView ArticleGoogle Scholar
- Sibley LD, Khan A, Ajioka JW, Rosenthal BM: Genetic diversity of Toxoplasma gondii in animals and humans. Philos Trans R Soc Lond B Biol Sci. 2009, 364 (1530): 2749-2761. 10.1098/rstb.2009.0087.PubMed CentralPubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.