In Leishmania major, the Homolog of the Oncogene PES1 May Play a Critical Role in Parasite Infectivity

Leishmaniasis is a neglected tropical disease caused by Leishmania spp. The improvement of existing treatments and the discovery of new drugs remain ones of the major goals in control and eradication of this disease. From the parasite genome, we have identified the homologue of the human oncogene PES1 in Leishmania major (LmjPES). It has been demonstrated that PES1 is involved in several processes such as ribosome biogenesis, cell proliferation and genetic transcription. Our phylogenetic studies showed that LmjPES encodes a highly conserved protein containing three main domains: PES N-terminus (shared with proteins involved in ribosomal biogenesis), BRCT (found in proteins related to DNA repair processes) and MAEBL-type domain (C-terminus, related to erythrocyte invasion in apicomplexan). This gene showed its highest expression level in metacyclic promastigotes, the infective forms; by fluorescence microscopy assay, we demonstrated the nuclear localization of LmjPES protein. After generating mutant parasites overexpressing LmjPES, we observed that these clones displayed a dramatic increase in the ratio of cell infection within macrophages. Furthermore, BALB/c mice infected with these transgenic parasites exhibited higher footpad inflammation compared to those inoculated with non-overexpressing parasites.

Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of “pseudoschizonts,” which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

Plasmodium falciparum Cysteine-Rich Protective Antigen (CyRPA) Elicits Detectable Levels of Invasion-Inhibitory Antibodies during Natural Infection in Humans

Plasmodium falciparum Cysteine-Rich Protective Antigen (CyRPA) is a conserved component of an essential erythrocyte invasion complex (RH5/Ripr/CyRPA) and a target of potent cross-strain parasite-neutralizing antibodies. While, naturally acquired human RH5 antibodies have been functionally characterized, there are no similar reports on CyRPA. Thus, we analyzed the parasite neutralizing activity of naturally acquired human CyRPA antibodies. In this regard, CyRPA human antibodies were measured and purified from malaria infected sera obtained from central India and analyzed for their parasite neutralizing activity in in vitro growth inhibition assays (GIA). We report that despite being susceptible to antibody, CyRPA being a highly conserved antigen does not appear to be under substantial immune selection pressure as a very low acquisition of anti-CyRPA antibodies was reported in malaria-exposed Indians. We demonstrate for the first time that the low amounts of natural CyRPA antibodies exhibited functional parasite-neutralizing activity and that a CyRPA based vaccine formulation induces highly potent antibodies in rabbits. Importantly, the vaccine induced CyRPA antibodies exhibited a robust IC 50 of 21.96 μg/ml that is comparable to IC 50 of antibodies against the leading blood stage vaccine candidate, RH5. Our data support CyRPA as a unique vaccine target that is highly susceptible to immune attack but highly conserved compared to other leading candidates such as MSP-1, AMA-1, further substantiating its promise as a leading blood-stage vaccine candidate.

The genome of the zoonotic malaria parasite Plasmodium simium reveals adaptations to host switching

Background Plasmodium simium, a malaria parasite of non-human primates (NHP), was recently shown to cause zoonotic infections in humans in Brazil. We sequenced the P. simium genome to investigate its evolutionary history and to identify any genetic adaptions that may underlie the ability of this parasite to switch between host species. Results Phylogenetic analyses based on whole genome sequences of P. simium from humans and NHPs reveals that P. simium is monophyletic within the broader diversity of South American Plasmodium vivax, suggesting P. simium first infected NHPs as a result of a host switch of P. vivax from humans. The P. simium isolates show the closest relationship to Mexican P. vivax isolates. Analysis of erythrocyte invasion genes reveals differences between P. vivax and P. simium, including large deletions in the Duffy-binding protein 1 (DBP1) and reticulocyte-binding protein 2a genes of P. simium. Analysis of P. simium isolated from NHPs and humans revealed a deletion of 38 amino acids in DBP1 present in all human-derived isolates, whereas NHP isolates were multi-allelic. Conclusions Analysis of the P. simium genome confirmed a close phylogenetic relationship between P. simium and P. vivax, and suggests a very recent American origin for P. simium. The presence of the DBP1 deletion in all human-derived isolates tested suggests that this deletion, in combination with other genetic changes in P. simium, may facilitate the invasion of human red blood cells and may explain, at least in part, the basis of the recent zoonotic infections.

Short-Term Cryopreservation and Thawing have Minimal Effects on Plasmodium Falciparum Ex Vivo Invasion Profile

Ex vivo phenotyping of P. falciparum erythrocyte invasion diversity is important in the identification and down selection of potential malaria vaccine targets. However, due to the lack of appropriate laboratory facilities in remote areas of endemic countries, direct processing of P. falciparum clinical isolates is usually not feasible. Here, we investigated the combined effect of short-term cryopreservation and thawing processes on the ex vivo invasion phenotypes of P. falciparum isolates. Ex-vivo or in vitro invasion phenotyping assays were performed with P. falciparum clinical isolates prior to or following culture adaptation, respectively. All isolates were genotyped at Day 0 for parasite clonality. Subsequently, isolates that were successfully culture-adapted were genotyped again at Days 7, 15, 21, and 28-post adaptation. Invasion phenotyping assays were performed in isogenic isolates revived at different time points (3, 6, and 12 months) post-cryopreservation and the resulting data were compared to that from ex-vivo invasion data of matched isogenic parental isolates. Our findings indicate that natural P. falciparum infections mostly occur as polyclonal infections. We also show that short-term culture adaptation selects for parasite clonality and could be a driving force for variation in invasion phenotypes as compared to ex vivo data where almost all parasite clones of a given isolate are present. Interestingly, our data show little variation in the parasites’ invasion phenotype following short-term cryopreservation. Altogether, our data suggest that short-term cryopreservation of uncultured P. falciparum clinical isolates is a reliable mechanism for storing parasites for future use.

A unique GCN5 histone acetyltransferase complex controls erythrocyte invasion and virulence in the malaria parasite Plasmodium falciparum

The histone acetyltransferase GCN5-associated SAGA complex is evolutionarily conserved from yeast to human and functions as a general transcription co-activator in global gene regulation. In this study, we identified a divergent GCN5 complex in Plasmodium falciparum, which contains two plant homeodomain (PHD) proteins (PfPHD1 and PfPHD2) and a plant apetela2 (AP2)-domain transcription factor (PfAP2-LT). To dissect the functions of the PfGCN5 complex, we generated parasite lines with either the bromodomain in PfGCN5 or the PHD domain in PfPHD1 deleted. The two deletion mutants closely phenocopied each other, exhibiting significantly reduced merozoite invasion of erythrocytes and elevated sexual conversion. These domain deletions caused dramatic decreases not only in histone H3K9 acetylation but also in H3K4 trimethylation, indicating synergistic crosstalk between the two euchromatin marks. Domain deletion in either PfGCN5 or PfPHD1 profoundly disturbed the global transcription pattern, causing altered expression of more than 60% of the genes. At the schizont stage, these domain deletions were linked to specific down-regulation of merozoite genes involved in erythrocyte invasion, many of which contain the AP2-LT binding motif and are also regulated by AP2-I and BDP1, suggesting targeted recruitment of the PfGCN5 complex to the invasion genes by these specific factors. Conversely, at the ring stage, PfGCN5 or PfPHD1 domain deletions disrupted the mutually exclusive expression pattern of the entire var gene family, which encodes the virulent factor PfEMP1. Correlation analysis between the chromatin state and alteration of gene expression demonstrated that up- and down-regulated genes in these mutants are highly correlated with the silent and active chromatin states in the wild-type parasite, respectively. Collectively, the PfGCN5 complex represents a novel HAT complex with a unique subunit composition including an AP2 transcription factor, which signifies a new paradigm for targeting the co-activator complex to regulate general and parasite-specific cellular processes in this low-branching parasitic protist.

Strain-Dependent Inhibition of Erythrocyte Invasion by Monoclonal Antibodies Against Plasmodium falciparum CyRPA

The highly conserved Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is a key target for next-generation vaccines against blood-stage malaria. PfCyRPA constitute the core of a ternary complex, including the reticulocyte binding-like homologous protein 5 (PfRh5) and the Rh5-interacting protein (PfRipr), and is fundamental for merozoite invasion of erythrocytes. In this study, we show that monoclonal antibodies (mAbs) specific to PfCyRPA neutralize the in vitro growth of Ghanaian field isolates as well as numerous laboratory-adapted parasite lines. We identified subsets of mAbs with neutralizing activity that bind to distinct sites on PfCyRPA and that in combination potentiate the neutralizing effect. As antibody responses against multiple merozoite invasion proteins are thought to improve the efficacy of blood-stage vaccines, we also demonstrated that combinations of PfCyRPA- and PfRh5 specific mAbs act synergistically to neutralize parasite growth. Yet, we identified prominent strain-dependent neutralization potencies, which our results suggest is independent of PfCyRPA expression level and polymorphism, demonstrating the importance of addressing functional converseness when evaluating blood-stage vaccine candidates. Finally, our results suggest that blood-stage vaccine efficacy can be improved by directing the antibody response towards defined protective epitopes on multiple parasite antigens.

Aqueous extract of Phyllanthus niruri protects against severe malaria by blocking erythrocyte invasion and modulating the host immune response

Plasmodium falciparum parasites are the major cause of malaria across Africa. Due to the appearance of multi-drug resistant parasites, new antimalarial drugs are needed. The medicinal plant Phyllanthus niruri is being used to treat fever and other symptoms of malaria in Nigeria; however, little is known about its antimalarial mechanisms. Here, we show that aqueous extract of P. niruri (PE) has multiple antimalarial effects, including anti-parasitic and host immunomodulatory activity. We found that co-culture of P. falciparum with PE drastically reduced parasite number, but PE did not inhibit parasite development or rupture; rather, it blocked erythrocytes invasion. In addition, we identified Astragalin as one of the antimalarial compounds which are contained in PE. Moreover, we found that PE suppresses the inflammatory activity and apoptosis of immune cells (T cells) and astrocytes and neurons in the central nervous system (CNS). Furthermore, we confirmed that oral administration of PE to mice suppressed parasite growth, excessive inflammation, CNS dysfunction, and the development of experimental cerebral malaria in an in vivo murine malaria model. Our findings demonstrate that PE has multiple effects on malaria progression, targeting both parasite and host cells.

The Plasmodium falciparum Rh5 invasion protein complex reveals an excess of rare variant mutations

Background The invasion of the red blood cells by Plasmodium falciparum merozoites involves the interplay of several proteins that are also targets for vaccine development. The proteins PfRh5-PfRipr-PfCyRPA-Pfp113 assemble into a complex at the apical end of the merozoite and are together essential for erythrocyte invasion. They have also been shown to induce neutralizing antibodies and appear to be less polymorphic than other invasion-associated proteins, making them high priority blood-stage vaccine candidates. Using available whole genome sequencing data (WGS) and new capillary sequencing data (CS), this study describes the genetic polymorphism in the Rh5 complex in P. falciparum isolates obtained from Kilifi, Kenya. Methods 162 samples collected in 2013 and 2014 were genotyped by capillary sequencing (CS) and re-analysed WGS from 68 culture-adapted P. falciparum samples obtained from a drug trial conducted from 2005 to 2007. The frequency of polymorphisms in the merozoite invasion proteins, PfRh5, PfRipr, PfCyRPA and PfP113 were examined and where possible polymorphisms co-occurring in the same isolates. Results From a total 70 variants, including 2 indels, 19 SNPs [27.1%] were identified by both CS and WGS, while an additional 15 [21.4%] and 36 [51.4%] SNPs were identified only by either CS or WGS, respectively. All the SNPs identified by CS were non-synonymous, whereas WGS identified 8 synonymous and 47 non-synonymous SNPs. CS identified indels in repeat regions in the p113 gene in codons 275 and 859 that were not identified in the WGS data. The minor allele frequencies of the SNPs ranged between 0.7 and 34.9% for WGS and 1.1–29.6% for CS. Collectively, 12 high frequency SNPs (> 5%) were identified: four in Rh5 codon 147, 148, 203 and 429, two in p113 at codons 7 and 267 and six in Ripr codons 190, 259, 524, 985, 1003 and 1039. Conclusion This study reveals that the majority of the polymorphisms are rare variants and confirms a low level of genetic polymorphisms in all proteins within the Rh5 complex.

Naturally induced humoral response against Plasmodium vivax reticulocyte binding protein 2P1

Background Plasmodium vivax is the most prevalent malaria parasite in many countries. A better understanding of human immunity to this parasite can provide new insights for vaccine development. Plasmodium vivax Reticulocyte Binding Proteins (RBPs) are key parasite proteins that interact with human proteins during erythrocyte invasion and are targets of the human immune response. The aim of this study is to characterize the human antibody response to RBP2P1, the most recently described member of the RBP family. Methods The levels of total IgG and IgM against RBP2P1 were measured using plasmas from 68 P. vivax malaria patients and 525 villagers in a malarious village of western Thailand. The latter group comprises asymptomatic carriers and healthy uninfected individuals. Subsets of plasma samples were evaluated for anti-RBP2P1 IgG subtypes and complement-fixing activity. Results As age increased, it was found that the level of anti-RBP2P1 IgG increased while the level of IgM decreased. The main anti-RBP2P1 IgG subtypes were IgG1 and IgG3. The IgG3-seropositive rate was higher in asymptomatic carriers than in patients. The higher level of IgG3 was correlated with higher in vitro RBP2P1-mediated complement fixing activity. Conclusions In natural infection, the primary IgG response to RBP2P1 was IgG1 and IgG3. The predominance of these cytophilic subtypes and the elevated level of IgG3 correlating with complement fixing activity, suggest a possible role of anti-RBP2P1 antibodies in immunity against P. vivax.