Whole genome sequencing and microsatellite analysis of the Plasmodium falciparum E5 NF54 strain show that the var, rifin and stevor gene families follow Mendelian inheritance

Background: Plasmodium falciparum exhibits a high degree of inter-isolate genetic diversity in its variant surface antigen (VSA) families: P. falciparum erythrocyte membrane protein 1, repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR). The role of recombination for the generation of this diversity is a subject of ongoing research. Here the genome of E5, a sibling of the 3D7 genome strain is presented. Short and long read whole genome sequencing (WGS) techniques (Ilumina, Pacific Bioscience) and a set of 84 microsatellites (MS) were employed to characterize the 3D7 and non-3D7 parts of the E5 genome. This is the first time that VSA genes in sibling parasites were analysed with long read sequencing technology. Results: Of the 5733 E5 genes only 278 genes, mostly var and rifin/stevor genes, had no orthologues in the 3D7 genome. WGS and MS analysis revealed that chromosomal crossovers occurred at a rate of 0–3 per chromosome. var, stevor and rifin genes were inherited within the respective non-3D7 or 3D7 chromosomal context. 54 of the 84 MS PCR fragments correctly identified the respective MS as 3D7- or non-3D7 and this correlated with var and rifin/stevor gene inheritance in the adjacent chromosomal regions. E5 had 61 var and 189 rifin/stevor genes. One large non-chromosomal recombination event resulted in a new var gene on chromosome 14. The remainder of the E5 3D7-type subtelomeric and central regions were identical to 3D7. Conclusions: The data show that the rifin/stevor and var gene families represent the most diverse compartments of the P. falciparum genome but that the majority of var genes are inherited without alterations within their respective parental chromosomal context. Furthermore, MS genotyping with 54 MS can successfully distinguish between two sibling progeny of a natural P. falciparum cross and thus can be used to investigate identity by descent in field isolates

cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites

Characterization of the repertoire diversity of the Plasmodium falciparum stevor multigene family in laboratory and field isolates

BACKGROUND: The evasion of host immune response by the human malaria parasite Plasmodium falciparum has been linked to expression of a range of variable antigens on the infected erythrocyte surface. Several genes are potentially involved in this process with the var, rif and stevor multigene families being the most likely candidates and coding for rapidly evolving proteins. The high sequence diversity of proteins encoded by these gene families may have evolved as an immune evasion strategy that enables the parasite to establish long lasting chronic infections. Previous findings have shown that the hypervariable region (HVR) of STEVOR has significant sequence diversity both within as well as across different P. falciparum lines. However, these studies did not address whether or not there are ancestral stevor that can be found in different parasites. METHODS: DNA and RNA sequences analysis as well as phylogenetic approaches were used to analyse the stevor sequence repertoire and diversity in laboratory lines and Kilifi (Kenya) fresh isolates. RESULTS: Conserved stevor genes were identified in different P. falciparum isolates from different global locations. Consistent with previous studies, the HVR of the stevor gene family was found to be highly divergent both within and between isolates. Importantly phylogenetic analysis shows some clustering of stevor sequences both within a single parasite clone as well as across different parasite isolates. CONCLUSION: This indicates that the ancestral P. falciparum parasite genome already contained multiple stevor genes that have subsequently diversified further within the different P. falciparum populations. It also confirms that STEVOR is under strong selection pressure

The Plasmodium falciparum STEVOR Multigene Family Mediates Antigenic Variation of the Infected Erythrocyte

Modifications of the Plasmodium falciparum–infected red blood cell (iRBC) surface have been linked to parasite-associated pathology. Such modifications enable the parasite to establish long-lasting chronic infection by evading antibody mediate immune recognition and splenic clearance. With the exception of the well-demonstrated roles of var-encoded PfEMP1 in virulence and immune evasion, the biological significance of other variant surface antigens (rif and stevor) is largely unknown. While PfEMP1 and RIFIN have been located on the iRBC surface, recent studies have located STEVOR at the iRBC membrane where it may be exposed on the erythrocyte surface. To investigate the role of STEVOR in more detail, we have developed antibodies against two putative STEVOR proteins and used a combination of indirect immunofluorescence assays (IFA), live IFA, flow cytometry, as well as agglutination assays, which enable us to demonstrate that STEVOR is clonally variant at the surface of schizont stage parasites. Crucially, expression of different STEVOR on the surface of the iRBC changes the antigenic property of the parasite. Taken together, our data for the first time demonstrate that STEVOR plays a role in creating antigenic diversity of schizont stage parasites, thereby adding additional complexity to the immunogenic properties of the iRBC. Furthermore, it clearly demonstrates that to obtain a complete understanding of how parasite-induced pathology is linked to variation on the surface of the iRBC, focusing the interactions of multiple multigene families needs to be considered

RSpred, a set of Hidden Markov Models to detect and classify the RIFIN and STEVOR proteins of Plasmodium falciparum

<p>Abstract</p> <p>Background</p> <p>Many parasites use multicopy protein families to avoid their host's immune system through a strategy called antigenic variation. RIFIN and STEVOR proteins are variable surface antigens uniquely found in the malaria parasites <it>Plasmodium falciparum </it>and <it>P. reichenowi</it>. Although these two protein families are different, they have more similarity to each other than to any other proteins described to date. As a result, they have been grouped together in one Pfam domain. However, a recent study has described the sub-division of the RIFIN protein family into several functionally distinct groups. These sub-groups require phylogenetic analysis to sort out, which is not practical for large-scale projects, such as the sequencing of patient isolates and meta-genomic analysis.</p> <p>Results</p> <p>We have manually curated the <it>rif </it>and <it>stevor </it>gene repertoires of two <it>Plasmodium falciparum </it>genomes, isolates DD2 and HB3. We have identified 25% of mis-annotated and ~30 missing <it>rif </it>and <it>stevor </it>genes. Using these data sets, as well as sequences from the well curated reference genome (isolate 3D7) and field isolate data from Uniprot, we have developed a tool named RSpred. The tool, based on a set of hidden Markov models and an evaluation program, automatically identifies STEVOR and RIFIN sequences as well as the sub-groups: A-RIFIN, B-RIFIN, B1-RIFIN and B2-RIFIN. In addition to these groups, we distinguish a small subset of STEVOR proteins that we named STEVOR-like, as they either differ remarkably from typical STEVOR proteins or are too fragmented to reach a high enough score. When compared to Pfam and TIGRFAMs, RSpred proves to be a more robust and more sensitive method. We have applied RSpred to the proteomes of several <it>P. falciparum </it>strains, <it>P. reichenowi, P. vivax</it>, <it>P. knowlesi </it>and the rodent malaria species. All groups were found in the <it>P. falciparum </it>strains, and also in the <it>P. reichenowi </it>parasite, whereas none were predicted in the other species.</p> <p>Conclusions</p> <p>We have generated a tool for the sorting of RIFIN and STEVOR proteins, large antigenic variant protein groups, into homogeneous sub-families. Assigning functions to such protein families requires their subdivision into meaningful groups such as we have shown for the RIFIN protein family. RSpred removes the need for complicated and time consuming phylogenetic analysis methods. It will benefit both research groups sequencing whole genomes as well as others working with field isolates. RSpred is freely accessible via <url>http://www.ifm.liu.se/bioinfo/</url>.</p

Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum </it>STEVOR proteins, encoded by the multicopy <it>stevor </it>gene family have no known biological functions. Their expression and unique locations in different parasite life cycle stages evoke multiple functionalities. Their abundance and hypervariability support a role in antigenic variation.</p> <p>Methods</p> <p>Immunoblotting of total parasite proteins with an anti-STEVOR antibody was used to identify variant antigens of this gene family and to follow changes in STEVOR expression in parasite populations panned on CSA or CD36 receptors. Immunofluorescence assays and immunoelectron microscopy were performed to study the subcellular localization of STEVOR proteins in different parasite stages. The capacity of the antibody to inhibit merozoite invasion of erythrocytes was assessed to determine whether STEVOR variants were involved in the invasion process.</p> <p>Results</p> <p>Antigenic variation of STEVORs at the protein level was observed in blood stage parasites. STEVOR variants were found to be present on the merozoite surface and in rhoptries. An insight into a participation in erythrocyte invasion was gained through an immunofluorescence analysis of a sequence of thin slides representing progressive steps in erythrocyte invasion. An interesting feature of the staining pattern was what appeared to be the release of STEVORs around the invading merozoites. Because the anti-STEVOR antibody did not inhibit invasion, the role of STEVORs in this process remains unknown.</p> <p>Conclusion</p> <p>The localization of STEVOR proteins to the merozoite surface and the rhoptries together with its prevalence as a released component in the invading merozoite suggest a role of these antigens in adhesion and/or immune evasion in the erythrocyte invasion process. These observations would also justify STEVORs for undergoing antigenic variation. Even though a role in erythrocyte invasion remains speculative, an association of members of the STEVOR protein family with invasion-related events has been shown.</p

The Gates Malaria Partnership: a consortium approach to malaria research and capacity development.

Recently, there has been a major increase in financial support for malaria control. Most of these funds have, appropriately, been spent on the tools needed for effective prevention and treatment of malaria such as insecticide-treated bed nets, indoor residual spraying and artemisinin combination therapy. There has been less investment in the training of the scientists from malaria-endemic countries needed to support these large and increasingly complex malaria control programmes, especially in Africa. In 2000, with support from the Bill & Melinda Gates Foundation, the Gates Malaria Partnership was established to support postgraduate training of African scientists wishing to pursue a career in malaria research. The programme had three research capacity development components: a PhD fellowship programme, a postdoctoral fellowship programme and a laboratory infrastructure programme. During an 8-year period, 36 African PhD students and six postdoctoral fellows were supported, and two research laboratories were built in Tanzania. Some of the lessons learnt during this project--such as the need to improve PhD supervision in African universities and to provide better support for postdoctoral fellows--are now being applied to a successor malaria research capacity development programme, the Malaria Capacity Development Consortium, and may be of interest to other groups involved in improving postgraduate training in health sciences in African universities

Transcriptional regulation of virulence gene families in "Plasmodium falciparum"

To date, malaria caused by Plasmodium falciparum is still a major health threat. It contributes to illness and severe disease and is responsible for up to one million deaths per year. The intra-erythrocytic asexual life cycle stage is responsible for the pathology associated with malaria. The major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1) is exposed at the surface of infected red blood cells (iRBC) and mediates binding to endothelial cells. This leads to sequestration of iRBC in the microvasculature and consequently to evasion of removal in the spleen. PfEMP1 is encoded by the 60-member var gene family, which undergoes antigenic variation by in-situ switching. Importantly, var genes are expressed in a mutually exclusive way, such that only one member is expressed whereas all other copies remain silenced. var genes as well as other gene families such as rif, stevor, phist and pfmc-2tm are located in subtelomeric heterochromatic regions. The function of these additional families is largely unknown, but they are thought to be implicated in host-parasite interactions and to contribute to antigenic variation. With this work, I provide deeper insights into the transcriptional regulation of virulence gene families in P. falciparum by using transfection-based approaches. We functionally identified autonomous cis-acting var promoter elements including an upstream activating sequence that is essential for promoter activation. Notably, an element downstream of the transcriptional start site determines mutually exclusive locus recognition. Further, I used comparative transcriptional profiling to show that mutually exclusive expression is restricted to the var gene family and is not used in the transcription of other subtelomeric gene families. I show for the first time that knock-down of endogenous var gene transcription is also conferred by promoters of a var gene subfamily that is implicated in severe malaria. Taken together, this work provides important insight into the mechanisms involved in the regulation of virulence gene families and antigenic variation in P. falciparum. Moreover, the findings presented here are consistent with a novel mechanism of mutually exclusive gene choice in eukaryotes

Characterisation of the rif and stevor Multigene Families in Plasmodium falciparum Isolates Sampled From Natural Infections

Parasite-derived variant surface antigens (VSA) expressed on the infected erythrocyte surface are important targets of naturally acquired protective immune responses against malaria. The three major VSA-encoding multigene families of Plasmodium falciparum, var, rif and stevor, exhibit high inter- and intra-strain genomic variability. The VSA component encoded by the var gene family, PfEMP1, comprises the major cytoadhesive ligand and the major antigenic target of protective antibodies. However, other VSA families, including those encoded by the rif and stevor multigene families, may also play important roles in malaria pathogenesis and immunity. However, the biological relevance of non-PfEMP1 VSA remains largely unknown. This thesis represents the first extensive analysis of sequence diversity and expression patterns of rif and stevor variant gene families in African field isolates of P. falciparum. The work details the characterization of rif and stevor gene repertoires of P. falciparum parasites from diverse geographical locations and identification of conserved genes in both P. falciparum and the related chimpanzee malaria parasite P. reichenowi. Both capillary sequencing and clone-free, 454 deep sequencing methods have been used to study changes in variant gene expression during asexual development of wild and culture-adapted isolates. Isolate- and stage-specific transcription patterns of rif and stevor genes were observed, and the major sets of transcripts in multiple isolates, including parasites that have been selected for different cytoadhesive phenotypes (resetting and adhesion to human brain endothelial cell lines) identified. The role of RIFIN antigens in the natural acquisition of antibodies to malaria was tested using a strain transcendent variant PF3D7_1253700 (PFL2585c). The hypervariable region of this RIFIN, expressed as a recombinant protein was used to purify naturally acquired human antibodies from sera from malaria-immune African adults. These antibodies recognized native RIFIN antigen on the surface of intact trophozoites showing that RIFINs for additional targets of naturally acquired antibodies that recognize the surface of parasite-infected red blood cells

Antigenic variation in Plasmodium falciparum : understanding the RIFIN protein family

RIFIN proteins are variable surface antigens, which have a central role in the survival and virulence of the malaria parasite Plasmodium falciparum. Antigenic variation is a mean for these parasites to avoid clearance by the host s immune system. However, this is often a secondary function to the main role of these proteins. In the case of RIFIN, P. falciparum s largest multicopy protein family, the main functions remain unknown. In order to elucidate a protein s function, it is crucial to understand its basic properties, including the structure of the protein family, their localization and the protein s topology. Through different methods, we have strived to simplify the RIFIN protein family into manageable entities. We have started with a simple classification of RIFIN proteins into meaningful sub-groups. We have predicted that these sub-groups are functionally distinct, although they probably share a related function. We then designed RSPred, an automatic method, based on hidden Markov models and a sorting program, to detect and classify RIFIN and STEVOR sequences according to their sub-group. Finally, using an in vitro method to determine protein topology, we have analyzed both A-RIFIN and B-RIFIN proteins for their number of transmembrane segments and their topologies. We show that both protein groups have a signal sequence targeting them to lipid bilayers and only one transmembrane domain. They both share a common topology where the bulk of the protein is exposed to the extracellular environment. With the current knowledge of RIFIN protein localizations, as well as the loss of expression of A-RIFIN but not B-RIFIN proteins in a splenectomized patient, it seems increasingly clear that B-RIFIN proteins are good targets for future studies, to decipher the functions of these variable proteins