Molecular Characterization of the Carboxypeptidase B1 of Anopheles stephensi and Its Evaluation as a Target for Transmission-Blocking Vaccines

Malaria is one of the most important infectious diseases in the world, and it has many economic and social impacts on populations, especially in poor countries. Transmission-blocking vaccines (TBVs) are valuable tools for malaria eradication. A study on Anopheles gambiae revealed that polyclonal antibodies to carboxypeptidase B1 of A. gambiae can block sexual parasite development in the mosquito midgut. Hence, it was introduced as a TBV target in regions where A. gambiae is the main malaria vector. However, in Iran and neighboring countries as far as China, the main malaria vector is Anopheles stephensi. Also, the genome of this organism has not been sequenced yet. Therefore, in this study, carboxypeptidase B1 of A. stephensi was characterized by genomic and proteomic approaches. Furthermore, its expression pattern after ingestion of Plasmodium falciparum gametocytes and the effect of anti-CPBAs1 antibodies on sexual parasite development were evaluated. Our results revealed that the cpbAs1 expression level was increased after ingestion of the mature gametocytes of P. falciparum and that anti-CPBAs1 directed antibodies could significantly reduce the mosquito infection rate in the test group compared with the control group. Therefore, according to our findings and with respect to the high similarity of carboxypeptidase enzymes between the two main malaria vectors in Africa (A. gambiae) and Asia (A. stephensi) and the presence of other sympatric vectors, CPBAs1 could be introduced as a TBV candidate in regions where A. stephensi is the main malaria vector, and this will broaden the scope for the potential wider application of CPBAs1 antigen homologs/orthologs.     

A transgenic Plasmodium falciparum NF54 strain that expresses GFP–luciferase throughout the parasite life cycle

Plasmodium falciparum is the pathogenic agent of the most lethal of human malarias. Transgenic P. falciparum parasites expressing luciferase have been created to study drug interventions of both asexual and sexual blood stages but luciferase-expressing mosquito stage and liver stage parasites have not been created which has prevented the easy quantification of mosquito stage development (e.g. for transmission blocking interventions) and liver stage development (for interventions that prevent infection). To overcome this obstacle, we have created a transgenic P. falciparum NF54 parasite that expresses a GFP–luciferase transgene throughout the life cycle. Luciferase expression is robust and measurable at all life cycle stages, including midgut oocyst, salivary gland sporozoites and liver stages, where in vivo development is easily measurable using humanized mouse infections in conjunction with an in vivo imaging system. This parasite reporter strain will accelerate testing of interventions against pre-erythrocytic life cycle stages.     

2-Cys Peroxiredoxin TPx-1 is involved in gametocyte development in Plasmodium berghei

Peroxiredoxins (Prxs) constitute a ubiquitous family of antioxidant enzymes involved in diverse cellular functions including cell proliferation and differentiation. To investigate the physiologic role of typical 2-Cys Prx in malaria parasites (TPx-1), we disrupted this gene in the rodent malaria parasite Plasmodium berghei (pbtpx-1). The gene-disrupted parasite (Prx KO) developed normally in mouse erythrocytes and multiplied at a rate similar to that of the parent strain (WT) during the experimental period. The normal growth rate was not altered after 10 passages, and the level of 8-hydroxy-2'-deoxyguanosine, which accumulates in the parasite genome during the cell cycle, was similar between Prx KO and WT. These results suggest that TPx-1 does not prevent parasite DNA oxidation, in contrast to mammalian Prx, and that it is not essential for asexual parasite growth in mouse erythrocytes. However, Prx KO produced up to 60% fewer gametocytes, sexual-stage parasites involved in the transition between the mammalian host and the mosquito, than WT did. The peak of gametocytemia was also delayed; however, the male/female ratio of gametocytes and the exflagellation activity of male gametocytes were normal. These results suggest that TPx-1 is required for normal gametocyte development but does not affect the male/female gametocyte ratio or male gametogenesis. Although the mechanism by which PbTPx-1 contributes to gametocyte development remains unknown, these findings suggest, for the first time, the involvement of Prx in the sexual development of the malaria parasite.     

Induction of Plasmodium falciparum transmission-blocking antibodies in nonhuman primates by a combination of DNA and protein immunizations

Malaria transmission-blocking vaccination can effectively reduce and/or eliminate transmission of parasites from the human host to the mosquito vector. The immunity achieved by inducing an antibody response to surface antigens of male and female gametes and parasite stages in the mosquito. Our laboratory has developed DNA vaccine constructs, based on Pfs25 (a Plasmodium falciparum surface protein of 25 kDa), that induce a transmission-blocking immune response in mice (C. A. Lobo, R. Dhar, and N. Kumar, Infect. Immun. 67:1688-1693, 1999). To evaluate the safety, immunogenicity, and efficacy of the Pfs25 DNA vaccine in nonhuman primates, we immunized rhesus macaques (Macaca mulatta) with a DNA vaccine plasmid encoding Pfs25 or a Pfg27-Pfs25 hybrid or with the plasmid (empty plasmid) alone. Immunization with four doses of these DNA vaccine constructs elicited antibody titers that were high but nonetheless unable to reduce the parasite's infectivity in membrane feeding assays. Further boosting of the antibody response with recombinant Pfs25 formulated in Montanide ISA-720 increased antibody titers (30-fold) and significantly blocked transmission of P. falciparum gametocytes to Anopheles mosquitoes (~90% reduction in oocyst numbers in the midgut). Our data show that a DNA prime-protein boost regimen holds promise for achieving transmission-blocking immunity in areas where malaria is endemic and could be effective in eradicating malaria in isolated areas where the level of malaria endemicity is low.     

Plasmodium helical interspersed subtelomeric family—an enigmatic piece of the Plasmodium biology puzzle

Plasmodium falciparum (Pf) refurbishes the infected erythrocytes by exporting a myriad of parasite proteins to the host cell. A novel exported protein family ‘Plasmodium Helical Interspersed Subtelomeric’ (PHIST) has gained attention for its significant roles in parasite biology. Here, we have collected and analysed available information on PHIST members to enhance understanding of their functions, varied localization and structure-function correlation. Functional diversity of PHIST proteins is highlighted by their involvement in PfEMP1 (Pf erythrocyte membrane protein 1) expression, trafficking and switching. This family also contributes to cytoadherence, gametocytogenesis, host cell modification and generation of extracellular vesicles. While the PHIST domain forms the hallmark of this family, existence and functions of additional domains (LyMP, TIGR01639) and the MEC motif underscores its diversity further. Since specific PHIST proteins seem to form pairs with PfEMP1 members, we have used in silico tools to predict such potential partners in Pf. This information and our analysis of structural data on a PHIST member provide important insights into their functioning. This review overall enables readers to view the PHIST family comprehensively, while highlighting key knowledge gaps in the field.

     

Fine mapping of Plasmodium falciparum ribosomal phosphoprotein PfP0 revealed sequences with highly specific binding activity to human red blood cells

The Plasmodium falciparum P0 ribosomal phosphoprotein (PfP0) was identified for the first time by screening a cDNA expression library of P. falciparum parasites with sera from malaria-immune individuals. Due to its localization on the surface of different parasite life-cycle stages (merozoites and gametocytes) and its recognition by invasion-blocking antibodies, PfP0 has been considered a potential malaria-vaccine component. In this study, 16 20-mer-long synthetic peptides spanning the entire PfP0 sequence were evaluated by means of receptor–ligand assays with human red blood cells (RBCs) in order to determine the role played by these peptides in the invasion process. Four RBC high-activity binding peptides (HABPs), located mostly toward the N-terminal region, were identified: HABP 33898 (¹MAKLSKQQKKQMYIEKLSSL²⁰), HABP 33900 (⁴¹ASVRKSLRGKATILMGKNTRY⁶⁰), HABP 33901 (⁶¹IRTALKKNLQAVPQIEKLLPY ⁸⁰), and HABP 33906 (¹⁶¹LIKQGEKVTASSATLLRKFNY¹⁸⁰). The binding pattern of HABPs 33898 and 33906 to enzyme-treated RBCs suggests receptors of protein nature for these two HABPs, one of which could correspond to a common 58-kDa RBC membrane protein, as indicated by results of cross-linking assays. Both HABPs exhibited high content of α-helical features and prevented P. falciparum merozoite invasion to RBCs in vitro by up to 91%. The invasion-blocking ability reported here for these PfP0 HABPs supports their inclusion in immunological studies with the aim of assessing their potential as candidates for a vaccine against P. falciparum malaria.     

expression of members of the heat-shock protein 70 family in the exoerythrocytic stages ofPlasmodium berghei andPlasmodium falciparum

Exoerythrocytic stages ofPlasmodium berghei cultured in HepG2-A16 hepatoma cells and those ofP. falciparum in human hepatocytes transplanted under the kidney capsule of CB-17/ICr scid/scid mice were used to evaluate expression of heat-shock-related stress proteins. Although undetectable in the sporozoites, the expression of proteins similar in sequence of a heat-shock protein of 70 kDa and a glucose-regulated protein of 78 kDa was markedly induced in the hepatic stages of malaria parasites. Expression of these proteins in the exoerythrocytic stages of the malaria parasite warrants a systematic evaluation of their potential role in eliciting cellular immune responses directed against infected hepatocytes.     

Stage-specific gametocytocidal effect in vitro of the antimalaria drug qinghaosu onPlasmodium falciparum

Qinghaosu, an anti-malaria drug, has been found to kill not only asexual blood stages but also the early stages of gametocytes ofPlasmodium falciparum. The effect of qinghaosu in vitro depends on the concentration of the drug as well as on the initial parasitemia level (IC₅₀=10–20 nM with 1% initial parasitemia). Resistance ofP. falciparum to other anti-malaria drugs, e.g., chloroquine and pyrimethamine, did not affect susceptibility of its asexual and sexual stages to qinghaosu. Gametocytocidal effect of qinghaosu may play a role in the interruption of malaria transmission.     

A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites

Pore-forming proteins are employed by many pathogens to achieve successful host colonization. Intracellular pathogens use pore-forming proteins to invade host cells, survive within and productively interact with host cells, and finally egress from host cells to infect new ones. The malaria-causing parasites of the genus Plasmodium evolved a number of life cycle stages that enter and replicate in distinct cell types within the mosquito vector and vertebrate host. Despite the fact that interaction with host-cell membranes is a central theme in the Plasmodium life cycle, little is known about parasite proteins that mediate such interactions. We identified a family of five related genes in the genome of the rodent malaria parasite Plasmodium yoelii encoding secreted proteins all bearing a single membrane-attack complex/perforin (MACPF)-like domain. Each protein is highly conserved among Plasmodium species. Gene expression analysis in P. yoelii and the human malaria parasite Plasmodium falciparum indicated that the family is not expressed in the parasites blood stages. However, one of the genes was significantly expressed in P. yoelii sporozoites, the stage transmitted by mosquito bite. The protein localized to the micronemes of sporozoites, organelles of the secretory invasion apparatus intimately involved in host-cell infection. MACPF-like proteins may play important roles in parasite interactions with the mosquito vector and transmission to the vertebrate host.     

Kinase signalling in Plasmodium sexual stages and interventions to stop malaria transmission

The symptoms of malaria, one of the infectious diseases with the highest mortality and morbidity world-wide, are caused by asexual parasites replicating inside red blood cells. Disease transmission, however, is effected by non-replicating cells which have differentiated into male or female gametocytes. These are the forms infectious to mosquito vectors and the insects are the only hosts where parasite sexual reproduction can take place. Malaria is thus a complex infection in which pharmacological treatment of symptoms may still allow transmission for long periods, while pharmacological blockage of infectivity may not cure symptoms. The process of parasite sexual differentiation and development is still being revealed but it is clear that kinase-mediated signalling mechanisms play a significant role. This review attempts to summarise our limited current knowledge on the signalling mechanisms involved in the transition from asexual replication to sexual differentiation and reproduction, with a brief mention to the effects of current treatments on the sexual stages and to some of the difficulties inherent in developing pharmacological interventions to curtail disease transmission.     

A fine functional homology between chitinases from host and parasite is relevant for malaria transmissibility

High levels of plasma chitotriosidase are a marker of macrophage activation in several pathologies and, in particular, in human malaria. Plasmodium falciparum, during its maturative cycle in the midgut of the Anopheles mosquito, secretes a chitinase to disrupt the peritrophic membrane, a necessary step in the migration of the parasite from the midgut to the salivary glands of malaria’s vector. The cooperation between human chitotriosidase (Chit) and the chitinase from P. falciparum in attacking the peritrophic membranes in the Anopheles midgut has been recently demonstrated by in vivo experiments. The present study confirms, by computational methods, this functional homology. A simple sequence analysis method, potentially useful to assess fine textual closeness in families of homologous proteins, is reported here and applied to a set of chitinases from mammals and plasmodia. This analysis confirms the clustering and the phylogenetic relationships obtained with well-known alignment methods, but also shows that the sequences of chitinases from malaria hosts and malaria parasites are correlated. This correlation, a sign of functional homology, is discussed as a condition for the spreading of different forms of malaria. From this perspective, one can get insight into the origins of malaria and its genetic or pharmacological control. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Interaction between Host Complement and Mosquito-Midgut-Stage Plasmodium berghei

After ingestion by mosquitoes, gametocytes of malaria parasites become activated and form extracellular gametes that are no longer protected by the red blood cell membrane against immune effectors of host blood. We have studied the action of complement on Plasmodium developmental stages in the mosquito blood meal using the rodent malaria parasite Plasmodium berghei and rat complement as a model. We have shown that in the mosquito midgut, rat complement components necessary to initiate the alternative pathway (factor B, factor D, and C3) as well as C5 are present for several hours following ingestion of P. berghei-infected rat blood. In culture, 30 to 50% of mosquito midgut stages of P. berghei survived complement exposure during the first 3 h of development. Subsequently, parasites became increasingly sensitive to complement lysis. To investigate the mechanisms involved in their protection, we tested for C3 deposition on parasite surfaces and whether host CD59 (a potent inhibitor of the complement membrane attack complex present on red blood cells) was taken up by gametes while emerging from the host cell. Between 0.5 and 22 h, 90% of Pbs21-positive parasites were positive for C3. While rat red and white blood cells stained positive for CD59, Pbs21-positive parasites were negative for CD59. In addition, exposure of parasites to rat complement in the presence of anti-rat CD59 antibodies did not increase lysis. These data suggest that parasite or host molecules other than CD59 are responsible for the protection of malaria parasites against complement-mediated lysis. Ongoing research aims to identify these molecules.     

Immunity against sexual stage Plasmodium falciparum and Plasmodium vivax parasites

The efficient spread of malaria from infected humans to mosquitoes is a major challenge for malaria elimination initiatives. Gametocytes are the only Plasmodium life stage infectious to mosquitoes. Here, we summarize evidence for naturally acquired anti-gametocyte immunity and the current state of transmission blocking vaccines (TBV). Although gametocytes are intra-erythrocytic when present in infected humans, developing Plasmodium falciparum gametocytes may express proteins on the surface of red blood cells that elicit immune responses in naturally exposed individuals. This immune response may reduce the burden of circulating gametocytes. For both P. falciparum and Plasmodium vivax, there is a solid evidence that antibodies against antigens present on the gametocyte surface, when co-ingested with gametocytes, can influence transmission to mosquitoes. Transmission reducing immunity, reducing the burden of infection in mosquitoes, is a well-acknowledged but poorly quantified phenomenon that forms the basis for the development of TBV. Transmission enhancing immunity, increasing the likelihood or intensity of transmission to mosquitoes, is more speculative in nature but is convincingly demonstrated for P. vivax. With the increased interest in malaria elimination, TBV and monoclonal antibodies have moved to the center stage of malaria vaccine development. Methodologies to prioritize and evaluate products are urgently needed.     

Plasmodium (Haemamoeba) cathemerium gene sequences for phylogenetic analysis of malaria parasites

The DNA sequence information on avian malaria parasites of the genus Plasmodium is quite limited. At present, sequences of only 6 out of 34 valid species are available. However, sequence data of avian malaria parasites are particularly important with regard to the resolution of the phylogenetic relationships of the most virulent human malaria agent, Plasmodium falciparum. The question as to whether P. falciparum originates from avian or from mammalian parasites would contribute to our understanding of its biology and would probably facilitate the interpretation of experimental results. To add to the body of molecular data, we sequenced three genes (cytochrome b, 18 SSU rRNA, caseinolytic protease C) of different organellar origin of one of the most widespread avian malaria parasites, Plasmodium (Haemamoeba) cathemerium, which once used to be an important laboratory in vivo model in human malaria research. The analysis of the new P. cathemerium sequences in direct comparison with the rodent parasite P. berghei and the four human malaria parasites by pairwise distance calculation do not suggest a closer relationship of P. cathemerium to P. falciparum than to the other species involved.     

Carrageenans inhibit the in vitro growth of Plasmodium falciparum and cytoadhesion to CD36

Carbohydrates are implicated in many of the invasive and adhesive interactions that occur between Plasmodium falciparum malaria parasites and human host cells, including invasion of sporozoites into hepatocytes, entry of merozoites into new host erythrocytes during asexual blood-stage replication, adhesion of infected erythrocytes to uninfected erythrocytes (rosetting) and to a number of host endothelial receptors including ICAM-1, CD36 and chondroitin-4-sulphate. In addition to increasing our understanding of host–parasite interactions, the investigation of carbohydrates with differing levels and patterns of sulphation as inhibitors may contribute to the development of novel therapeutics targeting malaria. Here we show that three polysaccharides derived from seaweed (carrageenans) with differing sulphation levels and patterns can inhibit the in vitro erythrocytic invasion and growth of both drug sensitive and drug resistant P. falciparum lines and the adhesion of parasitized erythrocytes to the human glycoprotein CD36.     

Comparative and functional genomics of the innate immune system in the malaria vector Anopheles gambiae

Summary: In much of Africa, the mosquito Anopheles gambiae is the major vector of human malaria, a devastating infectious disease caused by Plasmodium parasites. Vector and parasite interact at multiple stages and locations, and the nature and effectiveness of this reciprocal interaction determines the success of transmission. Many of the interactions engage the mosquito's innate immunity, a primitive but very effective defense system. In some cases, the mosquito kills the parasite, thus blocking the transmission cycle. However, not all interactions are antagonistic; some represent immune evasion. The sequence of the A. gambiae genome revealed numerous potential components of the innate immune system, and it established that they evolve rapidly, as summarized in the present review. Their rapid evolution by gene family expansion diversification as well as the prevalence of haplotype alleles in the best‐studied families may reflect selective adaptation of the immune system to the exigencies of multiple immune challenges in a variety of ecologic niches. As a follow‐up to the comparative genomic analysis, the development of functional genomic methodologies has provided novel opportunities for understanding the immune system and the nature of its interactions with the parasite. In this context, identification of both Plasmodium antagonists and protectors in the mosquito represents a significant conceptual advance. In addition to providing fundamental understanding of primitive immune systems, studies of mosquito interactions with the parasite open unprecedented opportunities for novel interventions against malaria transmission. The generation of transgenic mosquitoes that resist malaria infection in the wild and the development of antimalarial ‘smart sprays’ capable of disrupting interactions that are protective of the parasite, or reinforcing others that are antagonistic, represent technical challenges but also immense opportunities for improvement of global health.