Host Erythrocyte Environment Influences the Localization of Exported Protein 2, an Essential Component of the Plasmodium Translocon

Malaria parasites replicating inside red blood cells (RBCs) export a large subset of proteins into the erythrocyte cytoplasm to facilitate parasite growth and survival. PTEX, the parasite-encoded translocon, mediates protein transport across the parasitophorous vacuolar membrane (PVM) in Plasmodium falciparum-infected erythrocytes. Proteins exported into the erythrocyte cytoplasm have been localized to membranous structures, such as Maurer''s clefts, small vesicles, and a tubovesicular network. Comparable studies of protein trafficking in Plasmodium vivax-infected reticulocytes are limited. With Plasmodium yoelii-infected reticulocytes, we identified exported protein 2 (Exp2) in a proteomic screen of proteins putatively transported across the PVM. Immunofluorescence studies showed that P. yoelii Exp2 (PyExp2) was primarily localized to the PVM. Unexpectedly, PyExp2 was also associated with distinct, membrane-bound vesicles in the reticulocyte cytoplasm. This is in contrast to P. falciparum in mature RBCs, where P. falciparum Exp2 (PfExp2) is exclusively localized to the PVM. Two P. yoelii-exported proteins, PY04481 (encoded by a pyst-a gene) and PY06203 (PypAg-1), partially colocalized with these PyExp2-positive vesicles. Further analysis revealed that with P. yoelii, Plasmodium berghei, and P. falciparum, cytoplasmic Exp2-positive vesicles were primarily observed in CD71⁺ reticulocytes versus mature RBCs. In transgenic P. yoelii 17X parasites, the association of hemagglutinin-tagged PyExp2 with the PVM and cytoplasmic vesicles was retained, but the pyexp2 gene was refractory to deletion. These data suggest that the localization of Exp2 in mouse and human RBCs can be influenced by the host cell environment. Exp2 may function at multiple points in the pathway by which parasites traffic proteins into and through the reticulocyte cytoplasm.     

Antibody Responses to a Novel Plasmodium falciparum Merozoite Surface Protein Vaccine Correlate with Protection against Experimental Malaria Infection in Aotus Monkeys

The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals.     

Time-Lapse Imaging of Red Blood Cell Invasion by the Rodent Malaria Parasite Plasmodium yoelii

In order to propagate within the mammalian host, malaria parasites must invade red blood cells (RBCs). This process offers a window of opportunity in which to target the parasite with drugs or vaccines. However, most of the studies relating to RBC invasion have analyzed the molecular interactions of parasite proteins with host cells under static conditions, and the dynamics of these interactions remain largely unstudied. Time-lapse imaging of RBC invasion is a powerful technique to investigate cell invasion and has been reported for Plasmodium knowlesi and Plasmodium falciparum. However, experimental modification of genetic loci is laborious and time consuming for these species. We have established a system of time-lapse imaging for the rodent malaria parasite Plasmodium yoelii, for which modification of genetic loci is quicker and simpler. We compared the kinetics of RBC invasion by P. yoelii with that of P. falciparum and found that the overall kinetics during invasion were similar, with some exceptions. The most striking of these differences is that, following egress from the RBC, the shape of P. yoelii merozoites gradually changes from flat elongated ovals to spherical bodies, a process taking about 60 sec. During this period merozoites were able to attach to and deform the RBC membrane, but were not able to reorient and invade. We propose that this morphological change of P. yoelii merozoites may be related to the secretion or activation of invasion-related proteins. Thus the P. yoelii merozoite appears to be an excellent model to analyze the molecular dynamics of RBC invasion, particularly during the morphological transition phase, which could serve as an expanded window that cannot be observed in P. falciparum.     

Plasmodium: Mammalian codon optimization of malaria plasmid DNA vaccines enhances antibody responses but not T cell responses nor protective immunity

We have evaluated the effect of mammalian codon optimization on the immunogenicity and protective efficacy of plasmid DNA vaccines encoding pre-erythrocytic stage Plasmodium falciparum and Plasmodium yoelii antigens in mice. Codon optimization significantly enhanced in vitro expression and in vivo antibody responses for P. falciparum circumsporozoite protein (PfCSP) and P. yoelii hepatocyte erythrocyte protein 17 kDa (PyHEP17) but not for P. yoelii circumsporozoite protein (PyCSP). Unexpectedly, more robust CD4⁺ and CD8⁺ T cell responses as measured by IFN-γ ELIspot, lymphoproliferation, and cytotoxic T lymphocyte assays were noted with native as compared with codon optimization constructs. Codon optimization also failed to enhance CD8⁺ T cell dependent protection against P. yoelii sporozoite challenge as measured by liver-stage parasite burden. These data demonstrate that the effect of mammalian codon optimization is antigen-dependent and may not be beneficial for vaccines designed to induce T cell dependent protective immunity in this malaria model.     

约氏疟原虫与伯氏疟原虫诱导感染小鼠产生保护性抗体的特点研究

比较约氏疟原虫(Plasmodium yoelii)与伯氏疟原虫(Plasmodium berghei)再次感染模型特异性抗体产生的差异,追溯相应虫体抗原的表达特点.建立约氏疟原虫与伯氏疟原虫再次感染鼠疟模型,ELISA检测特异性抗体水平;Western Blot检测血清中优势抗体反应特点;检测两种虫株的MSP-1重组...     

Plasmodium berghei circumvents immune responses induced by merozoite surface protein 1- and apical membrane antigen 1-based vaccines

Background

Two current leading malaria blood-stage vaccine candidate antigens for Plasmodium falciparum, the C-terminal region of merozoite surface protein 1 (MSP1₁₉) and apical membrane antigen 1 (AMA1), have been prioritized because of outstanding protective efficacies achieved in a rodent malaria Plasmodium yoelii model. However, P. falciparum vaccines based on these antigens have had disappointing outcomes in clinical trials. Discrepancies in the vaccine efficacies observed between the P. yoelii model and human clinical trials still remain problematic.

Methodology and Results

In this study, we assessed the protective efficacies of a series of MSP1₁₉- and AMA1-based vaccines using the P. berghei rodent malarial parasite and its transgenic models. Immunization of mice with a baculoviral-based vaccine (BBV) expressing P. falciparum MSP1₁₉ induced high titers of PfMSP1₁₉-specific antibodies that strongly reacted with P. falciparum blood-stage parasites. However, no protection was achieved following lethal challenge with transgenic P. berghei expressing PfMSP1₁₉ in place of native PbMSP1₁₉. Similarly, neither P. berghei MSP1₁₉- nor AMA1-BBV was effective against P. berghei. In contrast, immunization with P. yoelii MSP1₁₉- and AMA1-BBVs provided 100% and 40% protection, respectively, against P. yoelii lethal challenge. Mice that naturally acquired sterile immunity against P. berghei became cross-resistant to P. yoelii, but not vice versa.

Conclusion

This is the first study to address blood-stage vaccine efficacies using both P. berghei and P. yoelii models at the same time. P. berghei completely circumvents immune responses induced by MSP1₁₉- and AMA1-based vaccines, suggesting that P. berghei possesses additional molecules and/or mechanisms that circumvent the host''s immune responses to MSP1₁₉ and AMA1, which are lacking in P. yoelii. Although it is not known whether P. falciparum shares these escape mechanisms with P. berghei, P. berghei and its transgenic models may have potential as useful tools for identifying and evaluating new blood-stage vaccine candidate antigens for P. falciparum.     

Generation and functional characterisation of Plasmodium yoelii csp deletion mutants using a microhomology-based CRISPR/Cas9 method

CRISPR/Cas9 is a powerful genome editing method that has greatly facilitated functional studies in many eukaryotic organisms including malaria parasites. Due to the lack of genes encoding enzymes necessary for the non-homologous end joining DNA repair pathway, genetic manipulation of malaria parasite genomes is generally accomplished through homologous recombination requiring the presence of DNA templates. Recently, an alternative double-strand break repair pathway, microhomology-mediated end joining, was found in the Plasmodium falciparum parasite. Taking advantage of the MMEJ pathway, we developed a MMEJ-based CRISPR/Cas9 (mCRISPR) strategy to efficiently generate multiple mutant parasites simultaneously in genes with repetitive sequences. As a proof of principle, we successfully produced various size mutants in the central repeat region of the Plasmodium yoelii circumsporozoite surface protein without the use of template DNA. Monitoring mixed parasite populations and individual parasites with different sizes of CSP-CRR showed that the CSP-CRR plays a role in the development of mosquito stages, with severe developmental defects in parasites with large deletions in the repeat region. However, the majority of the csp mutant parasite clones grew similarly to the wild type P. yoelii 17XL parasite in mice. This study develops a useful technique to efficiently generate mutant parasites with deletions or insertions, and shows that the CSP-CRR plays a role in parasite development in mosquito.     

Plasmodium falciparum Carbonic Anhydrase is a Possible Target for Malaria Chemotherapy

Plasmodium falciparum is responsible for the majority of life-threatening cases of human malaria. The global emergence of drug-resistant malarial parasites necessitates identification and characterization of novel drug targets. Carbonic anhydrase (CA) is present at high levels in human red cells and in P. falciparum. Existence of at least three isozymes of the α class was demonstrated in P. falciparum and a rodent malarial parasite Plasmodium berghei. The major isozyme CA1 was purified and partially characterized from P. falciparum (PfCA1). A search of the malarial genome database yielded an open reading frame similar to the α-CAs from various organisms, including human. The primary amino acid sequence of the PfCA1 has 60% identity with a rodent parasite Plasmodium yoelii enzyme (PyCA). The single open reading frames encoded 235 and 252 amino acid proteins for PfCA1 and PyCA, respectively. The highly conserved active site residues were also found among organisms having α-CAs. The PfCA1 gene was cloned, sequenced and expressed in Escherichia coli. The purified recombinant PfCA1 enzyme was catalytically active. It was sensitive to acetazolamide and sulfanilamide inhibition. Kinetic properties of the recombinant PfCA1 revealed the authenticity to the wild type enzyme purified from P. falciparum in vitro culture. Furthermore, the PfCA1 inhibitors acetazolamide and sulfanilamide showed good antimalarial effect on the in vitro growth of P. falciparum. Our molecular tools developed for the recombinant enzyme expression will be useful for developing potential antimalarials directed at P. falciparum carbonic anhydrase.     

Quantitative Bioluminescent Imaging of Pre-Erythrocytic Malaria Parasite Infection Using Luciferase-Expressing Plasmodium yoelii

The liver stages of Plasmodium parasites are important targets for the development of anti-malarial vaccine candidates and chemoprophylaxis approaches that aim to prevent clinical infection. Analyzing the impact of interventions on liver stages in the murine malaria model system Plasmodium yoelii has been cumbersome and requires terminal procedures. In vivo imaging of bioluminescent parasites has previously been shown to be an effective and non-invasive alternative to monitoring liver stage burden in the Plasmodium berghei model. Here we report the generation and characterization of a transgenic P. yoelii parasite expressing the reporter protein luciferase throughout the parasite life cycle. In vivo bioluminescent imaging of these parasites allows for quantitative analysis of P. yoelii liver stage burden and parasite development, which is comparable to quantitative RT-PCR analysis of liver infection. Using this system, we show that both BALB/cJ and C57BL/6 mice show comparable susceptibility to P. yoelii infection with sporozoites and that bioluminescent imaging can be used to monitor protective efficacy of attenuated parasite immunizations. Thus, this rapid, simple and noninvasive method for monitoring P. yoelii infection in the liver provides an efficient system to screen and evaluate the effects of anti-malarial interventions in vivo and in real-time.     

Evolutionary analysis of circumsporozoite surface protein and merozoite surface protein-1 (CSP and MSP-1) sequences of malaria parasites

Malaria, one of the world''s most common diseases, is caused by the intracellular protozoan parasite known as Plasmodium. In this study, we have determined the evolutionary relationship of two single-copy proteins, circumsporozoite protein (CSP) and merozoite surface protein-1 (MSP-1), among Plasmodium species using various bioinformatics tools and softwares. These two proteins are major blood stage antigens of Plasmodium species. This study demonstrates that the circumsporozoite protein of Plasmodium falciparum shows similarity with Plasmodium cynomolgi and Plasmodium knowlesi. The merozoite surface protein-1 of Plasmodium coatneyi forms a monophyletic group with Plasmodium knowlesi, demonstrating their close relationship and these two species also reveal similarity between the human malaria Plasmodium vivax. This Plasmodium phylogenetic arrangement is evidently crucial to identify shared derived characters as well as particular adaptation of plasmodium species from inside and between monophyletic groups.     

Facilitation through altered resource availability in a mixed‐species rodent malaria infection

A major challenge in disease ecology is to understand how co‐infecting parasite species interact. We manipulate in vivo resources and immunity to explain interactions between two rodent malaria parasites, Plasmodium chabaudi and P. yoelii. These species have analogous resource‐use strategies to the human parasites Plasmodium falciparum and P. vivax: P. chabaudi and P. falciparum infect red blood cells (RBC) of all ages (RBC generalist); P. yoelii and P. vivax preferentially infect young RBCs (RBC specialist). We find that: (1) recent infection with the RBC generalist facilitates the RBC specialist (P. yoelii density is enhanced ~10 fold). This occurs because the RBC generalist increases availability of the RBC specialist's preferred resource; (2) co‐infections with the RBC generalist and RBC specialist are highly virulent; (3) and the presence of an RBC generalist in a host population can increase the prevalence of an RBC specialist. Thus, we show that resources shape how parasite species interact and have epidemiological consequences.     

Type II Fatty Acid Biosynthesis Is Essential for Plasmodium falciparum Sporozoite Development in the Midgut of Anopheles Mosquitoes

The prodigious rate at which malaria parasites proliferate during asexual blood-stage replication, midgut sporozoite production, and intrahepatic development creates a substantial requirement for essential nutrients, including fatty acids that likely are necessary for parasite membrane formation. Plasmodium parasites obtain fatty acids either by scavenging from the vertebrate host and mosquito vector or by producing fatty acids de novo via the type two fatty acid biosynthesis pathway (FAS-II). Here, we study the FAS-II pathway in Plasmodium falciparum, the species responsible for the most lethal form of human malaria. Using antibodies, we find that the FAS-II enzyme FabI is expressed in mosquito midgut oocysts and sporozoites as well as liver-stage parasites but not during the blood stages. As expected, FabI colocalizes with the apicoplast-targeted acyl carrier protein, indicating that FabI functions in the apicoplast. We further analyze the FAS-II pathway in Plasmodium falciparum by assessing the functional consequences of deleting fabI and fabB/F. Targeted deletion or disruption of these genes in P. falciparum did not affect asexual blood-stage replication or the generation of midgut oocysts; however, subsequent sporozoite development was abolished. We conclude that the P. falciparum FAS-II pathway is essential for sporozoite development within the midgut oocyst. These findings reveal an important distinction from the rodent Plasmodium parasites P. berghei and P. yoelii, where the FAS-II pathway is known to be required for normal parasite progression through the liver stage but is not required for oocyst development in the Anopheles mosquito midgut.     

The multiplicity of Plasmodium falciparum infections is associated with acquired immunity to asexual blood stage antigens

We evaluated the relationship between immune response markers and the multiplicity of Plasmodium falciparum infections in order to assess the validity of the latter as an indicator of the acquisition of anti-malarial immunity.Parasite populations present during malaria episodes of 64 Gabonese children who presented with at least 4 such attacks during active follow-up over a 7-year period were characterized using MSP-1 and MSP-2 PCR-based methods. Plasma samples taken at healthy and parasite-free phase were used to measure P. falciparum antigen-specific antibody and cytokine activity.We found evidence of intra- and inter-individual variation in the number of parasite genotypes present in different malaria episodes, although in 72% of isolates no more than 2 parasite genotypes were detectable. Samples with the highest multiplicity were from children with significantly lower (p < 0.03) antibody responses to specific asexual stage antigens. Additionally, the whole blood interferon-γ production capacity was significantly higher (p < 0.02) in those with lower infection multiplicity.Malaria episodes with multiple clones indeed reflect a low level of acquired immunity and a consequently poor capacity to control the infection. These findings suggest that the multiplicity of falciparum infection may be a potentially useful parameter in the evaluation of malaria control interventions.     

A Novel Malaria Vaccine Candidate Antigen Expressed in Tetrahymena thermophila

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens.     

Plasmodium falciparum Dynein Light Chain 1 Interacts with Actin/Myosin during Blood Stage Development

Dynein light chain 1 (LC1), a member of the leucine-rich repeat protein family, has been shown to be engaged in controlling flagellar motility in Chlamydomonas reinhardtii and Trypanosoma brucei via its interaction with the dynein γ heavy chain. In Plasmodium falciparum, we have identified the LC1 ortholog, designated Pfdlc1. Negative attempts to disrupt the dlc1 gene by reverse genetic approaches in both P. falciparum and P. berghei suggest either its essentiality for parasite survival or the inaccessibility of its locus. Expression studies revealed high levels of DLC1 protein in late trophozoites and schizonts, pointing to an unexpected role of this protein in blood-stage parasites as they do not have flagella. Interactions studies and co-immunoprecipitation experiments revealed that PfDLC1 was able to bind to P. falciparum myosin A and actin 1. The PfDLC1 interacting domains present in P. falciparum myosin A and actin 1 were mapped to sequences containing SDIE and/or EEMKT motifs present in the upper 50-kDa segment of the myosin A head domain and in the subdomain IV of actin 1, respectively. Detection of PfDLC1 by fluorescence tagging and immunofluorescence staining using specific antibodies showed a cytoplasmic location similar to actin and immunofluorescence studies showed a co-localization of PfDLC1 and myosin A. Taken together, these findings suggest that PfDLC1 might play an important role in P. falciparum erythrocytic stages by its interaction with myosin A and actin 1, known to be essential for parasite development.     

Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) interacts with the band 3 receptor to promote erythrocyte invasion by malaria parasites

Glycosylphosphatidylinositol-anchored micronemal antigen (GAMA) is an erythrocyte binding protein known to be involved in malarial parasite invasion. Although anti-GAMA antibodies have been shown to block GAMA attachment to the erythrocyte surface and subsequently inhibit parasite invasion, little is known about the molecular mechanisms by which GAMA promotes the invasion process. In this study, LC-MS analysis was performed on the erythrocyte membrane to identify the specific receptor that interacts with GAMA. We found that ankyrin 1 and the band 3 membrane protein showed affinity for GAMA, and characterization of their binding specificity indicated that both Plasmodium falciparum and Plasmodium vivax GAMA bound to the same extracellular loop of band 3 (loop 5). In addition, we show the interaction between GAMA and band 3 was sensitive to chymotrypsin. Furthermore, antibodies against band 3 loop 5 were able to reduce the binding activity of GAMA to erythrocytes and inhibit the invasion of P. falciparum merozoites into human erythrocytes, whereas antibodies against P. falciparum GAMA (PfGAMA)-Tr3 only slightly reduced P. falciparum invasion. The identification and characterization of the erythrocyte GAMA receptor is a novel finding that identifies an essential mechanism of parasite invasion of host erythrocytes.     

Allelic Diversity and Geographical Distribution of the Gene Encoding Plasmodium falciparum Merozoite Surface Protein-3 in Thailand

Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines.     

The Ig Domain Protein CD9P-1 Down-regulates CD81 Ability to Support Plasmodium yoelii Infection

Invasion of hepatocytes by Plasmodium sporozoites is a prerequisite for establishment of a malaria natural infection. The molecular mechanisms underlying sporozoite invasion are largely unknown. We have previously reported that CD81 is required on hepatocytes for infection by Plasmodium falciparum and Plasmodium yoelii sporozoites. CD81 belongs to the tetraspanin superfamily of transmembrane proteins. By interacting with each other and with other transmembrane proteins, tetraspanins may play a role in the lateral organization of membrane proteins. In this study, we investigated the role of the two major molecular partners of CD81 in hepatocytic cells, CD9P-1/EWI-F and EWI-2, two transmembrane proteins belonging to a novel subfamily of immunoglobulin proteins. We show that CD9P-1 silencing increases the host cell susceptibility to P. yoelii sporozoite infection, whereas EWI-2 knock-down has no effect. Conversely, overexpression of CD9P-1 but not EWI-2 partially inhibits infection. Using CD81 and CD9P-1 chimeric molecules, we demonstrate the role of transmembrane regions in CD81-CD9P-1 interactions. Importantly, a CD9P-1 chimera that no longer associates with CD81 does not affect infection. Based on these data, we conclude that CD9P-1 acts as a negative regulator of P. yoelii infection by interacting with CD81 and regulating its function.