Search

The biology of Toxoplasma gondii, the causative pathogen of one of the most widespread parasitic diseases (toxoplasmosis), remains poorly understood. Lactate, which is derived from glucose metabolism, is not only an energy source in a variety of organisms, including T. gondii, but also a regulatory molecule that participates in gene activation and protein function. Lysine lactylation (Kla) is a type of post-translational modifications (PTMs) that has been recently associated with chromatin remodeling; however, Kla of histone and non-histone proteins has not yet been studied in T. gondii. To examine the prevalence and function of lactylation in T. gondii parasites, we mapped the lactylome of proliferating tachyzoite cells and identified 1964 Kla sites on 955 proteins in the T. gondii RH strain. Lactylated proteins were distributed in multiple subcellular compartments and were closely related to a wide variety of biological processes, including mRNA splicing, glycolysis, aminoacyl-tRNA biosynthesis, RNA transport, and many signaling pathways. We also performed a chromatin immunoprecipitation sequencing (ChIP-seq) analysis using a lactylation-specific antibody and found that the histones H4K12la and H3K14la were enriched in the promoter and exon regions of T. gondii associated with microtubule-based movement and cell invasion. We further confirmed the delactylase activity of histone deacetylases TgHDAC2-4, and found that treatment with anti-histone acetyltransferase (TgMYST-A) antibodies profoundly reduced protein lactylation in T. gondii. This study offers the first dataset of the global lactylation proteome and provides a basis for further dissecting the functional biology of T. gondii., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Babesiosis poses a serious threat to immunocompromised individuals and the major etiological species of Babesia for human babesiosis is Babesia microti . Merozoites are a critical stage in the life cycle of Babesia microti . Several merozoite proteins have been demonstrated to play important roles in this process; however, most of the merozoite proteins of B. microti remain unknown. In the present study, we identified a novel merozoite protein of B. microti with similar structure to the thioredoxin (Trx)-like domain of the Trx family, which was named as B. microti Trx-like protein ( Bm TLP). Western blot assays demonstrated that this protein was expressed by B. microti during the erythrocytic infection process, and its expression peaked on day 7 post-infection in vivo . Immunofluorescence assay further showed that this protein is mainly expressed in B. microti merozoites. Bm TLP hold both heparin- and erythrocyte-binding properties, which are critical functions of invasion-related proteins. Immunization with recombinant Bm TLP imparted significant protection against B. microti infection in mice. Taken together, these results suggest that the novel merozoite protein, Bm TLP, is an important pathogenic molecule of B. microti and may be a possible target for the design of babesiosis control strategy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Piao, Ma, Liu, Hou and Chen.)

African trypanosomatid parasites escape host acquired immune responses through periodic antigenic variation of their surface coat. In this study, we describe a mechanism by which the parasites counteract innate immune responses. Two TatD DNases were identified in each of Trypanosoma evansi and Trypanosoma brucei. These DNases are bivalent metal-dependent endonucleases localized in the cytoplasm and flagella of the parasites that can also be secreted by the parasites. These enzymes possess conserved functional domains and have efficient DNA hydrolysis activity. Host neutrophil extracellular traps (NETs) induced by the parasites could be hydrolyzed by native and recombinant TatD DNases. NET disruption was prevented, and the survival rate of parasites was decreased, in the presence of the DNase inhibitor aurintricarboxylic acid. These data suggest that trypanosomes can counteract host innate immune responses by active secretion of TatD DNases to degrade NETs.

Malaria elimination is still pending on the development of novel tools that rely on a deep understanding of parasite biology. Proteins of all living cells undergo myriad posttranslational modifications (PTMs) that are critical to multifarious life processes. An extensive proteome-wide dissection revealed a fine PTM map of most proteins in both Plasmodium falciparum, the causative agent of severe malaria, and the infected red blood cells. More than two-thirds of proteins of the parasite and its host cell underwent extensive and dynamic modification throughout the erythrocytic developmental stage. PTMs critically modulate the virulence factors involved in the host-parasite interaction and pathogenesis. Furthermore, P. falciparum stabilized the supporting proteins of erythrocyte origin by selective demodification. Collectively, our multiple omic analyses, apart from having furthered a deep understanding of the systems biology of P. falciparum and malaria pathogenesis, provide a valuable resource for mining new antimalarial targets., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Haem is a key metabolic factor in the life cycle of the malaria parasite. In the blood stage, the parasite acquires host haemoglobin to generate amino acids for protein synthesis and the by-product haem for metabolic use. The malaria parasite can also synthesize haem de novo on its own. Plasmodium falciparum-specific histidine-rich protein 2 (PfHRP2) has a haem-binding site to mediate the formation of haemozoin, a biocrystallized form of haem aggregates. Notably, the gene regulates the mechanism of haemoglobin-derived haem metabolism and the de novo haem biosynthetic pathway in the Pfhrp2-disrupted parasite line during the intraerythrocytic stages., Methods: The CRISPR/Cas9 system was used to disrupt the gene locus of Pfhrp2. DNA was extracted from the transgenic parasite, and PCR, Southern blotting and Western blotting were used to confirm the establishment of transgenic parasites. RNA-sequencing and comparative transcriptome analysis were performed to identify differences in gene expression between 3D7 and Pfhrp2 - -3D7 parasites., Results: Pfhrp2 - transgenic parasites were successfully established by the CRISPR/Cas9 system. A total of 964, 1261, 3138, 1064, 2512 and 1778 differentially expressed genes (DEGs) were identified in the six comparison groups, respectively, with 373, 520, 1499, 353, 1253 and 742 of these DEGs upregulated and 591, 741, 1639, 711, 1259 and 1036 of them downregulated, respectively. Five DEGs related to haem metabolism and synthesis were identified in the comparison groups at six time points (0, 8, 16, 24, 32, and 40 h after merozoite invasion). The genes encoding delta-aminolevulinic acid synthetase and ferrochelatase, both related to haem biosynthesis, were found to be significantly upregulated in the comparison groups, and those encoding haem oxygenase, stromal-processing peptidase and porphobilinogen deaminase were found to be significantly downregulated. No GO terms were significantly enriched in haem-related processes (Q value = 1)., Conclusion: Our data revealed changes in the transcriptome expression profile of the Pfhrp2 - -3D7 parasite during the intraerythrocytic stages. The findings provide insight at the gene transcript level that will facilitate further research on and development of anti-malaria drugs.

Induction of humoural immunity is critical for clinical protection against malaria. More than 100 malaria vaccine candidates have been investigated at different developmental stages, but with limited protection. One of the roadblocks constrains the development of malaria vaccines is the poor immunogenicity of the antigens. The objective of this study was to map the linear B-cell epitopes of the Plasmodium falciparum erythrocyte invasion-associated antigens with a purpose of understanding humoural responses and protection. We conducted a large-scale screen using overlapping peptide microarrays of 37 proteins from the P. falciparum parasite, most of which are invasion-associated antigens which have been tested in clinical settings as vaccine candidates, with sera from individuals with various infection episodes. Analysis of the epitome of the antigens revealed that the most immunogenic epitopes were predominantly located in the low-complexity regions of the proteins containing repetitive and/or glutamate-rich motifs in different sequence contexts. However, in vitro assay showed the antibodies specific for these epitopes did not show invasion inhibitory effect. These discoveries indicated that the low-complexity regions of the parasite proteins might drive immune responses away from functional domains, which may be an instructive finding for the rational design of vaccine candidates., (Copyright © 2020 Hou, Jiang, Ma, Zou, Piao, Liu and Chen.)

Toxoplasma gondii is a unicellular protozoan parasite of the phylum Apicomplexa. The parasite repeatedly goes through a cycle of invasion, division and induction of host cell rupture, which is an obligatory process for proliferation inside warm-blooded animals. It is known that the biology of the parasite is controlled by a variety of mechanisms ranging from genomic to epigenetic to transcriptional regulation. In this study, we investigated the global protein posttranslational lysine crotonylation and 2-hydroxyisobutyrylation of two T. gondii strains, RH and ME49, which represent distinct phenotypes for proliferation and pathogenicity in the host. Proteins with differential expression and modification patterns associated with parasite phenotypes were identified. Many proteins in T. gondii were crotonylated and 2-hydroxyisobutyrylated, and they were localized in diverse subcellular compartments involved in a wide variety of cellular functions such as motility, host invasion, metabolism and epigenetic gene regulation. These findings suggest that lysine crotonylation and 2-hydroxyisobutyrylation are ubiquitous throughout the T. gondii proteome, regulating critical functions of the modified proteins. These data provide a basis for identifying important proteins associated with parasite development and pathogenicity., (© 2019 Yin et al.)

Toxoplasmosis is a worldwide zoonosis caused by the protozoan parasite Toxoplasma gondii, an obligate intracellular parasite. Currently, no viable vaccine or effective drug strategies exist to prevent and control toxoplasmosis. T. gondii microneme protein 3 (MIC3), rhoptry protein 9 (ROP9), and surface antigen 2 (SAG2) are related to active invasion of the parasite. Hence, we constructed recombinant adenoviruses expressing TgMIC3, TgSAG2, or TgROP9 and evaluated the recombinant adenoviruses as potential vaccines against acute T. gondii infection in BALB/c mice. Mice immunized with the recombinant adenoviruses were measured the antibody levels, percentages of lymphocytes and actived T lymphocytes, cytokine productions, and the survival rates and time to evaluate the protective efficacy. Results showed that immunization with the bivalent or trivalent recombiant adenoviruses could strongly stimulate humoral and cellular immune responses in mice, resulting in effective immune protections against lethal challenge with the tachyzoites of T. gondii RH. These results indicated that the divalent and trivalent adenoviruses, especially Ad-ROP9-MIC3-EGFP, may be promising vaccine candidates against acute T. gondii infection., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: In recent years, human infection by the simian malaria parasite Plasmodium knowlesi has increased in Southeast Asia, leading to growing concerns regarding the cross-species spread of the parasite. Consequently, a deeper understanding of the biology of P. knowlesi is necessary in order to develop tools for control of the emerging disease. TatD-like DNase expressed at the surface of P. falciparum has recently been shown to counteract host innate immunity and is thus a potential malaria vaccine candidate., Methods: The expression of the TatD DNase of P. knowlesi (PkTatD) was confirmed by both Western-blot and immunofluorescent assay. The DNA catalytic function of the PkTatD was confirmed by digestion of DNA with the recombinant PkTatD protein in the presence of various irons., Results: In the present study, we investigated the expression of the homologous DNase in P. knowlesi. The expression of TatD-like DNase in P. knowslesi (PkTatD) was verified by Western blot and indirect immunofluorescence assays. Like that of the P. falciparum parasite, PkTatD was also found to be located on the surface of erythrocytes infected by the parasites. Biochemical analysis indicated that PkTatD can hydrolyze DNA and this activity is magnesium-dependent., Conclusions: We identified that PkTatD expressed on the surface of P. knowlesi-infected RBCs is a Mg 2+ -dependent DNase and exhibits a stronger hydrolytic capacity than TatD from P. falciparum. The data support our previous findings that TatD-like DNase is a unanimously expressed virulence factor of Plasmodium parasites.

Background: Plasmodium falciparum is the most virulent parasite of the five Plasmodium species that cause human malaria, and biological analysis of the parasite is critical for the development of novel strategies for disease control. DNA endonucleases are important for maintaining the biological activity, gene stability of the parasite and interaction with host immune systems. In this study, ten sequences of DNA endonucleases were found in the genome of P. falciparum 3D7 clone, seven of them were predicted to contain an endonuclease/exonuclease/phosphatase (IPR005135) domain which plays an important role in DNA catalytic activity. The seven DNA endonucleases of P. falciparum were systematically investigated., Methods: Plasmodium falciparum 3D7 clone was cultured in human O + RBCs, RNA was extracted at 8, 16, 24, 32, 40, and 48 h post invasion and real-time quantitative PCR was carried out to analyse the transcription of the seven DNA endonuclease genes in asexual stages. Immunofluorescence assay was carried out to confirm the location of the encoded proteins expressed in the erythrocytic stages. Finally, the catalytic activity of the DNA nucleases were tested., Results: Of the seven proteins analysed, two proteins were not soluble. Fragments derived from the rest five endonuclease sequences were successfully expressed as soluble proteins, and which were used to generate antisera for protein localization. The proteins were all located in the nucleus at ring and trophozoite stages. While at schizont stage, proteins encoded by PF3D7_1238600, PF3D7_0107200 and PF3D7_0319200 were in the punctuated forms in the parasite most likely around nuclei of the merozoites. But the proteins encoded by PF3D7_0305600 and PF3D7_1363500 were distributed around the infected erythrocyte membrane. The enzymatic activity of the recombinant GST-PF3D7_1238600 was very efficient without divalent iron, while the activity of the rest four enzymes was iron dependent. Further, divalent irons did not show any specific enhancement on the activity of GST-PF3D7_1238600, but the activity of GST-PF3D7_0107200, GST-PF3D7_1363500 and GST-PF3D7_0319200 were Cu 2+ dependent. The activity of GST-PF3D7_0305600 was dependent on Mg 2+ and Mn 2+ . Except GST-PF3D7_1363500, four of the GST tagged recombinant proteins hydrolysed the supercoiled circular plasmid DNA with or without divalent metal ions. The GST-PF3D7_1363500 protein only changed the supercoiled circular plasmid DNA into nicked plasmids, even with Cu 2+ ., Conclusions: Fragments derived from five of the endonuclease sequences of P. falciparum 3D7 clone were successfully expressed. The proteins displayed diverse cell distribution, biochemical and enzymatic activities, which indicated that they carried different biological function in the development of the parasite in the erythrocytes. The DNA repair and DNA degradation capacity of the DNA endonucleases in the biology of the parasite remained further studied.

Background: Individuals living in endemic areas gradually acquire natural immunity to clinical malaria, largely dependent on antibodies against parasite antigens. There are many studies indicating that the variant antigen PfEMP1 at the surface of the parasitized red blood cell (pRBC) is one of the major targets of the immune response. It is believed that antibodies against PfEMP1 confer protection by blocking sequestration (rosetting and cytoadherence), inducing antibody-dependent cellular-inhibitory effect and opsonizing pRBCs for phagocytosis., Methods: A recombinant NTS-DBL1α domain from a rosette-mediating PfEMP1 was expressed in Escherichia coli. The resulting protein was purified and used for immunization to generate polyclonal (goat) and monoclonal (mouse) antibodies. The antibodies' ability to opsonize and induce phagocytosis in vitro was tested and contrasted with the presence of opsonizing antibodies naturally acquired during Plasmodium falciparum infection., Results: All antibodies recognized the recombinant antigen and the surface of live pRBCs, however, their capacity to opsonize the pRBCs for phagocytosis varied. The monoclonal antibodies isotyped as IgG2b did not induce phagocytosis, while those isotyped as IgG2a were in general very effective, inducing phagocytosis with similar levels as those naturally acquired during P. falciparum infection. These monoclonal antibodies displayed different patterns, some of them showing a concentration-dependent activity while others showed a prozone-like effect. The goat polyclonal antibodies were not able to induce phagocytosis., Conclusion: Immunization with an NTS-DBL1-α domain of PfEMP1 generates antibodies that not only have a biological role in rosette disruption but also effectively induce opsonization for phagocytosis of pRBCs with similar activity to naturally acquired antibodies from immune individuals living in a malaria endemic area. Some of the antibodies with high opsonizing activity were not able to disrupt rosettes, indicating that epitopes of the NTS-DBL1-α other than those involved in rosetting are exposed on the pRBC surface and are able to induce functional antibodies. The ability to induce phagocytosis largely depended on the antibody isotype and on the ability to recognize the surface of the pRBC regardless of the rosette-disrupting capacity.

Mutually exclusive gene expression, whereby only one member of a multi-gene family is selected for activation, is used by the malaria parasite Plasmodium falciparum to escape the human immune system and perpetuate long-term, chronic infections. A family of genes called var encodes the chief antigenic and virulence determinant of P. falciparum malaria. var genes are transcribed in a mutually exclusive manner, with switching between active genes resulting in antigenic variation. While recent work has shed considerable light on the epigenetic basis for var gene activation and silencing, how switching is controlled remains a mystery. In particular, switching seems not to be random, but instead appears to be coordinated to result in timely activation of individual genes leading to sequential waves of antigenically distinct parasite populations. The molecular basis for this apparent coordination is unknown. Here we show that var2csa, an unusual and highly conserved var gene, occupies a unique position within the var gene switching hierarchy. Induction of switching through the destabilization of var specific chromatin using both genetic and chemical methods repeatedly led to the rapid and exclusive activation of var2csa. Additional experiments demonstrated that these represent "true" switching events and not simply de-silencing of the var2csa promoter, and that activation is limited to the unique locus on chromosome 12. Combined with translational repression of var2csa transcripts, frequent "default" switching to this locus and detection of var2csa untranslated transcripts in non-pregnant individuals, these data suggest that var2csa could play a central role in coordinating switching, fulfilling a prediction made by mathematical models derived from population switching patterns. These studies provide the first insights into the mechanisms by which var gene switching is coordinated as well as an example of how a pharmacological agent can disrupt antigenic variation in Plasmodium falciparum.

Background: Erythrocyte invasion by merozoites is an essential step in Plasmodium falciparum infection and leads to subsequent disease pathology. Proteins both on the merozoite surface and secreted from the apical organelles (micronemes, rhoptries and dense granules) mediate the invasion of erythrocytes; some of the molecules have been regarded as targets in the development of an anti-malaria vaccine. Recently, a subgroup of rhoptry neck proteins (PfRON2, PfRON4 and PfRON5) associated with the microneme protein apical membrane antigen AMA1 has been described as components of the moving junction complex that assists merozoite invasion into erythrocytes. However, unlike PfRON2, PfRON4 and PfRON5, the latest study suggested that PfRON3 might be located in the rhoptry bulb and participates in a novel PfRON complex (PfRON2, 3 and 4), but does not form a complex with AMA1. Additionally, the full-length PfRON3 protein possesses three transmembrane regions at the N-terminus, which is highly conserved among RON3 orthologues in the genus Plasmodium, Toxoplasma gondii and Eimeria tenella. Overall, these findings suggest that PfRON3 may play an important role in merozoite invasion into erythrocytes., Results: PfRON3 was primarily expressed during the late trophozoite stage, with a peak in transcription levels at 40 hours post-invasion. The subcellular localization of PfRON3 was confirmed that it is a merozoite rhoptry bulb protein. Additionally, the recombinant form of PfRON3 protein bound to the erythrocyte and was recognized by sera collected from malaria endemic areas in Africa, and anti-PfRON3 antibodies significantly inhibited merozoite invasion into erythrocytes., Methods: The expression of PfRON3 was analysed via real-time quantitative PCR, and the recombinant PfRON3 proteins were generated with an Escherichia coli expression system. The subcellular localization of PfRON3 was assessed with immunoelectron microscopy and immunofluorescence assay (IFA). The recognition PfRON3 by malaria immune sera was analysed with an enzyme-linked immunosorbent assay (ELISA). Erythrocyte-binding assays were performed using recombinant PfRON3 proteins and invasion inhibition assays were carried out with PfRON3-specific antibodies., Conclusion: This study confirmed that PfRON3 is a rhoptry protein with an erythrocyte-binding property, which is likely associated red blood cell invasion. PfRON3 is a potential vaccine candidate.

Background: Vaccines are the most effective agents to control infections. However, recombinant vaccines often do not elicit strong immune responses. Protein antigens combined with proper adjuvants have been widely used to induce immune responses, especially the humoral immune responses, against various pathogens, including parasites. The extracellular matrix protein mindin has been recognised as an immune facilitator for initiating innate immune responses. It has therefore been expected to be a potentially potent adjuvant in the development of novel vaccines. The aim of this study was to investigate whether mindin could facilitate the induction of antigen-specific immune responses to recombinant antigens (rBAG1, rSRS4 and rSRS9) of Toxoplasma gondii in BALB/c mice., Methods: In this study, we explored the adjuvant effect of the recombinant mindin in the generation of specific Th1 and Th2 responses to each of three T. gondii antigens, BAG1, SRS4 and SRS9. All mice in the experimental groups received either antigen alone or in combination with Freund's adjuvant or with the recombinant mindin. The immune responses after immunisation were measured by ELISA and lymphoproliferative assays. The immunised mice were challenged with live T. gondii tachyzoites, and the protection efficiency was compared between the groups., Results: Our results revealed that mindin as an adjuvant could facilitate the recombinant proteins to efficiently stimulate humoral and cellular responses, including antigen-specific IgG1 and IgG2a, as well as lymphocyte proliferation. Furthermore, significantly improved protection against T. gondii infection was observed in the mindin group compared with that of Freund's adjuvant and no-adjuvant groups., Conclusions: The extracellular matrix protein mindin can effectively induce antigen-specific humoral and cell-mediated immune responses. Our study provides a valuable basis for the development of an efficient, safe, non-toxic vaccine adjuvant for future use in humans and animals.

Toxoplasma gondii is an obligatory intracellular apicomplexan parasite which exploits host cell surface components in cell invasion and intracellular parasitization. Sulfated glycans such as heparin and heparan sulfate have been reported to inhibit cell invasion by T. gondii and other apicomplexan parasites such as Plasmodium falciparum. The aim of this study was to investigate the heparin-binding proteome of T. gondii. The parasite-derived components were affinity-purified on the heparin moiety followed by MS fingerprinting of the proteins. The heparin-binding proteins of T. gondii and P. falciparum were compared based on functionality and affinity to heparin. Among the proteins identified, the invasion-related parasite ligands derived from tachyzoite/merozoite surface and the secretory organelles were prominent. However, the profiles of the proteins were different in terms of affinity to heparin. In T. gondii, the proteins with highest affinity to heparin were the intracellular components with functions of parasite development contrasted to that of P. falciparum, of which the rhoptry-derived proteins were prominently identified. The profiling of the heparin-binding proteins of the two apicomplexan parasites not only explained the mechanism of heparin-mediated host cell invasion inhibition, but also, to a certain extent, revealed that the action of heparin on the parasite extended after endocytosis., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Acquisition of antibodies against blood stage antigens is crucial in malaria immunity and the Plasmodium falciparum antigen Pf332, which is present in close association with the infected red blood cell membrane, is one such antigen. In this study, the antibody response to a Duffy binding like fragment of Pf332, termed Pf332-DBL was investigated in sera from naturally exposed individuals living in Dielmo village, Senegal, with regard to immunoglobulin classes (IgG, IgM, IgE) and IgG subclasses (IgG1-4). While the levels of IgM, IgG, IgG1 and IgG2 only displayed a moderate trend to increase with age, Pf332-DBL specific IgG3 levels increased significantly in the older villagers. In multivariate analysis, when controlling for confounding factors, and in a linear model with a Poisson distribution, anti-Pf332-DBL IgG3 as well as the ratio of cytophilic to non cytophilic anti-Pf332-DBL antibodies were found significantly associated with a reduced risk of malaria attack. This association was also present when the IgG3:IgG1 ratio was tested. Finally, two subgroups of villagers with the same mean age, were delineated by IgG3 concentrations either lower or higher than the median value. A total of 45.2% of the individuals with low anti-Pf332-DBL-IgG3 levels but only 21.4% of the villagers in the group with high levels of such antibodies had a clinical malaria attack during a period of 3 years of continuous follow-up after the blood sampling. In conclusion, Pf332-DBL induces naturally the acquisition of antibodies, and Pf332-DBL-specific IgG3 appears to be associated with protection against malaria in this endemic setting., (Copyright © 2013. Published by Elsevier B.V.)

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important virulence factor encoded by a family of 59 var genes, including 56 var genes plus 3 small var3 genes. The var genes are among the most diverse sequences in the P. falciparum genome, but the var3 genes are found conserved in most P. falciparum strains. Previous studies have been mainly focused on the typical var genes, while the biological characteristics of the var3 genes remain unknown. In this study, the three var3 genes, PF3D7_0100300, PF3D7_0600400, and PF3D7_0937600, were found to be transcribed in the erythrocytic stages of P. falciparum, with a peak in the transcription level at 16 h post-invasion, but terminated immediately after 16 h post-invasion. The encoded protein of PF3D7_0600400 could be detected in both the late trophozoite stage and schizont stage, while the encoded proteins of PF3D7_0100300 and PF3D7_0937600 could only be detected in the late trophozoite stage and schizont stage, respectively. Thus, the var3 genes of the P. falciparum 3D7 strain were differentially expressed during the erythrocytic development of the parasite., (© Y. Zhang et al., published by EDP Sciences, 2014.)

The malaria parasite Plasmodium falciparum utilizes host glycosaminoglycans (GAGs) as receptors for erythrocyte invasion and intravascular sequestration. Heparin and heparan sulfate (HS) are GAGs which can block erythrocyte invasion of the P. falciparum merozoite, albeit the molecular mechanisms remain poorly understood. Characterization of these heparin-binding merozoite proteins and key ligands in the host-parasite interplay will lead to a better understanding of the mechanism of erythrocyte invasion by malaria parasites. Here, schizont-derived proteins that bind heparin were enriched by affinity chromatography, and 6062 peptides from 811 P. falciparum-derived proteins were identified by two-dimensional liquid chromatography-mass spectrometry (LC/LC-MS/MS). The proteins were categorized into 14 functional groups ranging from pathogenesis, protein catabolic process to signal transduction. Proteins with predominant peptide counts were found to mainly originate from the rhoptry organelle of merozoites and the parasitized erythrocyte membrane. The profile of the heparin/HS-binding proteome of P. falciparum suggests they have important functions in the biology of the parasite.

During the intraerythrocytic development of Plasmodium falciparum, the malaria parasite remodels the host cell cytosol by inducing membranous structures termed Maurer's clefts and inserting parasite proteins into the red blood cell cytoskeleton and plasma membrane. Pf332 is the largest known asexual malaria antigen that is exported into the red blood cell cytosol where it associates with Maurer's clefts. In the current work, we have utilized a set of different biochemical assays to analyze the solubility of the endogenous Pf332 molecule during its trafficking from the endoplasmic reticulum within the parasite to the host cell cytosol. Solubilization studies demonstrate that Pf332 is synthesized and trafficked within the parasite as a peripheral membrane protein, which after export into the host cell cytosol associates with the cytoplasmic side of Maurer's clefts in a peripheral manner. By immunofluorescence microscopy and flow cytometry, we show that Pf332 persists in close association with Maurer's clefts throughout trophozoite maturation and schizogony, and does not become exposed at the host cell surface. Our data also indicate that Pf332 interacts with the host cell cytoskeleton, but only in very mature parasite stages. Thus, the present study describes Pf332 as a resident peripheral membrane protein of Maurer's clefts and suggests that the antigen participates in host cytoskeleton modifications at completion of the intraerythrocytic developmental cycle.

Background: The pathogenicity of Plasmodium falciparum is in part due to the ability of the parasitized red blood cell (pRBC) to adhere to intra-vascular host cell receptors and serum-proteins. Binding of the pRBC is mediated by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a large multi-variant molecule encoded by a family of ≈60 var genes., Methods: The study of var gene transcription in the parasite clone FCR3S1.2 was performed by semi-quantitative PCR and quantitative PCR (qPCR). The expression of the major PfEMP1 in FCR3S1.2 pRBC was analysed with polyclonal sera in rosette disruption assays and immunofluorescence., Results: Transcripts from var1 (FCR3S1.2(var1); IT4var21) and other var genes were detected by semi-quantitative PCR but results from qPCR showed that one var gene transcript dominated over the others (FCR3S1.2(var2); IT4var60). Antibodies raised in rats to the recombinant NTS-DBL1α of var2 produced in E. coli completely and dose-dependently disrupted rosettes (≈95% at a dilution of 1/5). The sera reacted with the Maurer's clefts in trophozoite stages (IFA) and to the infected erythrocyte surface (FACS) indicating that FCR3S1.2(var2) encodes the dominant PfEMP1 expressed in this parasite., Conclusion: The major transcript in the rosetting model parasite FCR3S1.2 is FCR3S1.2(var2) (IT4var60). The results suggest that this gene encodes the PfEMP1-species responsible for the rosetting phenotype of this parasite. The activity of previously raised antibodies to the NTS-DBL1α of FCR3S1.2(var1) is likely due to cross-reactivity with NTS-DBL1α of the var2 encoded PfEMP1.

The Plasmodium falciparum antigen 332 (Pf332) is a conserved blood-stage antigen, which has been suggested to play a role in parasite invasion. In the present study, we have investigated the immunogenicity of the Duffy-binding like (DBL)-domain of the Pf332 molecule in combination with different adjuvants in four animal species. Three of the adjuvants are applicable for human use (Montanide ISA 720, alum and levamisole), whilst Freund's adjuvant served as a positive control adjuvant. Montanide ISA 720 was able to generate a significant and Th2-biased IgG response in BALB/c and C57BL/6 mice. Alum was a strong inducer of a Th2-type immune response only in BALB/c mice, whereas it was a poor adjuvant together with Pf332-DBL in C57BL/6 mice, rabbits and rats. Levamisole did not show any obvious adjuvant effect in any of the immunized animals. Thus in the case with Pf332-DBL, Montanide ISA 720 may be an adjuvant to further explore in the development of a vaccine against malaria.

An infection with Plasmodium falciparum may lead to severe malaria as a result of excessive binding of infected erythrocytes in the microvasculature. Vascular adhesion is mediated by P. falciparum erythrocyte membrane protein-1 (PfEMP1), which is encoded for by highly polymorphic members of the var-gene family. Here, we profile var gene transcription in fresh P. falciparum trophozoites from Ugandan children with malaria through var-specific DBL1alpha-PCR amplification and sequencing. A method for subsectioning region alignments into homology areas (MOTIFF) was developed to examine collected sequences. Specific PfEMP1-DBL1alpha amino acid motifs correlated with rosetting and severe malaria, with motif location corresponding to distinct regions of receptor interaction. The method is potentially applicable to other families of variant proteins and may be useful in identifying sequence-phenotype relationships. The results suggest that certain PfEMP1 sequences are predisposed to inducing severe malaria.

Plasmodium falciparum malaria is brought about by the asexual stages of the parasite residing in human red blood cells (RBC). Contact between the erythrocyte surface and the merozoite is the first step for successful invasion and proliferation of the parasite. A number of different pathways utilised by the parasite to adhere and invade the host RBC have been characterized, but the complete biology of this process remains elusive. We here report the identification of an open reading frame (ORF) representing a hitherto unknown second exon of the Pf332 gene that encodes a cysteine-rich polypeptide with a high degree of similarity to the Duffy-binding-like (DBL) domain of the erythrocyte-binding-ligand (EBL) family. The sequence of this DBL-domain is conserved and expressed in all parasite clones/strains investigated. In addition, the expression level of Pf332 correlates with proliferation efficiency of the parasites in vitro. Antibodies raised against the DBL-domain are able to reduce the invasion efficiency of different parasite clones/strains. Analysis of the DBL-domain revealed its ability to bind to uninfected human RBC, and moreover demonstrated association with the iRBC surface. Thus, Pf332 is a molecule with a potential role to support merozoite invasion. Due to the high level of conservation in sequence, the novel DBL-domain of Pf332 is of possible importance for development of novel anti-malaria drugs and vaccines.

Malaria vaccine development has so far been largely focused on antigens involved in parasite invasion pathways rather than on antigens associated with severe disease and naturally acquired immunity. Individuals repeatedly exposed to Plasmodium falciparum will eventually become immune to severe disease. Parasite-derived antigens expressed on the infected red blood cell (iRBC) surface are the main targets of protective immunity and can be explored as a rational alternative in development of an anti-malaria vaccine.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important virulence factor on the surface of infected erythrocytes. Naturally acquired antibodies to PfEMP1 expressed by parasites causing severe malaria are suggested to be protective and of major interest for the development of a vaccine against severe disease. In this study, the PfEMP1 expressed by a parasite clone displaying a multiadhesive phenotype associated with severe malaria was well recognized by sera of malaria semi-immune children. The efficiency of the Duffy binding-like 1 alpha (DBL1 alpha) domain of this PfEMP1 was therefore, alone or in combination with two additional DBL1 alpha domains, evaluated as a potential vaccine candidate using both a rodent model and a primate model. Antibodies against the DBL1 alpha domain were generated by immunization with recombinant DBL1 alpha-Semliki Forest virus particles and recombinant protein and analyzed in vitro. The immunized animals were challenged in vivo with various parasite strains or clones. Immunization with the PfEMP1-DBL1 alpha domain abolished the PfEMP1-dependent sequestration of the homologous strain in immunized rats and substantially inhibited parasite adhesion in immunized monkeys. Protection against sequestration of heterologous parasite strains was also confirmed by direct or indirect challenge in the rat model. These results strongly support the use of the DBL1 alpha domain in the development of a vaccine targeting severe malaria.

The interactions of Plasmodium falciparum infected erythrocytes parasitized red blood cells (pRBC) with endothelial receptors and erythrocytes are mediated by multiple Duffy-binding like (DBL) and cysteine-rich interdomain region (CIDR) domains harboured in the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). The success of a subunit vaccine based on PfEMP1 depends on its ability to elicit cross-reactive responses to a substantial number of PfEMP1 variants. We have here evaluated serological PfEMP1 cross-reactivity by immunizing rats with phylogenetically diverse recombinant NTS-DBL-1alpha/x fusion domains from the 3D7 genome parasite emulsified in Montanide ISA 720. Cross-reactivity was elicited to these diverse DBL-1alpha/x domains as measured by ELISA and by immunoblotting. Employing a novel in vivo model of human infected erythrocyte sequestration, immunized animals were challenged with the FCR3S1.2 clone and cross-protection in terms of reduction in lung sequestration amounting to approximately 50% was demonstrated. Our results suggest that immunization with phylogenetically distant DBL-1alpha/x variants, can elicit partial cross-protection to challenge with the parasites harbouring a distant variant. These observations have implications for the design of multi-component vaccines against P. falciparum malaria.

Background: Cellular interactions elicited by Plasmodium falciparum erythrocyte membrane protein antigen 1 (PfEMP1) are brought about by multiple DBL (Duffy binding like), CIDR (cysteine-rich interdomain region) and C2 domain types. Elucidation of the functional and structural characteristics of these domains is contingent on the abundant availability of recombinant protein in a soluble form. A priori prediction of PfEMP1 domains of the 3D7 genome strain, most likely to be expressed in the soluble form in Escherichia coli was computed and proven experimentally., Methods: A computational analysis correlating sequence-dependent features to likelihood for expression in soluble form was computed and predictions were validated by the colony filtration blot method for rapid identification of soluble protein expression in E. coli., Results: Solubility predictions for all constituent PfEMP1 domains in the decreasing order of their probability to be expressed in a soluble form (% mean solubility) are as follows: ATS (56.7%) > CIDR1alpha (46.8%) > CIDR2beta (42.9%) > DBL2-4gamma (31.7%) > DBL2beta + C2 (30.6%) > DBL1alpha (24.9%) > DBL2-7epsilon (23.1%) > DBL2-5delta (14.8%). The length of the domains does not correlate to their probability for successful expression in the soluble form. Immunoblot analysis probing for soluble protein confirmed the differential in solubility predictions., Conclusion: The acidic terminal segment (ATS) and CIDR alpha/beta domain types are suitable for recombinant expression in E. coli while all DBL subtypes (alpha, beta, gamma, delta, epsilon) are a poor choice for obtaining soluble protein on recombinant expression in E. coli. This study has relevance for researchers pursuing functional and structural studies on PfEMP1 domains.

Background: The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells., Methods: Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage., Results and Conclusions: When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons.

Polyclonal B-cell activation and hypergammaglobulinemia are prominent features of human malaria. We report here that Plasmodium falciparum-infected erythrocytes directly adhere to and activate peripheral blood B cells from nonimmune donors. The infected erythrocytes employ the cysteine-rich interdomain region 1alpha (CIDR1alpha) of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to interact with the B cells. Stimulation with recombinant CIDR1alpha induces proliferation, an increase in B-cell size, expression of activation molecules, and secretion of immunoglobulins (immunoglobulin M) and cytokines (tumor necrosis factor alpha and interleukin-6). Furthermore, CIDR1alpha binds to Fab and Fc fragments of human immunoglobulins and to immunoglobulins purified from the sera of different animal species. This binding pattern is similar to that of the polyclonal B-cell activator Staphylococcus aureus protein A. Our findings shed light on the understanding of the molecular basis of polyclonal B-cell activation during malaria infections. The results suggest that the var gene family encoding PfEMP1 has evolved not only to mediate the sequestration of infected erythrocytes but also to manipulate the immune system to enhance the survival of the parasite.

A family of parasite antigens known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is believed to play an important role in the binding of infected erythrocytes to host receptors in the micro-vasculature. Available data advocates the existence of a subset of very adhesive (rosetting, auto-agglutinating) and antigenic PfEMP1s implicated as virulence factors. Serum antibodies that disrupt rosettes are rarely found in children with severe malaria but are frequent in those with mild disease suggesting that they may be protective. Here we have developed a Semliki forest virus (SFV) vaccine construct with a recombinant gene (mini-var gene) encoding a mini-PfEMP1 (DBL1alpha-TM-ATS) obtained from a particularly antigenic and rosetting parasite (FCR3S1.2). The mini-PfEMP1 is presented to the host mimicking the location of the native molecule at the infected erythrocyte surface. Antibodies generated by a regimen of priming with SFV RNA particles and boosting with a recombinant protein recognize the infected erythrocyte surface (immuno-fluorescence/rosette-disruption) and prevent the sequestration of P. falciparum-infected erythrocytes in an in vivo model of severe malaria. The data prove the involvement of DBL1alpha in the adhesion of infected- and uninfected erythrocytes and the role of rosette-disruptive antibodies in preventing these cellular interactions. The work supports the use of DBL1alpha in a vaccine again severe malaria.

Plasmodium falciparum being the most lethal plasmodiae is still a major cause of the disease burden and mortality in malaria endemic areas. Due to the wide spread drug resistance in combination with poor socio-economic situation in the vast majority of the endemic countries, malaria is today a great global challenge. The scientific community is, however, progressing. The 23 Mb genome of P. falciparum has been decoded and publicly available. Data of transcriptional profiling at certain developmental stages have already been generated. More than 50% of P. falciparum genes are transcribed constitutively in all the developmental stages of parasite life cycle. Functional disruption of these genes might have implications for parasite growth and development. Available microarray data indicate that P. falciparum preferentially expresses rif and stevor gene families at gametocyte and sporozoite stages while var genes are predominantly expressed at the erythrocytic stage. Gene regulation mechanisms of the variant gene families in P. falciparum are still not understood though some regulatory elements have been proposed. The occurrence of severe malaria is determined by both parasite and human host factors. Sequestration and antigenic variation are two of the evasion mechanisms utilized by P. falciparum in order to escape the human host defences. Understanding the molecular mechanisms underlying these phenomena is of a major importance and interest in malaria research. Here, we summarize and highlight the recent progress in molecular aspects of severe malaria.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is an important virulence factor encoded by a family of roughly 60 var genes and is used by the parasite to interact with the human host. The parasite regularly exchanges the expressed var gene generating antigenic variation of the infected RBCs (pRBC) surface which is crucial for successful proliferation and transmission. PfEMP1 is also an adhesive molecule that binds to an array of human receptors. By sequestration in the post-capillary venules, pRBCs are able to escape the spleen-mediated clearance but severe malaria may develop if the local binding is extensive. Anti-PfEMP1 immunity is important for preventing the development of both cerebral malaria and placental malaria, but more immunological studies on PfEMP1 antigens and their interaction with the human host are needed. Over the last few years our knowledge about var genes and PfEMP1s has increased dramatically through genetic, biochemical, immunological and epidemiological studies. In addition, the genome sequence has also provided us with a new platform for further dissecting its biological functions. This review highlights the recent analyses of var genes in the P. falciparum genome and postulates significance of genome recombination to the diversity of parasite virulence.

Intraerythrocytic Plasmodium falciparum exports proteins to the cytosol and to the plasma membrane of the host cell. We here present data revealing the existence of a unique common pathway for the surface bound traffic of the clonally variant antigens, repeated-interspersed-antigen (RIFINS) and P. falciparum erythrocyte-membrane-protein-1 (PfEMP1). RIFIN- and PfEMP1-specific antibodies were found to stain single small vesicles (SSV) that bud off from the parasitophorus vacuolar membrane (PMV) at 6-10 h post-invasion. Large multimeric vesicle (LMV) assemblies, composed of subunits each of a similar size to that of a SSV, appeared as the dominant vesicle type carrying the variant antigens in the cytosol as the parasites developed into early trophozoite stages (> or = 16 h post-invasion). Later, more than 24 h post-invasion, large spinle-like vesicles (LSLV) built up as the LMV approached and accumulated underneath the erythrocyte membrane. LMV were found to associate both with the Maurer's cleft antigen Pf332 and with lipids as seen by fluorescent BODIPY-Ceramide staining. Co-traffic of Pf332 with RIFINS and PfEMP1 occurred in sub-compartmentalized LMV, as the variant antigens co-localized at the outer rim while Pf332 occupied the core of the vesicle complex. Formation of LMV for the trafficking of RIFINS and PfEMP1 is a prominent feature of freshly isolated P. falciparum and of in vitro propagated K+ as well as K- parasites, seemingly independent of the knob-associated histidine-rich protein (KAHRP). In vitro cultured 3D7 clones lack LMV formation and traffic the variant antigens in vesicles of a similar size to that of the SSV.

Relapse variants in chronic Plasmodium falciparum infections are antigenically distinct from the parental parasites. The variable antigen PfEMP1 expressed at the surface of the infected erythrocyte (IE) is encoded by the var gene family with approximately 60 copies per haploid genome. Placental isolates commonly express DBLgamma containing subtypes of var genes with homology to either 3D7var5.2 (var(COMMON)) or FCR3var(CSA). Here we report that var(COMMON) related genes are constitutively transcribed in approximately 60% of malaria infected children in Gabon. var(COMMON) is conserved in field isolates over at least 2.1kb. In 3D7 parasites var(COMMON) is present on chromosome 5 (var5.2) and constitutively transcribed in the opposite direction to most other var genes. It lacks a regulatory intron, an acidic terminal segment and ends in telomeric repeat sequences. var(COMMON) encodes a large, hypothetical PfEMP1 of a structure similar to previous placenta-binding PfEMP1s but it is not present at the IE-surface. IE of a 3D7 clone (3D7S8) transcribe var(COMMON) but express a PfEMP1 distinct from var(COMMON) at the surface and adhere to placental tissues through var(COMMON) independent novel mechanisms. Our report suggests that expression of var(COMMON) type genes is not restricted to placental malaria.

Plasmodium falciparum may cause severe forms of malaria when excessive sequestration of infected and uninfected erythrocytes occurs in vital organs. The capacity of wild-type isolates of P falciparum-infected erythrocytes (parasitized red blood cells [pRBCs]) to bind glycosaminoglycans (GAGs) such as heparin has been identified as a marker for severe disease. Here we report that pRBCs of the parasite FCR3S1.2 and wild-type clinical isolates from Uganda adhere to heparan sulfate (HS) on endothelial cells. Binding to human umbilical vein endothelial cells (HUVECs) and to human lung endothelial cells (HLECs) was found to be inhibited by HS/heparin or enzymes that remove HS from cell surfaces. (35)S-labeled HS extracted from HUVECs bound directly to the pRBCs' membrane. Using recombinant proteins corresponding to the different domains of P falciparum erythrocyte membrane protein 1 (PfEMP1), we identified Duffy-binding-like domain-1alpha (DBL1alpha) as the ligand for HS. DBL1alpha bound in an HS-dependent way to endothelial cells and blocked the adherence of pRBCs in a dose-dependent manner. (35)S-labeled HS bound to DBL1alpha-columns and eluted as a distinct peak at 0.4 mM NaCl. (35)S-labeled chondroitin sulfate (CS) of HUVECs did not bind to PfEMP1 or to the pRBCs' membrane. Adhesion of pRBCs of FCR3S1.2 to platelet endothelial cell adhesion molecule-1 (PECAM-1)/CD31, mediated by the cysteine-rich interdomain region 1alpha (CIDR1alpha), was found be operative with, but independent of, the binding to HS. HS and the previously identified HS-like GAG on uninfected erythrocytes may act as coreceptors in endothelial and erythrocyte binding of rosetting parasites, causing excessive sequestration of both pRBCs and RBCs.

The var gene family of Plasmodium falciparum encodes the clonally variant adhesin PfEMP1 present on the surface of infected erythrocytes. A poorly understood mechanism of allelic exclusion controls the expression of PfEMP1. Transcription of var genes is developmentally and, most likely, epigenetically regulated. Here we have studied the transcriptional pattern of 28 members of this multigene family in individual parasites, early in the intraerythrocytic cycle. The results show unique patterns (type and number) of var transcripts in each individual PRBC, with 1-15 mRNA species detected per cell at 2-4 h post-invasion. When a panel of ten single PRBC was analyzed, the var gene coding for the expressed PfEMP1 was transcribed in more cells than any other, although transcripts from this gene did not give the strongest hybridization signal within each individual cell. Chromosomal mapping of transcriptionally active var genes indicated that their distribution reflects that of var loci in the genome, including a pronounced clustering in chromosome 4. These findings, taken together with existing data on var transcription at later developmental stages, suggest that the mosaic-like transcription of multiple var genes detected at the ring stage and the steady transcription of the gene encoding the expressed PfEMP1 are distinct although superimposed events, one of them random and the other taking place under some form of imprinting. With its unique features, the expression of P. falciparum var genes may reveal new principles of gene regulation.