Differential effects of translation inhibitors on Plasmodium berghei liver stage parasites.

Increasing numbers of antimalarial compounds are being identified that converge mechanistically at inhibition of cytoplasmic translation, regardless of the molecular target or mechanism. A deeper understanding of how their effectiveness as liver stage translation inhibitors relates to their chemoprotective potential could prove useful. Here, we probed that relationship using the Plasmodium berghei-HepG2 liver stage infection model. After determining translation inhibition EC50s for five compounds, we tested them at equivalent effective concentrations to compare the parasite response to, and recovery from, a brief period of translation inhibition in early schizogony, followed by parasites to 120 h post-infection to assess antiplasmodial effects of the treatment. We show compound-specific heterogeneity in single parasite and population responses to translation inhibitor treatment, with no single metric strongly correlated to the release of hepatic merozoites for all compounds. We also demonstrate that DDD107498 is capable of exerting antiplasmodial effects on translationally arrested liver stage parasites and uncover unexpected growth dynamics during the liver stage. Our results demonstrate that translation inhibition efficacy does not determine antiplasmodial efficacy for these compounds.

Characterization of merozoite-specific thrombospondin-related anonymous protein (MTRAP) in Plasmodium vivax and P. knowlesi parasites.

Plasmodium vivax, the most widespread human malaria parasite, and P. knowlesi, an emerging Plasmodium that infects humans, are the phylogenetically closest malarial species that infect humans, which may induce cross-species reactivity across most co-endemic areas in Southeast Asia. The thrombospondin-related anonymous protein (TRAP) family is indispensable for motility and host cell invasion in the growth and development of Plasmodium parasites. The merozoite-specific TRAP (MTRAP), expressed in blood-stage merozoites, is supposed to be essential for human erythrocyte invasion. We aimed to characterize MTRAPs in blood-stage P. vivax and P. knowlesi parasites and ascertain their cross-species immunoreactivity. Recombinant P. vivax and P. knowlesi MTRAPs of full-length ectodomains were expressed in a mammalian expression system. The MTRAP-specific immunoglobulin G, obtained from immune animals, was used in an immunofluorescence assay for subcellular localization and invasion inhibitory activity in blood-stage parasites was determined. The cross-species humoral immune responses were analyzed in the sera of patients with P. vivax or P. knowlesi infections. The MTRAPs of P. vivax (PvMTRAP) and P. knowlesi (PkMTRAP) were localized on the rhoptry body of merozoites in blood-stage parasites. Both anti-PvMTRAP and anti-PkMTRAP antibodies inhibited erythrocyte invasion of blood-stage P. knowlesi parasites. The humoral immune response to PvMTRAP showed high immunogenicity, longevity, and cross-species immunoreactivity with P. knowlesi. MTRAPs are promising candidates for development of vaccines and therapeutics against vivax and knowlesi malaria.

Characterisation of the merozoite thrombospondin related anonymous protein (MTRAP) of Plasmodium berghei as a transmission-blocking antigen.

BACKGROUND: Malaria is an infectious disease caused by protozoan parasites belonging to the genus Plasmodium. Human-to-human transmission depends on a mosquito vector; thus, the interruption of parasite transmission from humans to mosquitoes is an important approach in the fight against malaria. The parasite stages infectious to mosquitoes are the gametocytes, sexual stages that are ingested by the vector during a blood meal and transform into male and female gametes in the midgut. Immunity against sexual stage antigens expressed by gametocytes, gametes, and the zygote formed after fertilisation can interrupt the parasite sexual cycle in the mosquito. This transmission blocking immunity is mediated by specific antibodies ingested during the mosquito blood feed, inhibiting the parasite development in the midgut. Merozoite thrombospondin related anonymous protein (MTRAP) is a merozoite and gametocyte surface protein essential for gamete egress from erythrocytes and for parasite transmission to mosquitoes. OBJECTIVES: Here, we evaluated the potential of the P. berghei MTRAP to elicit antibodies with the ability to inhibit gamete fertilisation in vitro. METHODS: We expressed a soluble recombinant PbMTRAP and used it to immunise BALB/c mice. The transmission blocking activity of the anti-rPbMTRAP antibodies was tested through in vivo challenge experiments followed by in vitro conversion assays. FINDINGS: Immunisations with the rPbMTRAP induced a strong antibody response and the antibodies recognised the native protein by Western Blot and IFA. Anti-rPbMTRAP present in the blood stream of immunised mice partially inhibited gamete conversion into ookinetes. CONCLUSION: Our results indicate that antibodies to PbMTRAP may reduce but are not sufficient to completely block transmission.

Atypical memory B cell frequency correlates with antibody breadth and function in malaria immune adults.

Clinical immunity to malaria develops slowly after repeated episodes of infection and antibodies are essential in naturally acquired immunity against malaria. However, chronic exposure to malaria has been linked to perturbation in B-cell homeostasis with the accumulation of atypical memory B cells. It is unclear how perturbations in B cell subsets influence antibody breadth, avidity, and function in individuals naturally exposed to malaria. We show that individuals living in high malaria transmission regions in Ghana have higher Plasmodium falciparum merozoite antigen-specific antibodies and an increased antibody breadth score but lower antibody avidities relative to low transmission regions. The frequency of circulating atypical memory B cells is positively associated with an individual's antibody breadth. In vitro growth inhibition is independent of the ability to bind to free merozoites but associated with the breadth of antibody reactivity in an individual. Taken together, our data shows that repeated malaria episodes hamper the development of high avid antibodies which is compensated for by an increase in antibody breadth. Our results provide evidence to reinforce the idea that in regions with high malaria prevalence, repeated malaria infections lead to the broadening of antibody diversity and the continued presence of atypical memory B cell populations.

Identification of conserved cross-species B-cell linear epitopes in human malaria: a subtractive proteomics and immuno-informatics approach targeting merozoite stage proteins.

Human malaria, caused by five Plasmodium species (P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi), remains a significant global health burden. While most interventions target P. falciparum, the species associated with high mortality rates and severe clinical symptoms, non-falciparum species exhibit different transmission dynamics, remain hugely neglected, and pose a significant challenge to malaria elimination efforts. Recent studies have reported the presence of antigens associated with cross-protective immunity, which can potentially disrupt the transmission of various Plasmodium species. With the sequencing of the Plasmodium genome and the development of immunoinformatic tools, in this study, we sought to exploit the evolutionary history of Plasmodium species to identify conserved cross-species B-cell linear epitopes in merozoite proteins. We retrieved Plasmodium proteomes associated with human malaria and applied a subtractive proteomics approach focusing on merozoite stage proteins. Bepipred 2.0 and Epidope were used to predict B-cell linear epitopes using P. falciparum as the reference species. The predictions were further compared against human and non-falciparum databases and their antigenicity, toxicity, and allergenicity assessed. Subsequently, epitope conservation was carried out using locally sequenced P. falciparum isolates from a malaria-endemic region in western Kenya (n=27) and Kenyan isolates from MalariaGEN version 6 (n=131). Finally, physiochemical characteristics and tertiary structure of the B-cell linear epitopes were determined. The analysis revealed eight epitopes that showed high similarity (70-100%) between falciparum and non-falciparum species. These epitopes were highly conserved when assessed across local isolates and those from the MalariaGEN database and showed desirable physiochemical properties. Our results show the presence of conserved cross-species B-cell linear epitopes that could aid in targeting multiple Plasmodium species. Nevertheless, validating their efficacy in-vitro and in-vivo experimentally is essential.

Prodomain-driven enzyme dimerization: a pH-dependent autoinhibition mechanism that controls Plasmodium Sub1 activity before merozoite egress.

Malaria symptoms are associated with the asexual multiplication of Plasmodium falciparum within human red blood cells (RBCs) and fever peaks coincide with the egress of daughter merozoites following the rupture of the parasitophorous vacuole (PV) and the RBC membranes. Over the last two decades, it has emerged that the release of competent merozoites is tightly regulated by a complex cascade of events, including the unusual multi-step activation mechanism of the pivotal subtilisin-like protease 1 (Sub1) that takes place in three different cellular compartments and remains poorly understood. Following an initial auto-maturation in the endoplasmic reticulum (ER) between its pro- and catalytic domains, the Sub1 prodomain (PD) undergoes further cleavages by the parasite aspartic protease plasmepsin X (PmX) within acidic secretory organelles that ultimately lead to full Sub1 activation upon discharge into the PV. Here, we report the crystal structure of full-length P. falciparum Sub1 (PfS1FL) and demonstrate, through structural, biochemical, and biophysical studies, that the atypical Plasmodium-specific Sub1 PD directly promotes the assembly of inactive enzyme homodimers at acidic pH, whereas Sub1 is primarily monomeric at neutral pH. Our results shed new light into the finely tuned Sub1 spatiotemporal activation during secretion, explaining how PmX processing and full activation of Sub1 can occur in different cellular compartments, and uncover a robust mechanism of pH-dependent subtilisin autoinhibition that plays a key role in P. falciparum merozoites egress from infected host cells.IMPORTANCEMalaria fever spikes are due to the rupture of infected erythrocytes, allowing the egress of Plasmodium sp. merozoites and further parasite propagation. This fleeting tightly regulated event involves a cascade of enzymes, culminating with the complex activation of the subtilisin-like protease 1, Sub1. Differently than other subtilisins, Sub1 activation strictly depends upon the processing by a parasite aspartic protease within acidic merozoite secretory organelles. However, Sub1 biological activity is required in the pH neutral parasitophorous vacuole, to prime effectors involved in the rupture of the vacuole and erythrocytic membranes. Here, we show that the unusual, parasite-specific Sub1 prodomain is directly responsible for its acidic-dependent dimerization and autoinhibition, required for protein secretion, before its full activation at neutral pH in a monomeric form. pH-dependent Sub1 dimerization defines a novel, essential regulatory element involved in the finely tuned spatiotemporal activation of the egress of competent Plasmodium merozoites.

In vitro infection of bovine erythrocytes with Theileria annulata merozoites as a key step in completing the T. annulata life cycle in vitro.

Theileria annulata is a protozoan parasite with a complex life cycle involving a bovine host and a tick vector. It is transmitted by Hyalomma ticks and is the causative agent of tropical theileriosis, a debilitating and often fatal disease in southern Europe, northern Africa and large parts of Asia. Understanding the biology of different life cycle stages is critical for the control of tropical theileriosis and requires the use of experimental animals which poses an ethical concern. We present for the first time the in vitro infection of red blood cells (RBCs) with T. annulata differentiated schizonts. The Ankara cell line of T. annulata was cultured at 41 °C for nine days to induce merogony and subsequently incubated with purified RBCs for one to three days. Percentage of parasitized erythrocyte (PPE) over the short culture period was estimated by Giemsa staining (0.007-0.01%), Flow cytometry activated sorting (FACS) (0.02-1.1%) and observation of FACS sorted cells by confocal microscopy (0.05-0.4%). There was a significant difference in the PPE between FACS and the two other techniques (one-way ANOVA followed by Tukey test, P = 0.004) but no significant difference was observed between the confocal imaging and Giemsa staining methods (ANOVA one-way followed by Tukey test, P = 0.06). Importantly, all three complementary methods confirmed the invasion of RBCs by T. annulata merozoites in vitro. Although the experimental conditions will require further optimization to increase the PPE, the in vitro infection of RBCs by T. annulata merozoites is pivotal in paving the way for the eventual completion of the T. annulata life cycle in vitro when combined with artificial tick feeding.

The transcription factor AP2XI-2 is a key negative regulator of Toxoplasma gondii merogony.

Sexual development in Toxoplasma gondii is a multistep process that culminates in the production of oocysts, constituting approximately 50% of human infections. However, the molecular mechanisms governing sexual commitment in this parasite remain poorly understood. Here, we demonstrate that the transcription factors AP2XI-2 and AP2XII-1 act as negative regulators, suppressing merozoite-primed pre-sexual commitment during asexual development. Depletion of AP2XI-2 in type II Pru strain induces merogony and production of mature merozoites in an alkaline medium but not in a neutral medium. In contrast, AP2XII-1-depleted Pru strain undergoes several rounds of merogony and produces merozoites in a neutral medium, with more pronounced effects observed under alkaline conditions. Additionally, we identified two additional AP2XI-2-interacting proteins involved in repressing merozoite programming. These findings underscore the intricate regulation of pre-sexual commitment by a network of factors and suggest that AP2XI-2 or AP2XII-1-depleted Pru parasites can serve as a model for studying merogony in vitro.

Ponazuril: Clinical efficacy, ultrastructure, and histopathology studies of in vivo anticoccidial action against Eimeria tenella.

Ponazuril, a novel antiprotozoal drug in the class of triazine, has shown a promising application on apicomplexan infections in poultry and livestock. However, the effect and mechanism of action of ponazuril against Eimeria tenella (E. tenella) are unclear. The efficacy against E. tenella was initially studied by administering different doses of ponazuril in drinking water. The treated stage and site of ponazuril on E. tenella were observed through ultrastructural and histopathological analyses. Chicks were orally treated with a dose of 15 mg/kg body weight of ponazuril at different endogenous stages of E. tenella post-infection. According to the clinical study, the values of anticoccidial indices (ACI) were 157.0, 162.3, 196.9, 194.5, and 190.9, respectively, when the ponazuril was administered in drinking water at doses of 5, 10, 20, 40, and 50 mg/L for two consecutive days after infection. Among them, the 20 mg/L ponazuril group showed the best anticoccidial effect, which was superior to that of the toltrazuril treatment group, with an ACI value of 191.7. Histological analysis indicated that ponazuril effectively relieved cecal lesions, and decreased the number of merozoites. Transmission electron micrographs (TEM) observed that merozoites became irregular in shape, and some apparent protrusions of the outer membrane were presented especially the second-generation merozoites. Additionally, abnormalities in the development of WFBI and WFBII in the macrogametocyte were observed, which may affect the formation of the ovule wall. Moreover, merozoites in the treated group showed uneven and marginalized chromatin and mitochondrial swelling. These results suggested ponazuril is a potential anticoccidial drug, providing information on the mechanism of anticoccidial effects.

Research Note: Preliminary functional analysis of EGF-like domains of Eimeria tenella microneme protein 7 identified in sporozoites and merozoites.

Eimeria tenella, an obligate intracellular apicomplexan parasite, is the major causative agent of chicken coccidiosis. Some epidermal growth factor (EGF)-like domain-containing proteins of other members of apicomplexan parasites have been reported to contribute to parasite survival. To date, however, EGF-like domain-containing proteins of E. tenella are not well studied. In this study, a gene fragment that encodes 4 EGF-like domains of E. tenella microneme protein 7 (EGF-EtMIC7) was amplified and expressed using an Escherichia coli expression system. Following generation of polyclonal antibodies that recognize recombinant EGF-EtMIC7 (rEGF-EtMIC7), the expression of EtMIC7 in sporozoites and merozoites was examined. Moreover, its roles in cellular regulation were investigated. The native EtMIC7 in E. tenella sporozoites and merozoites was detected by using Western blot and indirect immunofluorescence assays. rEGF-EtMIC7 could activate Akt, whereas blockade of EGF receptor (EGFR) failed to induce Akt phosphorylation. Compared with the control group, LMH cells treated with rEGF-EtMIC7 showed increased cell proliferation and expressed higher levels of B cell leukemia/lymphoma 2 (BCL-2). These findings contribute to the better understanding of parasite-host interactions at the molecular level during E. tenella infection.

Proteomics analysis of Toxoplasma gondii merozoites reveals regulatory proteins involved in sexual reproduction.

Sexual reproduction plays a crucial role in the transmission and life cycle of toxoplasmosis. The merozoites are the only developmental stage capable of differentiation into male and female gametes, thereby initiating sexual reproduction to form oocysts that are excreted into the environment. Hence, our study aimed to perform proteomic analyses of T. gondii Pru strain merozoites, a pre-sexual developmental stage in cat IECs, and tachyzoites, an asexual developmental stage, using the tandem mass tag (TMT) method in order to identify the differentially expressed proteins (DEPs) of merozoites. Proteins functions were subjected to cluster analysis, and DEPs were validated through the qPCR method. The results showed that a total of 106 proteins were identified, out of which 85 proteins had quantitative data. Among these, 15 proteins were differentially expressed within merozoites, with four exhibiting up-regulation and being closely associated with the material and energy metabolism as well as the cell division of T. gondii. Two novel DEPs, namely S8GHL5 and A0A125YP41, were identified, and their homologous family members have been demonstrated to play regulatory roles in oocyte maturation and spermatogenesis in other species. Therefore, they may potentially exhibit regulatory functions during the differentiation of micro- and macro-gametophytes at the initiation stage of sexual reproduction in T. gondii. In conclusion, our results showed that the metabolic and divisional activities in the merozoites surpass those in the tachyzoites, thereby providing structural, material, and energetic support for gametophytes development. The discovery of two novel DEPs associated with sexual reproduction represents a significant advancement in understanding Toxoplasma sexual reproduction initiation and oocyst formation.

In vitro production of cat-restricted Toxoplasma pre-sexual stages.

Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.

The PfRCR complex bridges malaria parasite and erythrocyte during invasion.

The symptoms of malaria occur during the blood stage of infection, when parasites invade and replicate within human erythrocytes. The PfPCRCR complex1, containing PfRH5 (refs. 2,3), PfCyRPA, PfRIPR, PfCSS and PfPTRAMP, is essential for erythrocyte invasion by the deadliest human malaria parasite, Plasmodium falciparum. Invasion can be prevented by antibodies3-6 or nanobodies1 against each of these conserved proteins, making them the leading blood-stage malaria vaccine candidates. However, little is known about how PfPCRCR functions during invasion. Here we present the structure of the PfRCR complex7,8, containing PfRH5, PfCyRPA and PfRIPR, determined by cryogenic-electron microscopy. We test the hypothesis that PfRH5 opens to insert into the membrane9, instead showing that a rigid, disulfide-locked PfRH5 can mediate efficient erythrocyte invasion. We show, through modelling and an erythrocyte-binding assay, that PfCyRPA-binding antibodies5 neutralize invasion through a steric mechanism. We determine the structure of PfRIPR, showing that it consists of an ordered, multidomain core flexibly linked to an elongated tail. We also show that the elongated tail of PfRIPR, which is the target of growth-neutralizing antibodies6, binds to the PfCSS-PfPTRAMP complex on the parasite membrane. A modular PfRIPR is therefore linked to the merozoite membrane through an elongated tail, and its structured core presents PfCyRPA and PfRH5 to interact with erythrocyte receptors. This provides fresh insight into the molecular mechanism of erythrocyte invasion and opens the way to new approaches in rational vaccine design.

Progeny counter mechanism in malaria parasites is linked to extracellular resources.

Malaria is caused by the rapid proliferation of Plasmodium parasites in patients and disease severity correlates with the number of infected red blood cells in circulation. Parasite multiplication within red blood cells is called schizogony and occurs through an atypical multinucleated cell division mode. The mechanisms regulating the number of daughter cells produced by a single progenitor are poorly understood. We investigated underlying regulatory principles by quantifying nuclear multiplication dynamics in Plasmodium falciparum and knowlesi using super-resolution time-lapse microscopy. This confirmed that the number of daughter cells was consistent with a model in which a counter mechanism regulates multiplication yet incompatible with a timer mechanism. P. falciparum cell volume at the start of nuclear division correlated with the final number of daughter cells. As schizogony progressed, the nucleocytoplasmic volume ratio, which has been found to be constant in all eukaryotes characterized so far, increased significantly, possibly to accommodate the exponentially multiplying nuclei. Depleting nutrients by dilution of culture medium caused parasites to produce fewer merozoites and reduced proliferation but did not affect cell volume or total nuclear volume at the end of schizogony. Our findings suggest that the counter mechanism implicated in malaria parasite proliferation integrates extracellular resource status to modify progeny number during blood stage infection.

Acquisition of complement fixing antibodies targeting Plasmodium falciparum merozoites in infants and their mothers in Uganda.

Background: Antibody-mediated complement fixation has previously been associated with protection against malaria in naturally acquired immunity. However, the process of early-life development of complement-fixing antibodies in infants, both in comparison to their respective mothers and to other immune parameters, remains less clear. Results: We measured complement-fixing antibodies in newborns and their mothers in a malaria endemic area over 5 years follow-up and found that infants' complement-fixing antibody levels were highest at birth, decreased until six months, then increased progressively until they were similar to birth at five years. Infants with high levels at birth experienced a faster decay of complement-fixing antibodies but showed similar levels to the low response group of newborns thereafter. No difference was observed in antibody levels between infant cord blood and mothers at delivery. The same result was found when categorized into high and low response groups, indicating placental transfer of antibodies. Complement-fixing antibodies were positively correlated with total schizont-specific IgG and IgM levels in mothers and infants at several time points. At nine months, complement-fixing antibodies were negatively correlated with total B cell frequency and osteopontin concentrations in the infants, while positively correlated with atypical memory B cells and P. falciparum-positive atypical memory B cells. Conclusion: This study indicates that complement-fixing antibodies against P. falciparum merozoites are produced in the mothers and placentally-transferred, and they are acquired in infants over time during the first years of life. Understanding early life immune responses is crucial for developing a functional, long lasting malaria vaccine.

Plasmodium vivax merozoite-specific thrombospondin-related anonymous protein (PvMTRAP) interacts with human CD36, suggesting a novel ligand-receptor interaction for reticulocyte invasion.

BACKGROUND: The Plasmodium vivax merozoite restrictively invades immature erythrocytes, suggesting that its ligand(s) might interact with corresponding receptor(s) that are selectively abundant on reticulocytes to complete the invasion. Finding the ligand‒receptor interaction involved in P. vivax invasion is critical to vivax malaria management; nevertheless, it remains to be unraveled. METHODS: A library of reticulocyte receptors and P. vivax ligands were expressed by a HEK293E mammalian cell expression system and were then used to screen the interaction using enzyme-linked immunosorbent assay (ELISA). A flow cytometry-based erythrocyte binding assay and bio-layer interferometry experiment were further utilized to cellularly and quantitatively identify the ligand‒receptor interaction, respectively. RESULTS: Plasmodium vivax merozoite-specific thrombospondin-related anonymous protein (PvMTRAP) was found to interact with human CD36 using systematic screening. This interaction was specific at a molecular level from in vitro analysis and comparable to that of P. vivax Duffy binding protein (PvDBP) and Duffy antigen receptor for chemokines (DARC) (KD: 37.0 ± 1.4 nM and 7.7 ± 0.5 nM, respectively). Flow cytometry indicated that PvMTRAP preferentially binds to reticulocytes, on which CD36 is selectively present. CONCLUSIONS: Human CD36 is selectively abundant on reticulocytes and is able to interact specifically with PvMTRAP, suggesting that it may function as a ligand and receptor during the invasion of reticulocytes by P. vivax.

Genetic diversity of merozoite surface protein-5 (MSP-5) of Plasmodium vivax isolates from Malaria patients in Iran.

Malaria has not yet been eradicated in Iran, and Plasmodium vivax (P. vivax) is the main cause of malaria in the country. This study aimed to investigate and analyze the amount of genetic diversity of Plasmodium vivax merozoite surface protein-5 (PvMSP-5) exon 1 gene in the southeast of Iran.Thirty-five patients with clinical symptoms of P. vivax malaria participated. The exon 1 of PvMSP-5 was amplified by PCR, and the PCR product of all isolates was sequenced, and genetic polymorphisms were determined using various genetic software.The analysis showed that studied isolates are different from one another in the DnaSP software version. Out of the 612 sites, 477 were monomorphic and 135 were segregated. The total number of mutations was 143. The singleton variable and the parsimony informative sites were 23 and 112, respectively. There were 17 specific haplotypes with haplotype diversity equal to 0.943. Nucleotide diversity was equal to 0.06766 in the isolates. The ratio of nonsynonymous (0.06446) to synonymous (0.07909) mutations was 0.815020. Tajima's D, which expressed coding, and non-coding regions, was 0.72403, which was not deemed significant (P > 0.10).The analysis of intrapopulation diversity revealed nucleotide and haplotype diversity in the msp-5 gene of Iranian P. vivax isolates. In addition to balancing or purifying selection, intragenic recombination also contributed to the variation observed in exon 1 of PvMSP-5, according to the findings.

Genetic Diversity of Merozoite Surface Antigens in Global Babesia bovis Populations.

Cattle can be severely infected with the tick-borne protozoa Babesia bovis, giving rise to serious economic losses. Invasion of the host's RBCs by the parasite merozoite/sporozoites depends largely on the MSA (merozoite surface antigens) gene family, which comprises various fragments, e.g., MSA-1, MSA-2a1, MSA-2a2, MSA-2b and MSA-2c, highlighting the importance of these antigens as vaccine candidates. However, experimental trials documented the failure of some developed MSA-based vaccines to fully protect animals from B. bovis infection. One reason for this failure may be related to the genetic structure of the parasite. In the present study, all MSA-sequenced B. bovis isolates on the GenBank were collected and subjected to various analyses to evaluate their genetic diversity and population structure. The analyses were conducted on 199 MSA-1, 24 MSA-2a1, 193 MSA-2b and 148 MSA-2c isolates from geographically diverse regions. All these fragments displayed high nucleotide and haplotype diversities, but the MSA-1 was the most hypervariable and had the lowest inter- and intra-population gene flow values. This fragment also displayed a strong positive selection when testing its isolates for the natural selection, which suggests the potential occurrence of more genetic variations. On the contrary, the MSA-2c was the most conserved in comparison to the other fragments, and displayed the highest inter- and intra-population gene flow values, which was evidenced by a significantly negative selection and negative neutrality indices (Fu's Fs and Tajima's D). The majority of the MSA-2c tested isolates had two conserved amino acid repeats, and earlier reports have found these repeats to be highly immunogenic, which underlines the importance of this fragment in developing vaccines against B. bovis. Results of the MSA-2a1 analyses were also promising, but many more MSA-2a1 sequenced isolates are required to validating this assumption. The genetic analyses conducted for the MSA-2b fragment displayed borderline values when compared to the other fragments.

Bottoms up! Malaria parasite invasion the right way around.

A critical part of the malaria parasite's life cycle is invasion of red blood cells (RBCs) by merozoites. Inside RBCs, the parasite forms a schizont, which undergoes segmentation to produce daughter merozoites. These cells are released, establishing cycles of invasion. Traditionally, merozoites are represented as nonmotile, egg-shaped cells that invade RBCs 'narrower end' first and pack within schizonts with this narrower end facing outwards. Here, we discuss recent evidence and re-evaluate previous data which suggest that merozoites are capable of motility and have spherical or elongated-teardrop shapes. Furthermore, merozoites invade RBCs 'wider end' first and pack within schizonts with this wider end facing outwards. We encourage the field to review this revised model and consider its implications for future studies.

Immuno-bioinformatic approach for designing of multi-epitope merozoite surface antigen of Babesia bigemina and evaluation of its immunogenicity in inoculated calves.

Babesiosis is a parasitic disease caused by intraerythrocytic parasites of the genus Babesia, which infect both wild and domestic animals. Merozoite surface antigens (MSAs) have been identified as efficient immunogens in Babesia-infected animals. MSAs play a key role in the invasion process and have been proposed as potential targets for vaccine development. Epitope-based vaccines offer several advantages over whole protein vaccines as the immunogenic proteins are small and can induce both Th1 and Th2 immune responses, which are desirable for protection. However, the MSA, particularly gp45, is polymorphic in Babesia bigemina, posing a challenge to vaccine development. The purpose of this study was to develop a recombinant gpME (gp45-multi-epitope) for a vaccine against Babesia bigemina. B-cell, T-cell, and HLA epitope predictions were used to synthesize the gpME sequence from the consensus sequence of gp45. The gpME sequence was synthesized and cloned in the pET28α vector through the commercial biotechnology company to get pET28-gpME. The plasmid cloned with the gpME sequence comprising 1068 bp was expressed in a bacterial expression system. A band of 39 kDa of rec-gpME was obtained via SDS-PAGE and Western blotting. Rec-gpME @200ng was injected in calves 3 times at 2 weeks interval. The humoral response was evaluated through the indirect ELISA method. The ELISA with rec-gp45 protein showed a significant value of optical density. The recombinant protein containing multiple epitopes from the MSA gp45 may represent a promising candidate for a vaccine against Babesia bigemina.