Antigenic relationship between Plasmodium falciparum and Babesia bovis: reactivity with antibodies to culture-derived soluble exoantigens

Antigenic similarities between Plasmodium and Babesia parasites of the phylum Apicomplexa have been previously demonstrated primarily by the serological cross reactivity observed in the indirect fluorescent antibody (IFA) test. We have now studied the antigenic relationship between the human malaria parasite, Plasmodium falciparum, and the hemoparasitic agent of cattle, Babesia bovis, using rabbit monospecific antibodies produced against individual culture-derived P. falciparum polypeptides and bovine polyspecific antibodies to B. bovis exoantigens. These respective antibodies were found to be distinctly cross reactive in the IFA test using infected erythrocytes (squirrel monkey--P. falciparum; bovine--B. bovis) as antigen substrates. Immunofluorescence was shown to be highly specific for parasite surfaces. Additionally, the degree of reactivity with soluble exoantigens contained in Plasmodium and Babesia culture supernatants was monitored by a two-site enzyme immunoassay employing the cross-reactive antibodies. Further evidence for antigenic cross reactivity between P. falciparum and B. bovis parasites was shown with the in vitro inhibition assay. Antibodies to P. falciparum and B. bovis were found to be highly inhibitory for the in vitro growth of P. falciparum in human erythrocytes.     

Babesia rodhaini, Babesia bovis, and Babesia bigemina: analysis and sorting of red cells from infected mouse or calf blood by flow fluorimetry using 33258 Hoechst.

The DNA of Babesia spp. parasites within host intact red blood cells was labeled using the fluorescent bisbenzimidazole dye 33258 Hoechst. The labeled cells were sorted on a fluorescence activated cell sorter on the basis of cell fluorescence (proportional to DNA content) and the intensity of light scattered from the cells at low angles (related to cell size). The optimal conditions for dye uptake were established for the murine parasite Babesia rodhaini and the bovine parasites B. bovis and B. bigemina. Uninfected cells were nonfluorescent after incubation with the dye and could be completely separated from infected fluorescent cells. The fluorescence of cells infected with B. rodhaini was proportional to the number of parasite nuclei per cell. With saturation levels of dye, samples infected with B. bovis or B. bigemina in which erythrocytes contained one or two parasites, both exhibited only one fluorescent cell peak. Cell sorting did not eliminate the infectivity of B. rodhaini. The method may be used to separate populations of uninfected blood cells and cells infected with Babesia spp. for biochemical and immunochemical experiments.     

Babesia argentina, Plasmodium vivax and P. falciparum: antigenic cross-reactions

An indirect fluorescent antibody test was used to analyze the antigenic relationships between Babesia argentina, a parasite of cattle, and two human malaria parasites, Plasmodium falciparum and Plasmodium vivax. Elevated antibody titers to P. falciparum were found in cattle infected with B. argentina. Some persons infected with P. falciparum or P. vivax were found to produce antibodies to B. argentina. Explanations for the occurrence of these cross reactions are considered.     

The novel protein BboRhop68 is expressed by intraerythrocytic stages of Babesia bovis

The apical complex of intracellular hemoparasites contains organelles like micronemes and rhoptries, specialized structures required for adherence and invasion of host cells. Several molecules discharged from rhoptries have been identified from Plasmodium spp., but only a single rhoptry associated protein-1 (RAP-1) has been characterized from Babesia bovis. In silico search of the B. bovis genome allowed to identifying a sequence homologous to the gene that encodes a P. falciparum rhoptry protein PfRhop148. The intron-less 1830 bp novel gene, predicted a 68 kDa protein, and it was highly conserved among different B. bovis strains and isolates. The deducted protein from the B. bovis T2Bo strain, named BboRhop68, showed two putative transmembrane domains, at least seven B-cell epitopes, and a well conserved DUF501 super family domain. The bborhop68 gene was amplified, analyzed and compared among different B. bovis strains and isolates showing overall high sequence conservation. A fragment of bborhop68 was expressed as a recombinant fusion protein (rBboRhop68). The mice anti-rBboRhop68 serum identified the novel protein in intraerythrocytic trophozoites and merozoites by WB and ELISA, but not in free merozoites. Sera from naturally and experimentally infected bovines also recognized BboRhop68, suggesting that it is expressed and immunogenic during B. bovis infection. Fluorescence microscopy analysis using anti-rBboRhop68 antibodies showed a rod structure associated to trophozoites and merozoites infected erythrocytes, but this pattern of reactivity was not observed in free merozoites. The BboRhop68 was also not detected in ELISA based on solubilized merozoites. Thus, at least three independent lines of evidence support differential expression of BboRhop68 in intraerythrocytic stages of B. bovis and its possible functional role immediately after B. bovis erythrocyte invasion. The results of this work suggest that BboRhop68 could be considered as a novel additional target for developing improved methods to control bovine babesiosis.     

Plasmodium falciparum: Association with Erythrocytic Superoxide Dismutase1

Levels of superoxide dismutase (SOD) activity and its properties in Plasmodium falciparum-infected erythrocytes, isolated parasites, and noninfected erythrocytes were studied. A higher specific activity was found in P. falciparum-infected erythrocytes compared to noninfected erythrocytes, resulting from the lower protein content of infected cells and not enzyme synthesis by the parasite, as the superoxide dismutase activity expressed per number of cells was decreased. Superoxide dismutase from noninfected erythrocytes and isolated P. falciparum parasites showed similar sensitivities to various inhibitors and had identical molecular weights and electrophoretic mobilities. These results support the hypothesis of uptake and use of the erythrocytic SOD enzyme by the parasite as a possible mechanism of defense against oxidative stress.     

Inhibition of Plasmepsin V Activity Demonstrates Its Essential Role in Protein Export,PfEMP1 Display,and Survival of Malaria Parasites

The malaria parasite Plasmodium falciparum exports several hundred proteins into the infected erythrocyte that are involved in cellular remodeling and severe virulence. The export mechanism involves the Plasmodium export element (PEXEL), which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed PEXEL cleavage occurs cotranslationaly, similar to signal peptidase. Treatment of P. falciparum–infected erythrocytes with the inhibitor caused dose-dependent inhibition of PEXEL processing as well as protein export, including impaired display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp. and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target.     

Bovine babesiosis: Cattle protected in the field with a frozen vaccine containing Babesia bovis and Babesia bigemina cultured in vitro with a serum-free medium

An attenuated live vaccine containing Babesia bovis and B. bigemina cultured in vitro with a serum-free medium was assessed for its clinical protection conferred of naïve cattle, under natural tick-challenge in a high endemicity zone to Babesia spp. Three groups of six animals were treated as follows: group I (GI) received a vaccine derived from parasites cultured with a free-serum medium; group II (GII) were immunized with the standard vaccine, with parasites cultured in a medium supplemented with 40% (v/v) bovine serum; and a control group (GIII) inoculated with non-infected bovine erythrocytes. Inocula were administered by IM route. Experimental animals were kept during 23 days after vaccination in a cattle farm free of ticks and Babesia spp. Thereafter, cattle were moved to a high endemicity farm for natural exposure to Babesia spp. transmitted by Rhipicephalus microplus ticks. Protection against clinical babesiosis was observed in bovines belonging to GI (100%) and GII (83.33%), while the control animals (GIII) were not protected, and showed severe clinical signs, closely related to babesiosis, were observed for at least three consecutive days during the challenge. These were fever, anemia, which were measured simultaneously, and circulating parasites were detected by optic light microscopy. All cattle showed B. bovis and B. bigemina in stained blood films during the challenge; B. bovis antibody titers were higher than those to B. bigemina in GI and GII, and lower titers were determined in GIII. The protective capacity of the vaccine derived from B. bovis and B. bigemina cultured in vitro in a serum-free medium was demonstrated.     

Identifying novel Plasmodium falciparum erythrocyte invasion receptors using systematic extracellular protein interaction screens

The invasion of host erythrocytes by the parasite Plasmodium falciparum initiates the blood stage of infection responsible for the symptoms of malaria. Invasion involves extracellular protein interactions between host erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. Despite significant research effort, many merozoite surface ligands have no known erythrocyte binding partner, most likely due to the intractable biochemical nature of membrane‐tethered receptor proteins and their interactions. The few receptor–ligand pairs that have been described have largely relied on sourcing erythrocytes from patients with rare blood groups, a serendipitous approach that is unsatisfactory for systematically identifying novel receptors. We have recently developed a scalable assay called AVEXIS (for AVidity‐based EXtracellular Interaction Screen), designed to circumvent the technical difficulties associated with the identification of extracellular protein interactions, and applied it to identify erythrocyte receptors for orphan P. falciparum merozoite ligands. Using this approach, we have recently identified Basigin (CD147) and Semaphorin‐7A (CD108) as receptors for RH5 and MTRAP respectively. In this essay, we review techniques used to identify Plasmodium receptors and discuss how they could beapplied in the future to identify novel receptors both for Plasmodium parasites but also other pathogens.     

Characterization of the ATG8-conjugation system in 2 Plasmodium species with special focus on the liver stage

Plasmodium parasites successfully colonize different habitats within mammals and mosquitoes, and adaptation to various environments is accompanied by changes in their organelle composition and size. Previously, we observed that during hepatocyte infection, Plasmodium discards organelles involved in invasion and expands those implicated in biosynthetic pathways. We hypothesized that this process is regulated by autophagy. Plasmodium spp. possess a rudimentary set of known autophagy-related proteins that includes the ortholog of yeast Atg8. In this study, we analyzed the activity of the ATG8-conjugation pathway over the course of the lifecycle of Plasmodium falciparum and during the liver stage of Plasmodium berghei. We engineered a transgenic P. falciparum strain expressing mCherry-PfATG8. These transgenic parasites expressed mCherry-PfATG8 in human hepatocytes and erythrocytes, and in the midgut and salivary glands of Anopheles mosquitoes. In all observed stages, mCherry-PfATG8 was localized to tubular structures. Our EM and colocalization studies done in P. berghei showed the association of PbATG8 on the limiting membranes of the endosymbiont-derived plastid-like organelle known as the apicoplast. Interestingly, during parasite replication in hepatocytes, the association of PbATG8 with the apicoplast increases as this organelle expands in size. PbATG3, PbATG7 and PbATG8 are cotranscribed in all parasitic stages. Molecular analysis of PbATG8 and PbATG3 revealed a novel mechanism of interaction compared with that observed for other orthologs. This is further supported by the inability of Plasmodium ATG8 to functionally complement atg8Δ yeast or localize to autophagosomes in starved mammalian cells. Altogether, these data suggests a unique role for the ATG8-conjugation system in Plasmodium parasites.     

Life cycle studies of the hexose transporter of Plasmodium species and genetic validation of their essentiality

A Plasmodium falciparum hexose transporter (PfHT) has previously been shown to be a facilitative glucose and fructose transporter. Its expression in Xenopus laevis oocytes and the use of a glucose analogue inhibitor permitted chemical validation of PfHT as a novel drug target. Following recent re‐annotations of the P. falciparum genome, other putative sugar transporters have been identified. To investigate further if PfHT is the key supplier of hexose to P. falciparum and to extend studies to different stages of Plasmodium spp., we functionally analysed the hexose transporters of both the human parasite P. falciparum and the rodent parasite Plasmodium berghei using gene targeting strategies. We show here the essential function of pfht for the erythrocytic parasite growth as it was not possible to knockout pfht unless the gene was complemented by an episomal construct. Also, we show that parasites are rescued from the toxic effect of a glucose analogue inhibitor when pfht is overexpressed in these transfectants. We found that the rodent malaria parasite orthologue, P. berghei hexose transporter (PbHT) gene, was similarly refractory to knockout attempts. However, using a single cross‐over transfection strategy, we generated transgenic P. berghei parasites expressing a PbHT–GFP fusion protein suggesting that locus is amenable for gene targeting. Analysis of pbht‐gfp transgenic parasites showed that PbHT is constitutively expressed through all the stages in the mosquito host in addition to asexual stages. These results provide genetic support for prioritizing PfHT as a target for novel antimalarials that can inhibit glucose uptake and kill parasites, as well as unveiling the expression of this hexose transporter in mosquito stages of the parasite, where it is also likely to be critical for survival.     

Gene sequencing, modelling and immunolocalization of the protein disulfide isomerase from Plasmodium chabaudi

Malaria remains one of the major human parasitic diseases, particularly in subtropical regions. Most of the fatal cases are caused by Plasmodium falciparum. The rodent parasite Plasmodium chabaudi has been the model of choice in research due to its similarities to human malaria, including developmental cycle, preferential invasion of mature erythrocytes, synchrony of asexual development, antigenic variation, gene sinteny as well as similar resistance mechanisms. Protein disulfide isomerase (PDI) is an essential catalyst of the endoplasmic reticulum in different biological systems with folding and chaperone activities. Most of the proteins exported by parasites have to pass through the endoplasmic reticulum before reaching their final destination and their correct folding is critical for parasite survival. PDI constitutes a potential target for the development of alternative therapy strategies based on the inhibition of folding and chaperoning of exported proteins. We here describe the sequencing of the gene coding for the PDI from P. chabaudi and analyse the relationship to its counterpart enzymes, particularly with the PDI from other Plasmodium species. The model constructed, based on the recent model deduced from the crystallographic structure 2B5E, was compared with the previous theoretical model for the whole PDI molecule constructed by threading. A recombinant PDI from P. chabaudi was also produced and used as an antigen for monoclonal antibody production for application in PDI immunolocalization.     

A thrombospondin structural repeat containing rhoptry protein from Plasmodium falciparum mediates erythrocyte invasion

Host cell invasion by Plasmodium falciparum requires multiple molecular interactions between host receptors and parasite ligands. A family of parasite proteins, which contain the conserved thrombospondin structural repeat motif (TSR), has been implicated in receptor binding during invasion. In this study we have characterized the functional role of a TSR containing blood stage protein referred to as P. falciparum thrombospondin related apical merozoite protein (PfTRAMP). Both native and recombinant PfTRAMP bind untreated as well as neuraminidase, trypsin or chymotrypsin‐treated human erythrocytes. PfTRAMP is localized in the rhoptry bulb and is secreted during invasion. Adhesion of microneme protein EBA175 with its erythrocyte receptor glycophorin A provides the signal that triggers release of PfTRAMP from the rhoptries. Rabbit antibodies raised against PfTRAMP block erythrocyte invasion by P. falciparum suggesting that PfTRAMP plays an important functional role in invasion. Combination of antibodies against PfTRAMP with antibodies against microneme protein EBA175 provides an additive inhibitory effect against invasion. These observations suggest that targeting multiple conserved parasite ligands involved in different steps of invasion may provide an effective strategy forthe development of vaccines against blood stage malaria parasites.     

Inhibition of Plasmodium falciparum cysteine proteases by the sugarcane cystatin CaneCPI-4

Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well-studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC₅₀ values of 12 nM and 42 nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.     

Gliding motility of Babesia bovis merozoites visualized by time-lapse video microscopy

Background

Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite.

Methodology/Principal Findings

Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion.

Conclusions/Significance

This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding.     

Shared elements of host‐targeting pathways among apicomplexan parasites of differing lifestyles

Apicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal‐mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host‐targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal‐mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal‐mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM.     

Purinergic signalling is involved in the malaria parasite <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> invasion to red blood cells

Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca²⁺ levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca²⁺ increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca²⁺ levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca²⁺ in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca²⁺]_c. Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca²⁺]_c in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells.     

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.     

Identification of microsatellite markers in Plasmodium mexicanum,a lizard malaria parasite that infects nucleated erythrocytes

Reptile and bird hosts of malaria parasites (Plasmodium) have nucleated erythrocytes. Infected blood thus contains a mix of abundant host and scant parasite DNA which has prevented identification of Plasmodium microsatellites. We developed a protocol for isolation of microsatellite markers for Plasmodium mexicanum, a parasite of lizards. The ATT repeat was common in the genome of P. mexicanum, but most (87%) of these repeats were exceptionally long (50–206 + repeats). Seven microsatellite markers with polymerase chain reaction primers are described. The protocol should allow discovery of microsatellites of malaria parasites (with AT‐rich genomes) infecting bird and reptile hosts.     

In vivo uptake of a haem analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum‐infected red blood cells

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle‐like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake byP. falciparum‐infected erythrocytes shows that at R and S stages, a time‐increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time‐increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.