Ribozyme-mediated, multiplex CRISPR gene editing and CRISPR interference (CRISPRi) in rodent-infectious Plasmodium yoelii.

Malaria remains a major global health issue, affecting millions and killing hundreds of thousands of people annually. Efforts to break the transmission cycle of the causal Plasmodium parasite, and to cure those that are afflicted, rely upon functional characterization of genes essential to the parasite's growth and development. These studies are often based upon manipulations of the parasite genome to disrupt or modify a gene of interest to understand its importance and function. However, these approaches can be limited by the availability of selectable markers and the time required to generate transgenic parasites. Moreover, there also is a risk of disrupting native gene regulatory elements with the introduction of exogenous sequences. To address these limitations, we have developed CRISPR-RGR, a Streptococcus pyogenes (Sp)Cas9-based gene editing system for Plasmodium that utilizes a ribozyme-guide-ribozyme (RGR) single guide RNA (sgRNA) expression strategy with RNA polymerase II promoters. Using rodent-infectious Plasmodium yoelii, we demonstrate that both gene disruptions and coding sequence insertions are efficiently generated, producing marker-free parasites with homology arms as short as 80-100 bp. Additionally, we find that the common practice of using one sgRNA can produce both unintended plasmid integration and desired locus replacement editing events, whereas the use of two sgRNAs results in only locus replacement editing. Lastly, we show that CRISPR-RGR can be used for CRISPR interference (CRISPRi) by binding catalytically dead SpCas9 (dSpCas9) to the region upstream of a gene of interest, resulting in a position-dependent, but strand-independent reduction in gene expression. This robust and flexible system facilitates efficient genetic characterizations of rodent-infectious Plasmodium species.

cGAS-mediated control of blood-stage malaria promotes Plasmodium-specific germinal center responses.

Sensing of pathogens by host pattern recognition receptors is essential for activating the immune response during infection. We used a nonlethal murine model of malaria (Plasmodium yoelii 17XNL) to assess the contribution of the pattern recognition receptor cyclic GMP-AMP synthase (cGAS) to the development of humoral immunity. Despite previous reports suggesting a critical, intrinsic role for cGAS in early B cell responses, cGAS-deficient (cGAS-/-) mice had no defect in the early expansion or differentiation of Plasmodium-specific B cells. As the infection proceeded, however, cGAS-/- mice exhibited higher parasite burdens and aberrant germinal center and memory B cell formation when compared with littermate controls. Antimalarial drugs were used to further demonstrate that the disrupted humoral response was not B cell intrinsic but instead was a secondary effect of a loss of parasite control. These findings therefore demonstrate that cGAS-mediated innate-sensing contributes to parasite control but is not intrinsically required for the development of humoral immunity. Our findings highlight the need to consider the indirect effects of pathogen burden in investigations examining how the innate immune system affects the adaptive immune response.

Dynamic control of hepatic Plasmodium numbers by hepcidin despite elevated liver iron during iron supplementation.

Treatment of iron deficiency anemia in malaria endemic areas is complicated as iron supplementation increases malaria risk while malaria decreases iron absorption. Here we measured the influence of hepcidin expression and non-heme iron during iron supplementation on hepatic Plasmodium berghei numbers in anemic and non-anemic mice. Despite elevated hepatic non-heme iron on the high iron diet, elevated hepcidin expression is associated with less parasite bioavailable iron and lower hepatic parasite loads in anemic, iron deficient mice after both two and six weeks of supplementation. A marginal trend to lower parasite hepatic numbers was seen in non-anemic, iron replete mice. In a transgenic model of severe anemia, mice with a deletion in Sec15l1, which reportedly have normal liver iron and normal hepcidin expression, there were no changes in liver parasite numbers or blood stage numbers or outcome in the lethal Plasmodium yoelii model. In summary during iron supplementation the lower hepatic malaria numbers are regulated more by hepcidin than the absolute level of non-heme hepatic iron.

Isolation of invasive Plasmodium yoelii merozoites with a long half-life to evaluate invasion dynamics and potential invasion inhibitors.

Malaria symptoms and pathogenesis are caused by blood stage parasite burdens of Plasmodium spp., for which invasion of red blood cells (RBCs) by merozoites is essential. Successful targeting by either drugs or vaccines directed against the whole merozoite or its antigens during its transient extracellular status would contribute to malaria control by impeding RBC invasion. To understand merozoite invasion biology and mechanisms, it is desired to obtain merozoites that retain their invasion activity in vitro. Accordingly, methods have been developed to isolate invasive Plasmodium knowlesi and Plasmodium falciparum merozoites. Rodent malaria parasite models offer ease in laboratory maintenance and experimental genetic modifications; however, no methods have been reported regarding isolation of high numbers of invasive rodent malaria merozoites. In this study, Plasmodium yoelii-infected RBCs were obtained from infected mice, and mature schizont-infected RBCs enriched via Histodenz™ density gradients. Merozoites retaining invasion activity were then isolated by passing the preparations through a filter membrane. RBC-invaded parasites developed to mature stages in vitro in a synchronous manner. Isolated merozoites were evaluated for retention of invasion activity following storage at different temperatures prior to incubation with uninfected mouse RBCs. Isolated merozoites retained their invasion activity 4h after isolation at 10 or 15 °C, whereas their invasion activity reduced to 0-10% within 30 min when incubated on ice or at 37 °C prior to RBC invasion assay. Images of merozoites at successive steps during RBC invasion were captured by light and transmission electron microscopy. Synthetic peptides derived from the amino acid sequence of the P. yoelii invasion protein RON2 efficiently inhibited RBC invasion. The developed method to isolate and keep invasive P. yoelii merozoites for up to 4h is a powerful tool to study the RBC invasion biology of this parasite. This method provides an important platform to evaluate the mode of action of drugs and vaccine candidates targeting the RBC invasion steps using rodent malaria model.

Detection of Plasmodium berghei and Plasmodium yoelii Liver-Stage Parasite Burden by Quantitative Real-Time PCR.

Direct detection and quantification of liver-stage Plasmodium parasites became possible with the development of quantitative real-time PCR (qPCR). Here we describe the measurement of parasite burden in the livers of mice infected with the rodent malaria species, Plasmodium berghei and Plasmodium yoelii. This method is based on detection of expression of parasite ribosomal 18S RNA and can serve as an endpoint to accurately evaluate the efficacy of vaccines targeting the preerythrocytic stages of malaria. This approach is fast and highly reproducible and allows quantification of liver-stage parasite burden in different mouse strains and different Plasmodium species after infection with a range of sporozoite challenge doses.

Assessment of real-time method to detect liver parasite burden under different experimental conditions in mice infected with Plasmodium yoelii sporozoites.

Use of highly specific, sensitive and quantitative Real-Time PCR (qRT-PCR) based methods greatly facilitate the monitoring of experimental drug intervention and vaccination efficacy targeting liver stage malaria parasite. Here, in this study we have used qRT-PCR to detect the growing liver stage parasites following inoculation of Plasmodium yoelii sporozoite. Route of sporozoite administration and size of the sporozoite inoculums are two major determinants that affect the liver stage parasite load and therefore its detection and quantification. Thus, these factors need to be addressed to determine the accuracy of detection and quantification of Real-Time PCR method. Furthermore, applicability of quantitative RT-PCR system needs to be confirmed by analyzing the effect of different antimalarials on liver stage parasite burden. We have observed that parasite burden in mice infected via intravenous route was higher compared to that in subcutaneous, intradermal and intraperitoneal route infected mice. Moreover, this method detected liver stage parasite load with as low as 50 sporozoites. The inhibition studies with primaquine and atovaquone revealed inhibition of liver stage parasite and well correlated with patency and course of blood stage infection. This study characterized the simplicity, accuracy, and quantitative analysis of liver stage parasite development by real time PCR under different experimental conditions. Use of real time PCR method greatly improves the reproducibility and applicability to estimate the efficacy and potency of vaccine or drug candidates targeting liver stage parasite.

Co-infection restrains Litomosoides sigmodontis filarial load and plasmodial P. yoelii but not P. chabaudi parasitaemia in mice.

Infection with multiple parasite species is clearly the norm rather than the exception, in animals as well as in humans. Filarial nematodes and Plasmodium spp. are important parasites in human public health and they are often co-endemic. Interactions between these parasites are complex. The mechanisms underlying the modulation of both the course of malaria and the outcome of filarial infection are poorly understood. Despite increasing activity in recent years, studies comparing co- and mono-infections are very much in their infancy and results are contradictory at first sight. In this study we performed controlled and simultaneous co-infections of BALB/c mice with Litomosoides sigmodontis filaria and with Plasmodium spp. (Plasmodium yoelii 17 XNL or Plasmodium chabaudi 864VD). An analysis of pathological lesions in the kidneys and lungs and a parasitological study were conducted at different times of infection. Whatever the plasmodial species, the filarial recovery rate was strongly decreased. The peak of parasitaemia in the plasmodial infection was decreased in the course of P. yoelii infection but not in that of P. chabaudi. Regarding pathological lesions, L. sigmodontis can reverse lesions in the kidneys due to the presence of both Plasmodium species but does not modify the course of pulmonary lesions. The filarial infection induces granulomas in the lungs.

Larval nutrition differentially affects adult fitness and Plasmodium development in the malaria vectors Anopheles gambiae and Anopheles stephensi.

BACKGROUND: Mosquito fitness is determined largely by body size and nutritional reserves. Plasmodium infections in the mosquito and resultant transmission of malaria parasites might be compromised by the vector's nutritional status. We studied the effects of nutritional stress and malaria parasite infections on transmission fitness of Anopheles mosquitoes. METHODS: Larvae of Anopheles gambiae sensu stricto and An. stephensi were reared at constant density but with nutritionally low and high diets. Fitness of adult mosquitoes resulting from each dietary class was assessed by measuring body size and lipid, protein and glycogen content. The size of the first blood meal was estimated by protein analysis. Mosquitoes of each dietary class were fed upon a Plasmodium yoelii nigeriensis-infected mouse, and parasite infections were determined 5 d after the infectious blood meal by dissection of the midguts and by counting oocysts. The impact of Plasmodium infections on gonotrophic development was established by dissection. RESULTS: Mosquitoes raised under low and high diets emerged as adults of different size classes comparable between An. gambiae and An. stephensi. In both species low-diet females contained less protein, lipid and glycogen upon emergence than high-diet mosquitoes. The quantity of larval diet impacted strongly upon adult blood feeding and reproductive success. The prevalence and intensity of P. yoelii nigeriensis infections were reduced in low-diet mosquitoes of both species, but P. yoelii nigeriensis impacted negatively only on low-diet, small-sized An. gambiae considering survival and egg maturation. There was no measurable fitness effect of P. yoelii nigeriensis on An. stephensi. CONCLUSIONS: Under the experimental conditions, small-sized An. gambiae expressed high mortality, possibly caused by Plasmodium infections, the species showing distinct physiological concessions when nutrionally challenged in contrast to well-fed, larger siblings. Conversely, An. stephensi was a robust, successful vector regardless of its nutrional status upon emergence. The data suggest that small-sized An. gambiae, therefore, would contribute little to malaria transmission, whereas this size effect would not affect An. stephensi.

Insights into the preclinical treatment of blood-stage malaria by the antibiotic borrelidin.

BACKGROUND AND PURPOSE: Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria. EXPERIMENTAL APPROACH: Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed. KEY RESULTS: Borrelidin protected against lethal malaria at 0.25 mg·kg⁻¹·day⁻¹. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts. CONCLUSIONS AND IMPLICATIONS: Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.

A rapid and scalable density gradient purification method for Plasmodium sporozoites.

BACKGROUND: Malaria remains a major human health problem, with no licensed vaccine currently available. Malaria infections initiate when infectious Plasmodium sporozoites are transmitted by Anopheline mosquitoes during their blood meal. Investigations of the malaria sporozoite are, therefore, of clear medical importance. However, sporozoites can only be produced in and isolated from mosquitoes, and their isolation results in large amounts of accompanying mosquito debris and contaminating microbes. METHODS: Here is described a discontinuous density gradient purification method for Plasmodium sporozoites that maintains parasite infectivity in vitro and in vivo and greatly reduces mosquito and microbial contaminants. RESULTS: This method provides clear advantages over previous approaches: it is rapid, requires no serum components, and can be scaled to purify >107 sporozoites with minimal operator involvement. Moreover, it can be effectively applied to both human (Plasmodium falciparum, Plasmodium vivax) and rodent (Plasmodium yoelii) infective species with excellent recovery rates. CONCLUSIONS: This novel method effectively purifies viable malaria sporozoites by greatly reducing contaminating mosquito debris and microbial burdens associated with parasite isolation. Large-scale preparations of purified sporozoites will allow for enhanced in vitro infections, proteomics, and biochemical characterizations. In conjunction with aseptic mosquito rearing techniques, this purification technique will also support production of live attenuated sporozoites for vaccination.

Impact of intravascular hemolysis in malaria on liver dysfunction: involvement of hepatic free heme overload, NF-κB activation, and neutrophil infiltration.

We have investigated the impact of persistent intravascular hemolysis on liver dysfunction using the mouse malaria model. Intravascular hemolysis showed a positive correlation with liver damage along with the increased accumulation of free heme and reactive oxidants in liver. Hepatocytes overinduced heme oxygenase-1 (HO-1) to catabolize free heme in building up defense against this pro-oxidant milieu. However, in a condition of persistent free heme overload in malaria, the overactivity of HO-1 resulted in continuous transient generation of free iron to favor production of reactive oxidants as evident from 2',7'-dichlorofluorescein fluorescence studies. Electrophoretic mobility shift assay documented the activation of NF-κB, which in turn up-regulated intercellular adhesion molecule 1 as evident from chromatin immunoprecipitation studies. NF-κB activation also induced vascular cell adhesion molecule 1, keratinocyte chemoattractant, and macrophage inflammatory protein 2, which favored neutrophil extravasation and adhesion in liver. The infiltration of neutrophils correlated positively with the severity of hemolysis, and neutrophil depletion significantly prevented liver damage. The data further documented the elevation of serum TNFα in infected mice, and the treatment of anti-TNFα antibodies also significantly prevented neutrophil infiltration and liver injury. Deferoxamine, which chelates iron, interacts with free heme and bears antioxidant properties that prevented oxidative stress, NF-κB activation, neutrophil infiltration, hepatocyte apoptosis, and liver damage. Furthermore, the administration of N-acetylcysteine also prevented NF-κB activation, neutrophil infiltration, hepatocyte apoptosis, and liver damage. Thus, hepatic free heme accumulation, TNFα release, oxidative stress, and NF-κB activation established a link to favor neutrophil infiltration in inducing liver damage during hemolytic conditions in malaria.

mRNA expression of cytokines and its impact on outcomes after infection with lethal and nonlethal Plasmodium vinckei parasites.

Cytokines play an important role in the defense against malaria and some have long been documented to influence the course of malaria infection in rodents and humans. The present study was conducted to determine the mRNA expression pattern of a few prominent cytokines at different time points during the course of infection with a nonlethal and lethal Plasmodium vinckei rodent malaria parasite, using highly sensitive real-time PCR. Analysis of mRNA expression of cytokines in spleen from infected mice revealed that the principal difference was an early depletion in pro-inflammatory cytokine's mRNA expression in mice infected with lethal P. vinckei (PvAS) parasites. In addition, an increase in anti-inflammatory cytokines particularly IL-10 mRNA expression levels was found in the same group of mice. In contrast, the significant rise in pro-inflammatory cytokine's mRNA expression levels was recorded at day 1 onwards after infection with nonlethal P. vinckei (PvAR). The maximum fold change was recorded for IFN-γ and IL-10, when compared to baseline value. TGF-ß did not seem to play any major role in P. vinckei infection.

Detection of malarial byproduct hemozoin utilizing its unique scattering properties.

The scattering characteristics of the malaria byproduct hemozoin, including its scattering distribution and depolarization, are modeled using Discrete Dipole Approximation (DDA) and compared to those of healthy red blood cells. Scattering (or dark-field) spectroscopy and imaging are used to identify hemozoin in fresh rodent blood samples. A new detection method is proposed and demonstrated using dark-field in conjunction with cross-polarization imaging and spectroscopy. SNRs greater than 50:1 are achieved for hemozoin in fresh blood without the addition of stains or reagents. The potential of such a detection system is discussed.

Daily Plasmodium yoelii infective mosquito bites do not generate protection or suppress previous immunity against the liver stage.

BACKGROUND: Human populations that are naturally subjected to Plasmodium infection do not acquire complete protection against the liver stage of this parasite despite prolonged and frequent exposure. However, sterile immunity against Plasmodium liver stage can be achieved after repeated exposure to radiation attenuated sporozoites. The reasons for this different response remain largely unknown, but a suppressive effect of blood stage Plasmodium infection has been proposed as a cause for the lack of liver stage protection. METHODS: Using Plasmodium yoelii 17XNL, the response generated in mice subjected to daily infective bites from normal or irradiated mosquitoes was compared. The effect of daily-infected mosquito bites on mice that were previously immunized against P. yoelii liver stage was also studied. RESULTS: It was observed that while the bites of normal infected mosquitoes do not generate strong antibody responses and protection, the bites of irradiated mosquitoes result in high levels of anti-sporozoite antibodies and protection against liver stage Plasmodium infection. Exposure to daily infected mosquito bites did not eliminate the protection acquired previously with a experimental liver stage vaccine. CONCLUSIONS: Liver stage immunity generated by irradiated versus normal P. yoelii infected mosquitoes is essentially different, probably because of the blood stage infection that follows normal mosquito bites, but not irradiated. While infective mosquito bites do not induce a protective liver stage response, they also do not interfere with previously acquired liver stage protective responses, even if they induce a complete blood stage infection. Considering that the recently generated anti-malaria vaccines induce only partial protection against infection, it is encouraging that, at least in mouse models, immunity is not negatively affected by subsequent exposure and infection with the parasite.

Automated estimation of parasitaemia of Plasmodium yoelii-infected mice by digital image analysis of Giemsa-stained thin blood smears.

BACKGROUND: Parasitaemia, the percentage of infected erythrocytes, is used to measure progress of experimental Plasmodium infection in infected hosts. The most widely used technique for parasitaemia determination is manual microscopic enumeration of Giemsa-stained blood films. This process is onerous, time consuming and relies on the expertise of the experimenter giving rise to person-to-person variability. Here the development of image-analysis software, named Plasmodium AutoCount, which can automatically generate parasitaemia values from Plasmodium-infected blood smears, is reported. METHODS: Giemsa-stained blood smear images were captured with a camera attached to a microscope and analysed using a programme written in the Python programming language. The programme design involved foreground detection, cell and infection detection, and spurious hit filtering. A number of parameters were adjusted by a calibration process using a set of representative images. Another programme, Counting Aid, written in Visual Basic, was developed to aid manual counting when the quality of blood smear preparation is too poor for use with the automated programme. RESULTS: This programme has been validated for use in estimation of parasitemia in mouse infection by Plasmodium yoelii and used to monitor parasitaemia on a daily basis for an entire challenge infection. The parasitaemia values determined by Plasmodium AutoCount were shown to be highly correlated with the results obtained by manual counting, and the discrepancy between automated and manual counting results were comparable to those found among manual counts of different experimenters. CONCLUSIONS: Plasmodium AutoCount has proven to be a useful tool for rapid and accurate determination of parasitaemia from infected mouse blood. For greater accuracy when smear quality is poor, Plasmodium AutoCount, can be used in conjunction with Counting Aid.

Noninvasive real-time monitoring of liver-stage development of bioluminescent Plasmodium parasites.

BACKGROUND: The morbidity and mortality associated with malaria are heightened because of the spread of drug-resistant parasites and the lack of an effective vaccine. Plasmodium liver stages are the targets of new chemotherapeutics and vaccines, but there are limited tools available to study this stage in vivo. METHODS: To overcome this obstacle, we developed a method with which to study Plasmodium liver stages by means of bioluminescent imaging (BLI) of the rodent malaria parasite Plasmodium yoelii. We created a P. yoelii YM strain (PyLuc) that stably expresses firefly luciferase driven by a constitutive promoter. RESULTS: Using BLI, we performed imaging of the Plasmodium liver stages of mice infected with PyLuc sporozoites and monitored parasite dissemination during blood-stage infection. Because PyLuc luciferase activity is proportional to the number of parasites, BLI can be used to quantify the effect of drugs on liver-stage development. Moreover, using BLI, we demonstrated that immunization with blood-stage parasites confers partial protective immunity against the development of liver stages. CONCLUSIONS: BLI is a noninvasive technique that is useful for screening potential drugs and candidate vaccines with which to combat malaria. The prospect of cross-stage protective immunity increases the number of avenues to be explored in the development of an effective vaccine against malaria.

Hundreds of microsatellites for genotyping Plasmodium yoelii parasites.

Genetic crosses have been employed to study various traits of rodent malaria parasites and to locate loci that contribute to drug resistance, immune protection, and disease virulence. Compared with human malaria parasites, genetic crossing of rodent malaria parasites is more easily performed; however, genotyping methods using microsatellites (MSs) or large-scale single nucleotide polymorphisms (SNPs) that have been widely used in typing Plasmodium falciparum are not available for rodent malaria species. Here we report a genome-wide search of the Plasmodium yoelii yoelii (P. yoelii) genome for simple sequence repeats (SSRs) and the identification of nearly 600 polymorphic MS markers for typing the genomes of P. yoelii and Plasmodium berghei. The MS markers are randomly distributed across the 14 physical chromosomes assembled from genome sequences of three rodent malaria species, although some variations in the numbers of MS expected according to chromosome size exist. The majority of the MS markers are AT-rich repeats, similar to those found in the P. falciparum genome. The MS markers provide an important resource for genotyping, lay a foundation for developing linkage maps, and will greatly facilitate genetic studies of P. yoelii.

Thermal behaviour of Anopheles stephensi in response to infection with malaria and fungal entomopathogens.

BACKGROUND: Temperature is a critical determinant of the development of malaria parasites in mosquitoes, and hence the geographic distribution of malaria risk, but little is known about the thermal preferences of Anopheles. A number of other insects modify their thermal behaviour in response to infection. These alterations can be beneficial for the insect or for the infectious agent. Given current interest in developing fungal biopesticides for control of mosquitoes, Anopheles stephensi were examined to test whether mosquitoes showed thermally-mediated behaviour in response to infection with fungal entomopathogens and the rodent malaria, Plasmodium yoelii. METHODS: Over two experiments, groups of An. stephensi were infected with one of three entomopathogenic fungi, and/or P. yoelii. Infected and uninfected mosquitoes were released on to a thermal gradient (14 - 38 degrees C) for "snapshot" assessments of thermal preference during the first five days post-infection. Mosquito survival was monitored for eight days and, where appropriate, oocyst prevalence and intensity was assessed. RESULTS AND CONCLUSION: Both infected and uninfected An. stephensi showed a non-random distribution on the gradient, indicating some capacity to behaviourally thermoregulate. However, chosen resting temperatures were not altered by any of the infections. There is thus no evidence that thermally-mediated behaviours play a role in determining malaria prevalence or that they will influence the performance of fungal biopesticides against adult Anopheles.

Cross-protection between attenuated Plasmodium berghei and P. yoelii sporozoites.

An attenuated Plasmodium falciparum sporozoite (PfSPZ) vaccine is under development, in part, based on studies in mice with P. berghei. We used P. berghei and P. yoelii to study vaccine-induced protection against challenge with a species of parasite different from the immunizing parasite in BALB/c mice. One-hundred percent of mice were protected against homologous challenge. Seventy-nine percent immunized with attenuated P. berghei sporozoite (PbSPZ) (six experiments) were protected against challenge with P. yoelii sporozoite (PySPZ), and 63% immunized with attenuated PySPZ (three experiments) were protected against challenge with PbSPZ. Antibodies in sera of immunized mice only recognized homologous sporozoites and could not have mediated protection against heterologous challenge. Immunization with attenuated PySPZ or PbSPZ induced CD8+ T cell-dependent protection against heterologous challenge. Immunization with attenuated PySPZ induced CD8+ T cell-dependent protection against homologous challenge. However, homologous protection induced by attenuated PbSPZ was not dependent on CD8+ or CD4+ T cells, and depletion of both populations only reduced protection by 36%. Immunization of C57BL/10 mice with PbSPZ induced CD8+ T cell-dependent protection against P. berghei, but no protection against P. yoelii. The cross-protection data in BALB/c mice support testing a human vaccine based on attenuated PfSPZ for its efficacy against P. vivax.

Sub-cellular localization and post-translational modifications of the Plasmodium yoelii enolase suggest moonlighting functions.

BACKGROUND: Enolase (2-Phospho-D-glycerate hydrolase; EC 4.2.1.11) is one of the glycolytic enzymes, whose levels are highly elevated in malaria parasite infected red blood cells. In several organisms, enolases have been shown to have diverse non glycolytic (moonlighting) biological functions. As functional diversity of a protein would require diverse sub-cellular localization, the possibility of involvement of Plasmodium enolase in moonlighting functions was examined by investigating its sub-cellular distribution in the murine malarial parasite, Plasmodium yoelii. METHODS: Cellular extracts of P. yoelii were fractionated in to soluble (cytosolic) and particulate (membranes, nuclear and cytoskeletal) fractions and were analysed by one and two-dimensional gel electrophoresis. These were probed by Western blotting using antibodies raised against recombinant Plasmodium falciparum enolase. Immunofluorescence assay was used for in situ localization. Fe+3 based metal affinity chromatography was used to isolate the phospho-proteome fraction from P. yoelii extracts. RESULTS: Apart from the expected presence of enolase in cytosol, this enzyme was also found to be associated with membranes, nuclei and cytoskeletal fractions. Nuclear presence was also confirmed by in situ immunofluorescence. Five different post translationally modified isoforms of enolase could be identified, of which at least three were due to the phosphorylation of the native form. in situ phosphorylation of enolase was also evident from the presence of enolase in purified phosphor-proteome of P. yoelii. Different sub-cellular fractions showed different isoform profiles. CONCLUSION: Association of enolase with nuclei, cell membranes and cytoskeletal elements suggests non-glycolytic functions for this enzyme in P. yoelii. Sub-cellular fraction specific isoform profiles indicate the importance of post-translational modifications in diverse localization of enolase in P. yoelii. Further, it is suggested that post-translational modifications of enolase may govern the recruitment of enolase for non-glycolytic functions.