Identification and characterization of the Plasmodium falciparum RhopH2 ortholog in Plasmodium vivax

Plasmodium vivax is one of the most important human malaria species that is geographically widely endemic and potentially affects a larger number of people than its more notorious cousin, Plasmodium falciparum. During invasion of red blood cells, the parasite requires the intervention of high molecular weight complex rhoptry proteins (RhopH) that are also essential for cytoadherence. PfRhopH2, a member of the RhopH multigene family, has been characterized as being crucial during P. falciparum infection. This study describes identifying and characterizing the pfrhoph2 orthologous gene in P. vivax (hereinafter named pvrhoph2). The PvRhopH2 is a 1,369-amino acid polypeptide encoded by PVX_099930 gene, for which orthologous genes have been identified in other Plasmodium species by bioinformatic approaches. Both P. falciparum and P. vivax genes contain nine introns, and there is a high degree of similarity between the deduced amino acid sequences of the two proteins. Moreover, PvRhopH2 contains a signal peptide at its N-terminus and 12 cysteines predominantly in its C-terminal half. PvRhopH2 is localized in one of the apical organelles of the merozoite, the rhoptry, and the localization pattern is similar to that of PfRhopH2 in P. falciparum. The recombinant PvRhopH2 protein is recognized by serum antibodies of patients naturally exposed to P. vivax, suggesting that PvRhopH2 is immunogenic in humans.     

The Plasmodium vivax homologues of merozoite surface proteins 4 and 5 from Plasmodium falciparum are expressed at different locations in the merozoite

Merozoite surface proteins of Plasmodium falciparum are one major group of antigens currently being investigated and tested as malaria vaccine candidates. Two recently described P. falciparum merozoite surface antigens, MSP4 and MSP5, are GPI-anchored proteins that each contain a single EGF-like domain and appear to have arisen by an ancient gene duplication event. The genes are found in tandem on chromosome 2 of P. falciparum and the syntenic region of the genome was identified in the rodent malarias P. chabaudi, P. yoelii and P. berghei. In these species, there is only a single gene, designated MSP4/5 encoding a single EGF-like domain similar to the EGF-like domain in both PfMSP4 and PfMSP5. Immunization of mice with PyMSP4/5 provides mice with high levels of protection against lethal challenge with blood stage P. yoelii. In this study, we show that in P. vivax, which is quite phylogenetically distant from P. falciparum, both MSP4 and MSP5 homologues can be found with their relative arrangements with respect to the surrounding genes mostly preserved. However, the gene for MSP2, found between MSP5 and adenylosuccinate lyase (ASL) in P. falciparum, is absent from P. vivax. The PvMSP4 and PvMSP5 genes have a two-exon structure and encode proteins with potential signal and GPI anchor sequences and a single EGF-like domain near the carboxyl-terminus. Rabbit antisera raised against purified recombinant proteins show that each of the antisera react with distinct proteins of 62 kDa for PvMSP4 and 86 kDa for PvMSP5 in parasite lysates. Indirect immunofluorescence assays (IFA) localized PvMSP4 over the entire surface of P. vivax merozoites, as expected, whereas, the MSP5 homologue was found to be associated with an apical organellar location consistent with micronemes or over the polar prominence.     

Identification of Plasmodium knowlesi erythrocyte binding proteins

Plasmodium knowlesi, a malaria of Old World monkeys, invades all Duffy blood group positive human erythrocytes and various New World monkey erythrocytes except Cebus apella. We had previously identified a 135 kDa parasite protein in supernatants of P. knowlesi cultures that bound to Duffy positive but not to Duffy negative human erythrocytes [Haynes et al., J. Exp. Med. 167, 1873-1881 (1988)]. We now use New World monkey erythrocytes as a reagent to identify P. knowlesi proteins in culture supernatants that will bind to all New World monkey erythrocytes susceptible to invasion but not to C. apella erythrocytes, which are refractory to invasion. The 135 kDa protein binds to all New World monkey erythrocytes, including C. appella. Another protein of 155 kDa binds to all New World monkey erythrocytes except C. apella. The 155 kDa protein binds to Old World monkey erythrocytes, the natural host of P. knowlesi; it does not bind to human Duffy positive erythrocytes. This and the previous study are the beginning of the identification of parasite proteins of P. knowlesi that bind to erythrocytes in a receptor specific manner.     

Differential antibody responses to Plasmodium falciparum and P. vivax circumsporozoite proteins in a human population.

Papua New Guineans exposed to hyperendemic malaria in the Madang area showed different antibody responses to Plasmodium falciparum and Plasmodium vivax sporozoites despite comparable entomological inoculation rates. Although there was a significant trend of increasing prevalence of anti-P. falciparum circumsporozoite (CS) protein immunoglobulin G (IgG) with age, there was no significant increase in the antibody units of IgG recognizing P. falciparum CS proteins. Antibodies recognizing P. vivax CS proteins steadily increased in prevalence and antibody units with age. Significant trends of increasing prevalence of antibody responders (both IgG and IgM) with increasing splenic enlargement were found in the younger age groups for P. falciparum CS proteins but not for P. vivax CS proteins. When antibody responders were analyzed by quartiles, there was a trend of increasing antibody response with age against P. vivax CS peptide, but not for P. falciparum CS protein. There was no evidence for increasing protection against blood-stage infections with increasing antibody levels for either P. falciparum or P. vivax. Neither were any significant relationships found between entomological inoculation rates and either CS antibody prevalence or concentration among the villages studied.     

A Plasmodium falciparum blood stage antigen highly homologous to the glycophorin binding protein GBP.

We have isolated a gene coding for a protein highly homologous to an antigen known as the glycophorin binding protein (GBP) which was therefore called GBPH. The gene consists of 2 exons interrupted by an intron located at a position corresponding to that of the GBP gene. The deduced amino acid sequence of GBPH comprises 427 residues and is characterized by a signal sequence and by an extended repeat region consisting of 8 units of 40 amino acid residues. The comparison of the amino acid sequences of GBPH and GBP reveals an identity of 69%. Antisera raised against a GBPH fragment that carries part of the repetitive region cross-react with GBP (105 kDa) and additionally detect some bands between 40 and 70 kDa, one of which may correspond to GBPH. The genes coding for GBP and GBPH are located on chromosomes 10 and 14, respectively. The GBP gene is transcribed as a highly abundant 6.5 kb mRNA in the blood-stage form, whereas Northern blot analysis using a GBPH specific probe detects 2 less abundant mRNAs of 2.3 kb and 2.7 kb. Southern blot analysis of P. falciparum DNA identifies a third member of the GBP gene family.     

Functional Antibodies against VAR2CSA in Nonpregnant Populations from Colombia Exposed to Plasmodium falciparum and Plasmodium vivax

In pregnancy, parity-dependent immunity is observed in response to placental infection with Plasmodium falciparum. Antibodies recognize the surface antigen, VAR2CSA, expressed on infected red blood cells and inhibit cytoadherence to the placental tissue. In most settings of malaria endemicity, antibodies against VAR2CSA are predominantly observed in multigravid women and infrequently in men, children, and nulligravid women. However, in Colombia, we detected antibodies against multiple constructs of VAR2CSA among men and children with acute P. falciparum and Plasmodium vivax infection. The majority of men and children (>60%) had high levels of IgGs against three recombinant domains of VAR2CSA: DBL5ε, DBL3X, and ID1-ID2. Surprisingly, these antibodies were observed only in pregnant women, men, and children exposed either to P. falciparum or to P. vivax. Moreover, the anti-VAR2CSA antibodies are of high avidity and efficiently inhibit adherence of infected red blood cells to chondroitin sulfate A in vitro, suggesting that they are specific and functional. These unexpected results suggest that there may be genotypic or phenotypic differences in the parasites of this region or in the host response to either P. falciparum or P. vivax infection outside pregnancy. These findings may hold significant clinical relevance to the pathophysiology and outcome of malaria infections in this region.     

Identification in Plasmodium falciparum of a protein specifically binding human fibronectins

A fibronectin binding protein (FnBp) was identified in 3H isoleucine labeled P. falciparum schizonts using affinity chromatography on human fibronectin (Fn) coupled to Sepharose 4B. After incubation of Nonidet-P 40 parasite lysate with Fn-Sepharose, elution was performed with SDS-PAGE buffer. Analysis of FnBp by SDS-PAGE demonstrated a major band which migrated with an apparent Mr of 70,000 under reducing conditions. This band was not found when human or rabbit IgG coupled Sepharose 4B were used instead of Fn as control.     

Plasmodium falciparum homologue of the genes for Plasmodium vivax and Plasmodium yoelii adhesive proteins, which is transcribed but not translated

The 235-kDa family of rhoptry proteins in Plasmodium yoelii and the two reticulocyte binding proteins of P. vivax comprise a family of proteins involved in host cell selection and erythrocyte invasion. Here we described a member of the gene family found in P. falciparum (PfRH3) that is transcribed in its entirety, under stage-specific control, with correct splicing of the intron, but appears not to be translated, probably due to two reading frameshifts at the 5' end of the gene.     

Epitope-specific humoral immunity to Plasmodium vivax Duffy binding protein

Erythrocyte invasion by Plasmodium vivax is completely dependent on binding to the Duffy blood group antigen by the parasite Duffy binding protein (DBP). The receptor-binding domain of this protein lies within a cysteine-rich region referred to as region II (DBPII). To examine whether antibody responses to DBP correlate with age-acquired immunity to P. vivax, antibodies to recombinant DBP (rDBP) were measured in 551 individuals residing in a village endemic for P. vivax in Papua New Guinea, and linear epitopes mapped in the critical binding region of DBPII. Antibody levels to rDBP(II) increased with age. Four dominant linear epitopes were identified, and the number of linear epitopes recognized by semi-immune individuals increased with age, suggesting greater recognition with repeated infection. Some individuals had antibodies to rDBP(II) but not to the linear epitopes, indicating the presence of conformational epitopes. This occurred in younger individuals or subjects acutely infected for the first time with P. vivax, indicating that repeated infection is required for recognition of linear epitopes. All four dominant B-cell epitopes contained polymorphic residues, three of which showed variant-specific serologic responses in over 10% of subjects examined. In conclusion, these results demonstrate age-dependent and variant-specific antibody responses to DBPII and implicate this molecule in partial acquired immunity to P. vivax in populations in endemic areas.     

Low-complexity regions in Plasmodium falciparum proteins

Full-sequence data available for Plasmodium falciparum chromosomes 2 and 3 are exploited to perform a statistical analysis of the long tracts of biased amino acid composition that characterize the vast majority of P. falciparum proteins and to make a comparison with similarly defined tracts from other simple eukaryotes. When the relatively minor subset of prevalently hydrophobic segments is discarded from the set of low-complexity segments identified by current segmentation methods in P. falciparum proteins, a good correspondence is found between prevalently hydrophilic low-complexity segments and the species-specific, rapidly diverging insertions detected by multiple-alignment procedures when sequences of bona fide homologs are available. Amino acid preferences are fairly uniform in the set of hydrophilic low-complexity segments identified in the two P. falciparum chromosomes sequenced, as well as in sequenced genes from Plasmodium berghei, but differ from those observed in Saccharomyces cerevisiae and Dictyostelium discoideum. In the two plasmodial species, amino acid frequencies do not correlate with properties such as hydrophilicity, small volume, or flexibility, which might be expected to characterize residues involved in nonglobular domains but do correlate with A-richness in codons. An effect of phenotypic selection versus neutral drift, however, is suggested by the predominance of asparagine over lysine.     

Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination

Malaria remains a major cause of morbidity and mortality in the tropics, with Plasmodium falciparum responsible for the majority of the disease burden and P. vivax being the geographically most widely distributed cause of malaria. Gametocytes are the sexual-stage parasites that infect Anopheles mosquitoes and mediate the onward transmission of the disease. Gametocytes are poorly studied despite this crucial role, but with a recent resurgence of interest in malaria elimination, the study of gametocytes is in vogue. This review highlights the current state of knowledge with regard to the development and longevity of P. falciparum and P. vivax gametocytes in the human host and the factors influencing their distribution within endemic populations. The evidence for immune responses, antimalarial drugs, and drug resistance influencing infectiousness to mosquitoes is reviewed. We discuss how the application of molecular techniques has led to the identification of submicroscopic gametocyte carriage and to a reassessment of the human infectious reservoir. These components are drawn together to show how control measures that aim to reduce malaria transmission, such as mass drug administration and a transmission-blocking vaccine, might better be deployed.     

Low-complexity segments in Plasmodium falciparum proteins are primarily nucleic acid level adaptations

Protein segments that contain few of the possible 20 amino acids, sometimes in tandem repeat arrays, are referred to as containing "simple" or "low-complexity" sequence. Many Plasmodium falciparum proteins are longer than their homologs in other species by virtue of their content of such low-complexity segments that have no known function; these are interspersed among segments of higher complexity to which function can often be ascribed. If there is low complexity at the protein level, there is likely to be low complexity at the corresponding nucleic acid level (departure from equifrequency of the four bases). Thus, low complexity may have been selected primarily at the nucleic acid level and low complexity at the protein level may be secondary. In this case, the amino acid composition of low-complexity segments should be more reflective than that of high complexity segments on forces operating at the nucleic acid level, which include GC-pressure and AG-pressure. Consistent with this, for amino acid determining first and second codon positions, open reading frames containing low-complexity segments show increased contributions to downward GC-pressure (revealed as decreased percentage of G+C) and to upward AG-pressure (revealed as increased percentage A+G). When not countermanded by high contributions to AG-pressure, low-complexity segments can contribute to base order-dependent fold potential; in this respect, they resemble introns. Thus, in P. falciparum, low-complexity segments appear as adaptations primarily serving nucleic acid level functions.     

Differences in erythrocyte receptor specificity of different parts of the Plasmodium falciparum reticulocyte binding protein homologue 2a

The Plasmodium falciparum reticulocyte-binding-like protein homologue (RH) and erythrocyte binding-like (EBL) protein families play important roles during invasion, though their exact roles are not clear. Both EBL and RH proteins are thought to directly bind different receptors on the surface of the erythrocyte, and the binding properties for a number of EBLs and RHs have been described. While P. falciparum RH1 (PfRH1) and PfRH4 have been shown to act directly in two alternative invasion pathways used by merozoites, the functions of PfRH2a and PfRH2b during invasion are less defined. Here, using monoclonal antibodies raised against a unique region of PfRH2a, we show that PfRH2a moves from the rhoptry neck to the moving junction during merozoite invasion. The movement of PfRH2a to the junction is independent of the invasion pathway used by the merozoite, suggesting an additional function of the protein that is independent of receptor binding. We further show that PfRH2a is processed both in the schizont and during invasion, resulting in proteins with different erythrocyte binding properties. Our findings suggest that PfRH2a and, most likely, the other members of the RH family, depending on their processing stage, can engage different receptors at different stages of the invasion process.     

Anti-lymphocytotoxic antibodies in sera of Thai adults infected with Plasmodium falciparum or Plasmodium vivax.

Because of the potential for the elimination of lymphocytes through anti-lymphocytotoxic antibodies we examined individual sera of patients infected with falciparum or vivax malaria for the presence of antibodies against normal peripheral blood mononuclear cells. In assays done at 15 degrees C, 95% of the P. falciparum patients and 98% of the P. vivax patients showed evidence for antibody activity. Activity at 37 degrees C was significantly less than that at 15 degrees C. These studies suggest that infection with malaria induces anti-lymphocytotoxic antibodies which are predominantly cold-reactive. It is possible that this phenomenon plays a role in modulating the immune response of patients toward malaria.     

Identification of the Plasmodium chabaudi homologue of merozoite surface proteins 4 and 5 of Plasmodium falciparum

Previous studies of Plasmodium falciparum have identified a region of chromosome 2 in which are clustered three genes for glycosylphosphatidylinositol (GPI)-anchored merozoite surface proteins, MSP2, MSP5, and MSP4, arranged in tandem. MSP4 and MSP5 both encode proteins 272 residues long that contain hydrophobic signal sequences, GPI attachment signals, and a single epidermal growth factor (EGF)-like domain at their carboxyl termini. Nevertheless, the remainder of their protein coding regions are quite dissimilar. The locations and similar structural features of these genes suggest that they have arisen from a gene duplication event. Here we describe the identification of the syntenic region of the genome in the murine malaria parasite, Plasmodium chabaudi adami DS. Only one open reading frame is present in this region, and it encodes a protein with structural features reminiscent of both MSP4 and MSP5, including a single EGF-like domain. Accordingly, the gene has been designated PcMSP4/5. The homologue of the P. falciparum MSP2 gene could not be found in P. chabaudi; however, the amino terminus of the PcMSP4/5 protein shows similarity to that of MSP2. The PcMSP4/5 gene encodes a protein with an apparent molecular mass of 36 kDa, and this protein is detected in mature stages of the parasite. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localized to the surfaces of trophozoites and developing and free merozoites. The PcMSP4/5 gene is transcribed in both ring and trophozoite stages but appears to be spliced in a stage-specific manner such that the central intron is spliced from the mRNA in the parasitic stage in which the protein is expressed.     

Serological relationship of tumor necrosis factor-inducing exoantigens of Plasmodium falciparum and Plasmodium vivax.

Exoantigens of Plasmodium vivax-parasitized erythrocytes stimulated macrophages to secrete tumor necrosis factor, and antisera raised against the exoantigens inhibited this secretion. The antisera also inhibited the activity of Plasmodium falciparum and Plasmodium yoelii exoantigens, and conversely, antisera against the latter cross-reacted with the exoantigens of P. vivax.