Genome-wide expression patterns of calcium-dependent protein kinases in Toxoplasma gondii
© Wang et al. 2015
Received: 12 January 2015
Accepted: 27 May 2015
Published: 4 June 2015
Calcium-dependent protein kinases (CDPKs) are found in plants and some Apicomplexan parasites but not in animals or fungi. CDPKs have been shown to play important roles in various calcium-signaling pathways such as host cell invasion, egress and protein secretion in Toxoplasma gondii. The objectives of the present study were to examine the T. gondii CDPK genes expression patterns during different development stages and stress responses.
We carried out a comprehensive expression analysis of CDPK genes based on previously published microarray datasets, and we also used real time quantitative RT-PCR to study ten T. gondii CDPK genes expression patterns under acid, alkali, high temperature and low temperature conditions.
Microarrays analysis indicated that some TgCDPK genes exhibited different expression levels in IFN-γ stimuli conditions or at different developmental stages, suggesting that CDPK genes may play different roles in these processes. Expression profiles under low temperature, high temperature, acid and alkaline indicated that most CDPK may be involved in regulating high temperature, acid and alkaline signaling pathways.
We present a genome-wide expression analysis of CDPK genes in T. gondii for the first time, and the mRNA levels change with abiotic and biotic stresses, suggesting their functional roles in these processes. These results will provide a solid basis for future functional studies of the CDPK gene family in T. gondii.
KeywordsToxoplasma gondii Calcium-dependent protein kinases (CDPKs) Expression patterns
The protozoan phylum Apicomplexa comprises thousands of obligate intracellular parasites, many of which cause significant human and animal health problems. Toxoplasma gondii infects approximately one third of the global human population and causes severe disease in immunocompromised patients and pregnant women  and plasmodium falciparum, the causative agents of malaria cause over 1 million deaths per year . In order to perpetuate infection, parasites need to egress from infected cells and then reinvade uninfected cells. In response to these events, parasites have developed various remarkable regulatory mechanisms for proliferation. Among them, intracellular calcium, the second messenger, plays an important role in various signal transduction cascades, including protein secretion, gliding motility, invasion of and egress from host cell, proliferation and differentiation .
In T. gondii, treatment with a calcium ionophore increases the frequency of calcium transients, resulting in enhanced secretion of micronemal proteins which are required during both invasion and egress. Conversely, chelation of intracellular calcium inhibits microneme secretion, disrupts motility and cell invasion . Transient changes in intracellular calcium concentration are mediated by different calcium sensors or calcium-binding proteins. Comparison of Apicomplexan genomes reveals numerous EF-hand containing proteins including calmodulin-dependent kinases (CaMKs), centrin (CETN)-caltractin-like proteins, CaM-CETN-like proteins, calcium-dependent protein kinases (CDPKs) and CDPK-like proteins . Among them, CDPKs are the most interesting, because they are the most abundant class of calcium sensors in Apicomplexan parasites, at the same time they are also commonly found in plants and some ciliates, but absent from mammalian hosts . The essential functions of Apicomplexan CDPKs and absent from mammalian hosts have made CDPKs as potential drug targets for controlling Apicomplexan-based diseases.
Increasing evidence suggests that CDPKs control important physiological events in the complex life cycles of Apicomplexan parasites. For example, conditional suppression of TgCDPK1 resulted in a weakening of microneme secretion, parasites gliding motility, host cell invasion and egress abilities [7, 8]. CDPK4 in Plasmodium berghei, the orthologue of TgCDPK1, regulates cell cycle progression in the male gametocyte . Genetic disruption of TgCDPK3 has demonstrated that it has a regulatory function in parasite physiology in addition to ionophore induced egress [10–12]. In Plasmodium, CDPK1 (The orthologue of TgCDPK3) plays a key role in schizont development, microneme secretion, invasion of erythrocyte and regulating mRNAs to assure timely and stage-specific protein expression [13–15]. A recent study demonstrated that TgCDPK7 were crucial for parasites division, growth and proper maintenance of centrosome . Knock-out of PbCDPK3 leads to a pronounced defect in ookinete transmission to the mosquito midgut epithelium and terminates oocysts production [17, 18]. PfCDPK5 plays an essential role in regulating parasite egress from erythrocytes , whereas PbCDPK6 is critical for controlling the sporozoites switch from a migratory to an invasion phenotype . Finally, Sharma et al. demonstrated that PfCDPK7 bound to PI(4,5)P2 and controlled parasite development in the erythrocyte . Taken together, these studies suggest CDPKs regulate various biologic functions. However, the accurate CDPKs regulatory mechanisms and their targets remain unclear.
In plants, CDPKs constitute a large multigene family. Recently, genome-wide analyses have identified 34 CDPK isoforms in Arabidopsis genome , 31 genes in rice genome  and 25 CDPK genes in canola (Brassica napus L.) . Apicomplexan parasites also contain multiple CDPK genes . Sibley et al. indicated T. gondii contained 12 CDPKs gene , While Tallevich et al. identified TGME49_240390 on the Toxodb database as a novel CDPK . Through our unpublished data, this CDPK contains 3 EF-hand, and don’t have known orthologues in Apicomplexan parasites (except for Neospora caninum). Here, we named it as TgCDPK10. To our knowledge, only three TgCDPKs (CDPK1, CDPK3 and CDPK7) have been studied, yet the physiological functions of the majority of CDPKs remained unclear.
In the present study, we carried out a comprehensive expression analysis of calcium-dependent protein kinase genes based on previously published microarrays datasets. Furthermore, we also studied ten T. gondii CDPK genes expression patterns under acid, alkali, high temperature and low temperature conditions. Our results showed most TgCDPKs may regulate stress responses.
T. gondii RH strain were maintained by passage through Vero cell monolayers in Dulbecco’s modified Eagle’s medium (DMEM) with 2 % fetal calf serum (FCS), 10 mM HEPES (pH = 7.2), 100 U/ml penicillin and 100 Ug/ml streptomycin at 37 °C with 5 % CO2 as previously described .
Freshly released tachyzoites were harvested by centrifugation (1000 g × 4 min). Parasites were washed with phosphate-buffered saline (PBS) to remove impurities, and then replaced with fresh control or medium with different pH value. A low temperature treatment was carried out at 4 °C under the same conditions while the high temperature treatment was at 42 °C. Acid and alkaline solution treatment were carried out by cultivation in DMEM with acetate buffer (pH3.6) or 50 mM Hepes (pH 8.1) at 37 °C in the presence of air.
Real-time quantitative RT-PCR
Total RNA was isolated from parasites of different time periods using Trizol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The properties of RNA samples were examined by Goldview-stained agarose gel electrophoresis and spectrophotometric analysis. Total RNA samples were transcribed into cDNA using superscript First-Strand Synthesis System (Promega, USA). Real-time quantification RT-PCR was performed in 20 ml volume reaction mixture containing 2 × SYBR Green qPCR Supermix (Invitrogen, Carlsbad, CA, USA), 10 mM gene-specific primer and 0.1 mg of cDNA. The thermal cycling conditions were set as follows: 95 °C for 3 min followed by 40 cycles of amplification at 95 °C for 15 s, 60 °C for 31 s and 72 °C for 15 s. Lack of primer dimers or genomic DNA contamination of reagents was verified with melting curve analysis. The β-tubulin gene was used as internal reference for all the reactions analysis and the primers used for qRT-PCR are described in Additional file 1: Table S1 . The relative gene expression levels were determined using 2-△△CT method and each experiment was performed three biological replicates.
To examine the global expression profiles of CDPK genes among different invasion periods, cell cycle, developmental stages and on IFN-γ-dependent cell mediated immune response, expression analysis were generated using published microarrays data which were deposited at NCBI Gene Expression Omnibus (GEO) database under the series accession GSE20480, GSE19092, GSE32427, and ArrayExpress database E-MEXP-3579 respectively. Dendrogram and heatmap for display expression pattern were carried out using Cluster 3.0 for normalizing and hierarchical clustering with average linkage. The analyzing datasets was visualized by Java Tree-View 1.1 program.
Results and discussion
Expression analysis of CDPK genes by microarray
The published microarray data provided us abundant resources for studying TgCDPKs gene expression patterns. We obtained microarrays data from four microarrays experiments [29–33]. Fortunately, all of the T. gondii CDPK genes have the corresponding probe sets in these datasets.
To investigate the expression profiles of TgCDPK genes in the intracellular cell cycle, another microarray data set  was used for this analysis. In this experiment, T. gondii were synchronized by thymidine block, and then cell cycle expression profiles were studied after thymidine release. As shown in Fig. 1b, the expression levels of CDPK1, CDPK2, CDPK3, CDPK4A, CDPK5, CDPK6 and CDPK9 were rather higher from S phase to cytokinetic periods (S/M phases), and then became lower in the major G1 period, especially for CDPK6 and CDPK1 which decreased 5.9-fold and 4.4-fold, respectively. CDPK4 had a different expression pattern with a lower expression in S/M phases but a higher expression in G1 period. The expression levels of CDPK2A, CDPK2B, CDPK7, CDPK8 and CDPK10 genes were quite stable, and there was no obvious change during the intracellular cell cycle. Comparison of the results of two microarrays revealed an alignment between CDPK genes elevated in extracellular parasites and the S/M phases and down-regulated in the intracellular mRNA expression with the G1 period. The expression level changes of CDPK1, CDPK2, CDPK3, CDPK4A, CDPK5, CDPK6 and CDPK9 were correlated with known microneme proteins which are involved in host cell invasion, suggesting these CDPKs may also be involved in host cell invasion. In extracellular parasites, the expression of proteins involved in invasion, motility and signal transduction are increased and the parasites appear to be optimally primed for cell invasion. Conversely, the expressions of biosynthetic and metabolic genes are increased after T. gondii entry and in the G1 period suggesting parasites begin to proliferate . Thus, these results indicated that CDPK1, CDPK2, CDPK3, CDPK4A, CDPK5, CDPK6, and CDPK9 may be involved in host cell invasion, egress and motility, while CDPK4 may play novel roles in metabolism and DNA replication.
To study the function of TgCDPKs in specific parasite development stages, a third microarray data [31, 32] was used to carry out this study. As show in Fig. 1c, the expression levels of CDPK3 and CDPK6 were lower in unsporulated oocysts but higher in sporulated oocysts of 4 days, and the expression became lower as the development of oocysts increased. The expression levels of CDPK3 and CDPK6 were 6.6-fold and 2.8-fold in d4 oocysts comparing to d0 oocysts, respectively; the expression levels of CDPK3 and CDPK6 were 3.9-fold and 1.7-fold in d10 oocysts comparing to d0 oocysts, respectively. These results suggested that CDPK3 and CDPK6 may be involved in oocysts development or adjust oocysts to suit the nutrient-poor and stressing external environment. The expression levels of other CDPKs were almost unchanged during this process, and the expression changes of these CDPK genes may need some other stimuli.
Expression profiles of the T. gondii CDPK genes under different stresses
Most bacterial pathogens significantly alter their transcriptomes to fine-tune the infected host cell environment and thus avoid clearance by host-cell-intrinsic mechanisms and achieve efficient invasion to the next host. T. gondii has also evolved various strategies to modify the host environment, including altering the gene expression and reshaping the host genome expression to responses to intracellular environmental changes [34, 35].
To identify the effects of CDPKs on IFN-γ-dependent cell mediated immune response, the expression of the TgCDPK genes were analyzed in C57BL/6 mice, IFN-γ KO mice (Parasites were harvested from the peritoneal cavity of the mice after 4 days with 2 × 106 parasites intraperitoneally) and in vitro environment in HFF cells (Parasites were harvested 48 h later as parasites were beginning to egress) using the published microarray data with four biological replicates . The results indicated that the expression levels of all CDPK genes were quite similar in IFN-γ KO mice and in vitro samples, which the expression changes were less than 2-fold. The results suggest that the functions of TgCDPKs in IFN-γ KO mice and in HFF cells are not significantly different. CDPK2B, CDPK7 and CDPK10 were significantly up-regulated and CDPK6 was down-regulated in vitro comparing to that in WT mice. The up-regulated CDPK genes CDPK2B, CDPK7 and CDPK10 increased 2.8, 4.6 and 2.4-fold, respectively and the down-regulated CDPK6 gene declined 2.5-fold. Similar results were obtained from comparison between in vivo in WT and in IFN-γ KO mice. This indicates that the changes of four CDPK genes expression patterns are largely induced in response to the IFN-γ-dependent-innate immune response, which suppresses T. gondii proliferation. It has been reported that knock-down of TgCDPK7 protein resulted in pronounced defects in parasite division . These suggest that CDPK7 gene may increase expression in response to avoid IFN-γ-induced suppression of T. gondii proliferation. Thus, these results suggest that the four CDPK genes may play an important role in IFN-γ-dependent-innate immune response. Further studies are needed to explain the accurate processions.
In the present study, when T. gondii were treated under low temperature conditions, the results indicated that the expression levels of all TgCDPKs remain unchanged (As shown in Additional file 2: Figure S1). Many plant CDPKs have been reported to play crucial roles during cold stress . In contrast our results suggested that TgCDPKs does not play such a role. This may be due to the fact that T. gondii do not encounter a cold environment in the host.
We conducted a comprehensive expression analysis of the CDPK gene family in T. gondii for the first time. We found the following results: First, during the T. gondii invasion and in the intracellular cell cycle, the expression pattern of CDPK4 was distinctly different from others. Second, in parasite development stages, only CDPK3 and CDPK6 change their expression level during oocyst sporulation. Third, CDPK2B, CDPK7 and CDPK10 were significantly up-regulated and CDPK6 was down-regulated during the IFN-γ stimulate. Fourth, during the acid solution treatment, all CDPKs had a marked decrease at 3 h and then increased except CDPK1 which remain unchanged, and during the alkaline treatment, CDPK9 remain unchanged while the other CDPK genes had a marked increase in the transcript levels. Finally, during temperature stresses, all the CDPK (except CDPK9, which remain unchanged) were up-regulated in response to high temperature stress, while the expression patterns of all the CDPK genes remain unchanged in response to cold stress. Our results suggest CDPKs are a large family of multi-functional genes which may play essential roles in parasite development, abiotic and biotic stress responses. These results will provide a solid basis for future functional studies of the CDPK gene family in T. gondii.
Project support was provided, in part, by the National Natural Science Foundation of China (Grant Nos. 31472184, 31101812, 31230073), Natural Science Foundation of Gansu Province (Grant Nos. 1308RJYA092), and the Science Fund for Creative Research Groups of Gansu Province (Grant No. 1210RJIA006).
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