A Network Approach to Analyzing Highly Recombinant Malaria Parasite Genes

The var genes of the human malaria parasite Plasmodium falciparum present a challenge to population geneticists due to their extreme diversity, which is generated by high rates of recombination. These genes encode a primary antigen protein called PfEMP1, which is expressed on the surface of infected red blood cells and elicits protective immune responses. Var gene sequences are characterized by pronounced mosaicism, precluding the use of traditional phylogenetic tools that require bifurcating tree-like evolutionary relationships. We present a new method that identifies highly variable regions (HVRs), and then maps each HVR to a complex network in which each sequence is a node and two nodes are linked if they share an exact match of significant length. Here, networks of var genes that recombine freely are expected to have a uniformly random structure, but constraints on recombination will produce network communities that we identify using a stochastic block model. We validate this method on synthetic data, showing that it correctly recovers populations of constrained recombination, before applying it to the Duffy Binding Like-α (DBLα) domain of var genes. We find nine HVRs whose network communities map in distinctive ways to known DBLα classifications and clinical phenotypes. We show that the recombinational constraints of some HVRs are correlated, while others are independent. These findings suggest that this micromodular structuring facilitates independent evolutionary trajectories of neighboring mosaic regions, allowing the parasite to retain protein function while generating enormous sequence diversity. Our approach therefore offers a rigorous method for analyzing evolutionary constraints in var genes, and is also flexible enough to be easily applied more generally to any highly recombinant sequences

Characterization and gene expression analysis of the cir multi-gene family of plasmodium chabaudi chabaudi (AS)

Background: The pir genes comprise the largest multi-gene family in Plasmodium, with members found in P. vivax, P. knowlesi and the rodent malaria species. Despite comprising up to 5% of the genome, little is known about the functions of the proteins encoded by pir genes. P. chabaudi causes chronic infection in mice, which may be due to antigenic variation. In this model, pir genes are called cir s and may be involved in this mechanism, allowing evasion of host immune responses. In order to fully understand the role(s) of CIR proteins during P. chabaudi infection, a detailed characterization of the cir gene family was required. Results: The cir repertoire was annotated and a detailed bioinformatic characterization of the encoded CIR proteins was performed. Two major sub-families were identified, which have been named A and B. Members of each sub-family displayed different amino acid motifs, and were thus predicted to have undergone functional divergence. In addition, the expression of the entire cir repertoire was analyzed via RNA sequencing and microarray. Up to 40% of the cir gene repertoire was expressed in the parasite population during infection, and dominant cir transcripts could be identified. In addition, some differences were observed in the pattern of expression between the cir subgroups at the peak of P. chabaudi infection. Finally, specific cir genes were expressed at different time points during asexual blood stages. Conclusions: In conclusion, the large number of cir genes and their expression throughout the intraerythrocytic cycle of development indicates that CIR proteins are likely to be important for parasite survival. In particular, the detection of dominant cir transcripts at the peak of P. chabaudi infection supports the idea that CIR proteins are expressed, and could perform important functions in the biology of this parasite. Further application of the methodologies described here may allow the elucidation of CIR sub-family A and B protein functions, including their contribution to antigenic variation and immune evasion

Predicting Functional and Regulatory Divergence of a Drug Resistance Transporter Gene in the Human Malaria Parasite

Background: The paradigm of resistance evolution to chemotherapeutic agents is that a key coding mutation in a specific gene drives resistance to a particular drug. In the case of resistance to the anti-malarial drug chloroquine (CQ), a specific mutation in the transporter pfcrt is associated with resistance. Here, we apply a series of analytical steps to gene expression data from our lab and leverage 3 independent datasets to identify pfcrt-interacting genes. Resulting networks provide insights into pfcrt’s biological functions and regulation, as well as the divergent phenotypic effects of its allelic variants in different genetic backgrounds. Results: To identify pfcrt-interacting genes, we analyze pfcrt co-expression networks in 2 phenotypic states - CQ-resistant (CQR) and CQ-sensitive (CQS) recombinant progeny clones - using a computational approach that prioritizes gene interactions into functional and regulatory relationships. For both phenotypic states, pfcrt co-expressed gene sets are associated with hemoglobin metabolism, consistent with CQ’s expected mode of action. To predict the drivers of co-expression divergence, we integrate topological relationships in the co-expression networks with available high confidence protein-protein interaction data. This analysis identifies 3 transcriptional regulators from the ApiAP2 family and histone acetylation as potential mediators of these divergences. We validate the predicted divergences in DNA mismatch repair and histone acetylation by measuring the effects of small molecule inhibitors in recombinant progeny clones combined with quantitative trait locus (QTL) mapping. Conclusions: This work demonstrates the utility of differential co-expression viewed in a network framework to uncover functional and regulatory divergence in phenotypically distinct parasites. pfcrt-associated co-expression in the CQ resistant progeny highlights CQR-specific gene relationships and possible targeted intervention strategies. The approaches outlined here can be readily generalized to other parasite populations and drug resistances

Recent advances in malaria genomics and epigenomics

Malaria continues to impose a significant disease burden on low- and middle-income countries in the tropics. However, revolutionary progress over the last 3 years in nucleic acid sequencing, reverse genetics, and post-genome analyses has generated step changes in our understanding of malaria parasite (Plasmodium spp.) biology and its interactions with its host and vector. Driven by the availability of vast amounts of genome sequence data from Plasmodium species strains, relevant human populations of different ethnicities, and mosquito vectors, researchers can consider any biological component of the malarial process in isolation or in the interactive setting that is infection. In particular, considerable progress has been made in the area of population genomics, with Plasmodium falciparum serving as a highly relevant model. Such studies have demonstrated that genome evolution under strong selective pressure can be detected. These data, combined with reverse genetics, have enabled the identification of the region of the P. falciparum genome that is under selective pressure and the confirmation of the functionality of the mutations in the kelch13 gene that accompany resistance to the major frontline antimalarial, artemisinin. Furthermore, the central role of epigenetic regulation of gene expression and antigenic variation and developmental fate in P. falciparum is becoming ever clearer. This review summarizes recent exciting discoveries that genome technologies have enabled in malaria research and highlights some of their applications to healthcare. The knowledge gained will help to develop surveillance approaches for the emergence or spread of drug resistance and to identify new targets for the development of antimalarial drugs and perhaps vaccines

Var gene diversity and their serological recognition by naturally exposed individuals

Plasmodium falciparum causes the worst form of human malaria and leads to 1-2 million deaths annually, most of them children below the age of 5 living in subsaharan Africa. Morbidity varies from asymptomatic infections with no symptoms to severe malaria accompanied by organ failure, severe anemia and coma. Most of these clinical presentations are associated with sequestration of infected red blood cells (iRBC) on host endothelium. By attaching the parasitized erythrocyte to host receptors such as CD36, ICAM or CSA the parasite prevents the cell from being cleared by the spleen and therefore prolongs its own survival. A key protein involved in this process is the variant surface antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) which is a parasite derived protein transported to the RBC surface to mediate cytoadherence. With this process exposes the parasite itself to the host immune system leading to the production of specific antibodies. In order to evade this host immune response the parasite undergoes antigenic variation by switching to another member of the same protein family. PfEMP1 is encoded by approximately 60 var genes per haploid genome and is expressed at the surface in a mutually exclusive manner, i.e. only 1 of the 60 proteins is expressed and exposed at any one time whilst the others remain silenced. Protection against severe malaria is thought to be mediated to a large degree by the piecemeal acquisition of anti-PfEMP1 antibodies during early childhood, since adults still get infected but rarely develop severe malaria symptoms. Recent observations suggest that not all PfEMP1 proteins expressed by a parasite are equally virulent, but only a subset of distinct var genes might render a parasite more pathogenic than parasites expressing different var gene variants. To generate potential anti-severe disease interventions members of this particular subset need to be identified. To date, only 6 studies have been published investigating the repertoire of expressed var genes in vivo. We have further used samples collected in Papua New Guinea from a case control study and analyzed var transcripts by RT-PCR followed by cloning and sequencing. We determined the 3 main groups of 5’UTR and analysed the data with respect to the clinical presentation of the children they were collected from. The detected number of different var group B and C transcipts was not significantly different between asymptomatic, mild or severe malaria cases, whereas an increase of group A var genes was observed in symptomatic cases when compared to children without any malaria symptoms. We identified an amino acid substitution mainly occurring in asymptomatic children with high parasitemia that might influence the binding affinity of parasites expressing these variants. However, using phylogenetic analyses we were not able to identify other distinct var genes or subsets associated with severe malaria. Blasting DBL1α domains against the 3D7 genome to obtain information on the upstream region was found to be suitable for group A var genes only, whereas 28% of group B and 62% of group C sequences were assigned to the wrong subgroup using this method. Even though we observed a 7% sequence overlap, bioinformatic analyses estimated the var gene repertoire in this region of PNG to be unlimited. It has previously been shown, that isolates causing severe disease are recognized more frequently than those causing mild malaria. In the second part of this thesis, we wanted to obtain information on the importance of distinct PfEMP1 domains in the recognition by the host immune system. For that purpose, fragments of 2 representative var genes shown to be associated with severe malaria were recombinantly expressed in E.coli and analyzed for their recognition by naturally exposed sera of different origin. Analysis of synthetic peptides using the same sera served to complement the results of ELISAs using recombinant proteins if expression of distinct domains was not possible. ELISA and Western blot analysis determined that 3 recombinant fragments and 2 synthetic peptides harbor epitopes that might play a role in the generation of protective antibodies. However, since sample size was small further investigations are required to confirm these findings. In the third part of this thesis, we tested the usefulness of the GeneMapper® analysis software to genotype var genes. It has been successfully established for genotyping the polymorphic marker gene msp2 and since var genes also show some length polymorphism it was investigated whether this technique could replace tedious cloning and sequencing approaches, used so far to dissect var gene diversity. Therefore, purified PCR products of UTR-DBL domains generated during the sequence analysis were reamplified with fluorescently labeled DBL-specific primers and analyzed by GeneMapper®. The results were then compared to the sequencing data. GeneMapper® sizing was highly accurate with a mean deviation of 1bp and showed a high consistency with sequencing data. Furthermore, GeneMapper® detected 141 sequences which were not identified with the sequencing approach, whereas vice verca, this was only the case for 16 sequences. However, a significant proportion of var genes could not be distinguished because the analyzed DBL domains were identical in size. Despite this shortcoming, we belive that GeneMapper® would greatly facilitate the analysis of expressed var genes and their dynamics

Profiling invasive Plasmodium falciparum merozoites using an integrated omics approach

The symptoms of malaria are brought about by blood-stage parasites, which are established when merozoites invade human erythrocytes. Our understanding of the molecular events that underpin erythrocyte invasion remains hampered by the short-period of time that merozoites are invasive. To address this challenge, a Plasmodium falciparum gamma-irradiated long-lived merozoite (LLM) line was developed and investigated. Purified LLMs invaded erythrocytes by an increase of 10–300 fold compared to wild-type (WT) merozoites. Using an integrated omics approach, we investigated the basis for the phenotypic difference. Only a few single nucleotide polymorphisms within the P. falciparum genome were identified and only marginal differences were observed in the merozoite transcriptomes. By contrast, using label-free quantitative mass-spectrometry, a significant change in protein abundance was noted, of which 200 were proteins of unknown function. We determined the relative molar abundance of over 1100 proteins in LLMs and further characterized the major merozoite surface protein complex. A unique processed MSP1 intermediate was identified in LLM but not observed in WT suggesting that delayed processing may be important for the observed phenotype. This integrated approach has demonstrated the significant role of the merozoite proteome during erythrocyte invasion, while identifying numerous unknown proteins likely to be involved in invasion

B-Cell Epitopes in NTS-DBL1 alpha of PfEMP1 Recognized by Human Antibodies in Rosetting Plasmodium falciparum

Plasmodium falciparum is the most lethal of the human malaria parasites. the virulence is associated with the capacity of the infected red blood cell (iRBC) to sequester inside the deep microvasculature where it may cause obstruction of the blood-flow when binding is excessive. Rosetting, the adherence of the iRBC to uninfected erythrocytes, has been found associated with severe malaria and found to be mediated by the NTS-DBL1 alpha-domain of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1). Here we show that the reactivity of plasma of Cameroonian children with the surface of the FCR3S1.2-iRBC correlated with the capacity to disrupt rosettes and with the antibody reactivity with a recombinant PfEMP1 (NTS-DBL1 alpha of IT4(var60)) expressed by parasite FCR3S1.2. the plasma-reactivity in a microarray, consisting of 96 overlapping 15-mer long peptides covering the NTS-DBL1 alpha domain from IT4var60 sequence, was compared with their capacity to disrupt rosettes and we identified five peptides where the reactivity were correlated. Three of the peptides were localized in subdomain-1 and 2. the other two peptide-sequences were localized in the NTS-domain and in subdomain-3. Further, principal component analysis and orthogonal partial least square analysis generated a model that supported these findings. in conclusion, human antibody reactivity with short linear-peptides of NTS-DBL1 alpha of PfEMP1 suggests subdomains 1 and 2 to hold anti-rosetting epitopes recognized by anti-rosetting antibodies. the data suggest rosetting to be mediated by the variable areas of PfEMP1 but also to involve structurally relatively conserved areas of the molecule that may induce biologically active antibodies.Swedish Research Council (VR)Swedish Academy of Sciences (KVA, Soderberg Foundation)Karolinska Institutet-DPAEU Network of Excellence EviMalarKarolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Stockholm, SwedenKarolinska Inst, Dept Lab Med, Therapeut Immunol TIM, Stockholm, SwedenKarolinska Univ Hosp, CAST, Huddinge, SwedenUniv Estadual Campinas, Dept Biochem, Campinas, SP, BrazilWeb of Scienc