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Background.γ-irradiation is commonly used to create attenuation in Plasmodium parasites. However, there are no systematic studies on the survival, reversion of virulence, and molecular basis for γ-radiation–induced cell death in malaria parasites.
Methods.The effect of γ-irradiation on the growth of asexual Plasmodium falciparum was studied in erythrocyte cultures. Cellular and ultrastructural changes within the parasite were studied by fluorescence and electron microscopy, and genome-wide transcriptional profiling was performed to identify parasite biomarkers of attenuation and cell death.
Results.γ-radiation induced the death of P. falciparum in a dose-dependent manner. These parasites had defective mitosis, sparse cytoplasm, fewer ribosomes, disorganized and clumped organelles, and large vacuoles—observations consistent with “distressed” or dying parasites. A total of 185 parasite genes were transcriptionally altered in response to γ-irradiation (45.9% upregulated, 54.1% downregulated). Loss of parasite survival was correlated with the downregulation of genes encoding translation factors and with upregulation of genes associated with messenger RNA–sequestering stress granules. Genes pertaining to cell-surface interactions, host-cell remodeling, and secreted proteins were also altered.
Conclusions.These studies provide a framework to assess the safety of γ-irradiation attenuation and promising targets for genetic deletion to produce whole parasite-based attenuated vaccines.
Exposure to ionizing radiation has a profound effect on the viability and further replication of living cells. Radiation is used for the treatment of cancer , generation of attenuated vaccines , and in food safety . Radiation attenuation has also been one of the earliest methods used to generate whole parasite–based malaria vaccines [4–6]. Given the importance of ionizing radiation in the treatment of different forms of cancer, a large body of scientific data has accumulated on the effects of ionizing radiation on the cell cycle . Such studies have shed light on the molecular and biochemical aspects of radiation-mediated cell death and survival mechanisms [1, 8]. These studies have also provided insight into the cell cycle checkpoints and the complex signaling pathways and associated molecules that ensure that each phase of the cell cycle is completed without errors [7, 9, 10].
Ionizing radiation is detrimental to the growth and survival of Plasmodium parasites. In murine models, asexual blood-stage parasites exposed to high doses of radiation failed to cause blood-stage parasite infection , and immunization with radiation-attenuated asexual-stage parasites protected against parasitemia [11–13] and experimental cerebral malaria . In preerythrocytic-stage malaria, exposure of Plasmodium sporozoites to a threshold level of γ-radiation allows their invasion into liver cells and early development. However, these parasites undergo arrested development and fail to produce infectious liver-stage merozoites and, consequently, blood-stage parasite infection . Irradiation-attenuated malaria sporozoites protect mice [4, 15] and humans [5, 16] against malaria in experimental challenge studies. Accordingly, despite the limited success of subunit-based malaria vaccines in clinical testing , there has been considerable interest in using radiation exposure to create an attenuated sporozoite-stage vaccine [14, 16].
In this study, we measured the effects of different doses of γ-irradiation on P. falciparum in short-term and long-term cultures. These studies were performed to evaluate both the immediate effect on survival and the ability of rare parasite populations to recover and repopulate. We also studied the effect of γ-irradiation on parasite structure, by fluorescence microscopy and transmission electron microscopy. In addition, we performed extensive genome-wide transcriptional profiling in P. falciparum exposed to increasing doses of γ-radiation, to identify parasite proteins that could be correlated with survival and cell death, and, on the basis of the data set, created a functionally diverse molecular signature of growth attenuation. Last, bioinformatic analyses were performed to decipher the biological systems altered by γ-irradiation that are associated with growth regulation and survival.
Cultivation, γ-irradiation treatment, and survival and growth studies of P. falciparum were performed as described in the Supplementary Materials.
Detailed procedures for immunofluorescence and electron microscopy studies to determine the effect of γ-radiation–induced ultrastructural changes in parasites are provided in the Supplementary Materials.
RNA preparation and microarray analysis of untreated and γ-radiation–treated parasites were performed as previously described . Details of these procedures can be found in the Supplementary Materials.
Details of gene annotation, ontology, and bioinformatic analyses to determine the biological pathways associated with γ-radiation–induced growth attenuation and cell death can be found as described earlier [18–20] and in the materials and methods and results sections of the Supplementary Materials.
Real-time PCR to create a molecular signature of γ-radiation–induced growth attenuation of P. falciparum was performed as previously described . A further description can also be found in the Supplementary Materials.
We used logistic regression to analyze the effect of γ-irradiation on nuclear division and microneme development in asexual blood-stage P. falciparum. The adjacent categorical model was used to assess the cell cycle progression on the basis of the number of nuclei counted during schizogony in γ-radiation–treated and untreated P. falciparum cultures. The logit model was used to measure the effect of γ-radiation treatment on microneme development by comparing the percentage of RAP1- and EBA175-positive parasites between the treated and untreated P. falciparum cultures.
Duplicate in vitro cultures of asynchronous parasites at 0.5% initial parasitemia were treated with 15, 30, and 60 K of γ-radiation (where K is equal to 1 kilorad of γ-radiation delivered by a Cesium-137 source), and parasitemia was then monitored every 24 hours for 3 days. Parasitemia continued to be monitored on days 7 and 13 in the group treated with 60 K of γ-radiation.
In untreated control cultures, parasitemia increased from 0.5% to 0.73% ± 0.15% and 6.20% ± 0.30%, after 24 and 48 hours, respectively, and then slightly dropped to 4.78% ± 0.32% after 72 hours of culture, an indication of culture overgrowth. Treatment with γ-irradiation resulted in a dose-dependent decrease in parasite growth and survival that was most evident at 48 and 72 hours after treatment. In cultures treated with 15 K of γ-radiation, while parasitemia remained unchanged during the initial 24 hours after treatment, parasite survival was markedly reduced by 84% and 73% at 48 and 72 hours, respectively, after treatment. The detrimental effect of γ-irradiation on parasite survival was even more dramatic at higher doses. Parasitemia was reduced by 13%, 91%, and 95% at 24, 48, and 72 hours, respectively, after treatment with 30 K of γ-radiation and by 29%, 93%, and 99.5% at 24, 48, and 72 hours, respectively, after treatment with 60 K of γ-irradiation (Figure 1).
To evaluate the ability of rare parasite populations to recover and repopulate, cultures treated with 60 K of γ-radiation were monitored for long-term parasite growth and survival. After 7 days of continuous culture, parasitemia continued to drop from 0.23% (at 72 hours) to 0.099% (at day 7) but by day 13 rose to 1.27% and thereafter continued to grow at a rate that was indistinguishable from that of untreated control cultures.
To better understand the cellular effects of ionizing radiation on the asexual blood-stage of P. falciparum, we magnetically enriched parasites from both irradiated (60 K) and nonirradiated control samples after 24 hours of culture and observed parasite organelles by fluorescence microscopy. We assessed the development and structural integrity of parasite nuclei, rhoptries, and micronemes.
In control cultures, 40% of the parasites (230 of 579) contained 1–4 nuclei, 33% (193 of 579) contained 5–8 nuclei, and 27% (156 of 579) contained ≥9 nuclei (Figure 2A). This distribution is typical for an unsynchronized blood-stage culture and indicated that the parasites were progressing through the multinuclear cycle of asexual development. In advanced schizonts from control cultures, the nuclei were sharply defined, condensed, and arranged in an orderly pattern around the parasite (Figure 2B and and22D).
In contrast to control cultures, irradiated cultures had fewer clearly distinguishable nuclei per parasite: 84% of irradiated parasites (569 of 675) contained 1–4 nuclei, 13% (88 of 675) contained 5–8 nuclei, and only 3% (18 of 675) contained ≥9 nuclei (Figure 2A). This distribution indicated that the irradiated parasite culture was not progressing through multinuclear development normally. In irradiated parasites, the nuclei often appeared to be clumped together in the cytoplasm and in some examples presented irregular shapes or sizes (Figure 2C and and22E). Furthermore, statistical analysis by adjacent categorical modeling predicted that, overall, irradiated parasites had an approximately 5-fold higher prospect of having fewer number of multinucleated P. falciparum schizonts, compared with untreated parasites.
We also evaluated development of blood-stage parasites by examining the morphology of rhoptries and micronemes, the vesicular organelles that develop during schizogony and that ultimately form part of the apical complex in daughter merozoites. Both organelles are important for establishing a new parasitophorous vacuole when the merozoite invades a new red blood cell . The rhoptry-associated protein 1 (RAP1) is a rhoptry component that appears in vesicles during the third nuclear division of schizogony and that remains visible as the parasites progress through asexual blood-stage development . In control cultures, 66% of the parasites (162 of 246) were positive for RAP1, and as expected, these RAP1-positive parasites contained at least 5 nuclei, and half of these parasites were more advanced schizonts containing ≥9 nuclei. As reported in other studies, the RAP1 vesicles in control parasites were arranged in a regular pattern around the nuclei (Figure 2B). In contrast to control parasites, only 18% of irradiated parasites (56 of 319) stained positively for RAP1. Approximately half of the RAP1-positive irradiated parasites contained 5–8 nuclei, yet half of the RAP1 positive parasites appeared to only contain 1–4 nuclei. Thus, the irradiated cultures had fewer RAP1-positive parasites than control cultures, and the RAP1-positive parasites from irradiated cultures appeared to have fewer nuclei, on average, than RAP1-positive parasites from control cultures. The RAP1 vesicles in irradiated parasites were sometimes clumped together, and some of these RAP1 structures appeared to be abnormally large (Figure 2C).
Micronemes can be detected in late-stage schizonts after the fourth nuclear division by the appearance of the protein EBA175 in these structures . In control cultures, 16% of the parasites (30 of 192) were positive for EBA175. EBA175 staining was visible as a ring around each nucleus, and the staining usually included a brighter spot at one end of the nucleus (Figure 2D). Consistent with previous reports, most of the EBA175-positive parasites in control cultures contained ≥9 nuclei and showed evidence of daughter cell budding, one of the final steps of blood-stage development. In irradiated cultures, only 7% of the parasites (17 of 230) were EBA175 positive, and the majority of these parasites contained only 5–8 nuclei. Similar to the observations with RAP1 staining, irradiated cultures had fewer EBA175-positive parasites than control cultures, and the EBA175-positive parasites from irradiated cultures appeared to have fewer nuclei than those from control cultures. The EBA175 staining in these irradiated parasites was generally disorganized and not always associated with budding parasites (Figure 2E, EBA175), indicating a disruption in microneme development and schizont development overall in irradiated cultures. The logit model yielded a P value of <2 × 10–16 for RAP1-positive parasites and a P value of 8.73 × 10–3 for EBA175-positive parasites, indicating that irradiation treatment interfered with organogenesis and development of P. falciparum (Figure 2F).
To further investigate the cellular effects of radiation on blood-stage parasites, magnetically enriched control parasites and 60 K–irradiated parasites were observed by thin section electron microscopy after 24 hours of culture (Figure 3). In the majority of control parasites (56 of 58), the cytoplasm was densely packed with ribosomes, and membranous organelles with distinct outlines were distributed around the cell, with an ordered structure (Figure 3A and and33C). In irradiated cultures, 49% of the parasites (36 of 73) had an abnormal appearance that included sparse cytoplasms with few ribosomes, disorganized and clumped organelles, and, in some cases, abnormally large vacuoles (Figure 3B and and33D). These attributes are consistent with distressed or dying cells. However, in the abnormal parasites from irradiated cultures, the parasite plasma membrane, the parasitophorous vacuole membrane, and the host red blood cell plasma membrane all appeared to be intact.
To elucidate the molecular mechanism of γ-radiation–induced attenuation of parasite growth and survival, microarray analysis was performed in duplicate on RNA isolated from nonirradiated (0 K) versus γ-radiation–treated (15, 30, 60 K) parasite cultures at 3% initial parasitemia. The criterion for altered gene expression in an individual gene induced by γ-irradiation was a statistically significant (P<.05, by 2-tailed Student t test) ≥2-fold increase (upregulation) or decrease (downregulation) in the average ratio of expression between nonirradiated and γ-irradiated cultures.
A total of 185 genes were transcriptionally altered in response to treatment with 15, 30, or 60 K of γ-radiation, with some overlap in gene expression between doses of γ-radiation (Table 1). Of these 185 genes, 85 (45.9%) were upregulated, and 100 (54.1%) were downregulated. This data set of 185 genes with statistically significant findings provides a platform for dissecting the molecular mechanism(s) of γ-radiation–induced attenuation of parasite growth and survival. Additional data can be found in the results section of the Supplementary Materials.
Next, we systematically categorized these 185 transcriptionally altered genes by function to further decipher the biological consequences of γ-irradiation on P. falciparum. A description of some of the biologically relevant pathways altered in response to γ-irradiation is provided below.
Our previous study of the response of P. falciparum to an unrelated stress, febrile temperature, presented evidence for a major downregulation of translation related genes, including those encoding ribosomal proteins . Interestingly, in the current study, at least 8 ribosomal proteins, 1 ribosome assembly GTPase (PFE1435c, Nug1 ortholog), and 1 ribosomal RNA-modifying pseudouridylate synthase were significantly downregulated by irradiation (Tables 1 and and2).2). In contrast, only 1 gene encoding a protein involved in ribosomal biogenesis, the Nop52 ortholog (PFI1510w), was upregulated by irradiation.
Five of the 185 parasite genes (<3% of the genes) showing altered expression in response to radiation are directly related to DNA replication or repair, and interestingly all of them are downregulated. The 5 downregulated genes in our experiments are the origin recognition complex subunit 2 (ORC2) ortholog (PF11_0332), the DNA replication GINS complex protein PSF1 (PFE1175w), one of the replication factor A paralogs (PF11_0332), the topoisomerase II ATPase subunit (PF14_0316), and the Rev1 nucleotidyltransferase (PFI0510c).
We found evidence for transcriptional upregulation of certain signaling mechanisms by γ-irradiation (Tables 1 and and2).2). Of these, the cyclic nucleotide phosphodiesterase (MAL13P1.119) and the EF-hand protein PFF0565c, which is well-conserved across apicomplexan organisms, showed elevated expression. This suggests that the response to γ-irradiation might be channelized via cyclic nucleotide and calcium-dependent signaling networks. Interestingly, we found the protein phosphatase PFI1245c and its activator protein (PF14_0280) to be elevated in expression, whereas 3 protein kinases (MAL7P1.18, PFI0160w, and PFI0100c) were downregulated. Hence, a potential reduction of the specific phosphorylation of proteins might be an aspect of the γ-irradiation response in P. falciparum. Both the phosphatidylinositol polyphosphate phosphatase (PF07_0024) and the inositol hexakisphosphate kinase were downregulated, suggesting reduction of inositol phosphate–based signaling in the γ-irradiation response.
Next, on the basis of our microarray data set, we created a molecular signature of γ-radiation–induced transcriptional changes that could be predictive of parasite growth and attenuation. Real-time PCR was performed in triplicate on pooled RNA isolated from untreated or γ-radiation–treated (60 K) parasite cultures on the following functionally diverse genes: protein phosphatase β (PFI1245c), OTU-like cysteine protease (PFI1135c), nucleolar preribosomal GTPase (PFE1435c), 40S ribosomal protein S7 (PF13_0014), AAA ATPase (PFI0355c), and RNA pseudouridylate synthase (PFL1350w). We found that measurements of changes in messenger RNA (mRNA) abundance by real-time PCR were in accordance with our microarray results (Table 3).
In this article, we provide the first comprehensive study on survival and growth and examine the molecular and cellular alterations in intraerythrocytic P. falciparum treated with high doses of γ-radiation. Exposure to high doses of γ-radiation causes the attenuation and eventual death of intraerythrocytic P. falciparum. At 72 hours after treatment with 60 K of γ-radiation, a 99.5% reduction in parasitemia was observed (Figure 1). However, the parasites that survived the irradiation treatment had a normal growth profile, suggesting that these parasites arose from a few parasite clones that were either unaffected by γ-irradiation or were able to repair the damage and repopulate.
The mechanism of cell death in Plasmodium parasites is poorly understood. For example, apoptosis, a form of programmed cell death, while common in eukaryotic cells, has never been conclusively demonstrated in asexual-stage Plasmodium parasites . In this study, we failed to observe any DNA fragmentation, a hallmark of cell death, using terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick end labeling (TUNEL) analysis in P. falciparum 2 hours after treatment with 60 K of γ-radiation (data not shown). Previously, the presence of TUNEL-positive P. falciparum was reported following treatment at 41°C for 2 hours .
Interestingly, we noted a delay of 24–48 hours before a significant decline in parasitemia in γ-radiation–treated parasites, raising the prospect that, prior to clearance, these parasites might have undergone mitotic catastrophe or senescence. The fact that γ-radiation–treated parasites had significantly fewer distinguishable nuclei per parasite suggested that the cycle of nuclear duplication was perturbed (Figure 2C and and22E, DAPI). γ-irradiation also had an adverse effect on parasite organogenesis and maturation. Irradiated cultures contained fewer RAP1-positive (Figure 2C) and EBA 175–positive parasites, indicating that rhoptry and microneme organelle formation may have been disrupted in these parasites (Figure 2E). Electron microscopy further revealed irradiation-associated structural abnormalities, such as sparse cytoplasms, reduced numbers of ribosomes, disorganized organelles, and, in some instances, large vacuoles (Figure 3B and and33D). While the observation of parasites with fewer nuclei on average may be indicative of mitotic catastrophe, the general disorder of organelles within the cytoplasm and the degree of cell and organelle swelling suggest that these parasites were progressing toward cell death. This interpretation is consistent with a published guideline defining stressed cells in higher eukaryotes .
We also performed the first in-depth molecular characterization of the irradiation response at the transcriptional level in P. falciparum that were treated with sublethal (15 K and 30 K) and lethal (60 K) doses of γ-radiation, and we created a molecular signature of γ-radiation–induced parasite growth attenuation by validating the microarray transcriptional changes of 6 novel biomarkers by real-time PCR. The significance of this signature is evident by the demonstrated role of Pf AAA ATPase in parasite cell death  and of Pf RNA pseudouridylate synthase in parasite growth attenuation .
A major effect of γ-irradiation treatment was a general downregulation of the parasite translation machinery (Tables 1 and and2).2). One such molecule with reduced transcription in response to irradiation was a ribosomal RNA–modifying pseudouridylate synthase. It was recently shown that a pseudouridine synthase is critical for stage conversion of tachyzoites to bradyzoites in Toxoplasma gondii . Similarly, disruption of at least 1 P. falciparum pseudouridylate synthase results in a 47% attenuation of the parasite in in vitro cultures . Hence, the pseudouridylate synthase recovered in this study might be an attractive target for development of mutant attenuated parasites.
One of the few genes related to RNA metabolism that was upregulated by irradiation (2.15 ± 0.06-fold in response to a 30-K dose) encoded Pf LSM12, an Sm superfamily protein with a functional link to translational arrest. LSM12 is a component of stress granules , which are composed of repositories of untranslated mRNAs and 40S ribosomal subunits that form during cellular stress [29, 30]. We propose that γ-irradiation may result in translational arrest with a concomitant accumulation of untranslated mRNAs that may accumulate in stress granules in conjunction with the Pf LSM12 protein.
Another major effect of γ-irradiation is the downregulation of genes directly related to the DNA replication machinery of P. falciparum (Tables 1 and and2).2). Among these, of particular interest is Pf topoisomerase II. Expression and activity of Pf topoisomerase II peaks during the trophozoite/schizont stage when DNA replication occurs , and the cleavage site of Pf topoisomerase II is a highly AT-rich element (approximately 97%) present once per chromosome that has previously been proposed as the site of the parasite centromere . Proliferation of parasites is inhibited by antisense oligonucleotides against the Pf topoisomerase II gene, indicating the importance of this molecule in parasite growth . The downregulation of the Rev1 ortholog is also of considerable interest because it is a key nucleotidyltransferase that is involved in repair of abasic lesions via template-independent addition of dCMP . The downregulation of the DNA replication genes could be a part of the general regression of growth and proliferation, a hypothesis further supported by the modulation of translation-related genes observed in our experiments.
A matter of concern in terms of the safety of γ-radiated vaccines might be that, in our data set, we observed a large set of genes pertaining to cell-surface interactions, host cell remodeling, and secreted proteins being both downregulated and upregulated (eg, rifins, plasmepsin IX, and Rex2; Tables 1 and and2).2). These alterations might result in parasites with different adhesive and host interaction capabilities whose pathological properties remain unclear.
The results reported here provide a platform for further defining the mechanism of γ-radiation–induced attenuation and cell death and the associated biomarkers of parasite attenuation and virulence.
Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org/). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.
Acknowledgments. We thank Dr Anthony Holder, for providing the anti-RAP1 mAb 7H8/50, and Ms Yukiko Kozakai, for help with statistical analysis.
Financial support.This work was supported by the intramural grants from the Food and Drug Administration and National Institutes of Health.
Potential conflicts of interest.All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.