Background
The life cycle of the
Plasmodium falciparum malaria parasite involves asexual and sexual phases. To maintain a persistent infection in the human host for successful transmission to mosquitoes, parasites express various polymorphic proteins that help evade human antibody responses and facilitate invasion of host cells. During asexual multiplication in the blood, parasites invade and multiply inside erythrocytes, apart from short periods as extracellular merozoites, which are released at erythrocyte rupture and then quickly re-invade fresh host cells. Polymorphic proteins like merozoite surface proteins 1 (MSP-1) and apical membrane antigen 1 (AMA-1)[
1,
2] are expressed on the merozoite surface and are known to play specific roles in erythrocyte invasion. The STEVOR family of variant antigens are also known to be expressed on the merozoite surface[
3] and to be associated with the plasma membrane of mature gametocyte-infected erythrocytes[
4]. The locations of the related, highly diverse RIFIN antigen family members are less well understood, but they have been reported to be present inside the merozoite[
5]. Each parasite carries approximately 150–200
rif and 30–35
stevor gene copies per genome, and it remains a possibility that their abundance and diversity also contribute to immune evasion by merozoites during their brief extra-cellular phase.
While it is uncertain whether
rif genes are expressed in a ‘relaxed’ or strictly mutually exclusive manner, multiple RIFIN variants have been reported in bulk cultures of parasites grown
in vitro[
6,
7]. Rifin variants can be divided into A- and B-types based on the presence or absence of a 25 amino acid motif in the semi-conserved domain[
8] and sub-structuring of RIFIIN protein sequence reflect functional divergence with A- and B-types serving different roles in distinct parasite stages[
5]. During intraerythrocytic multiplication B-type RIFIIN were reported to be retained inside the parasites while A type RIFIN were expressed on the infected erythrocyte surface, potentially contributing to the antigenic variation capacity of the parasite[
5].
Plasmodium falciparum pathology is profoundly influenced by the sequestration of infected erythrocytes to microvascular endothelium in various tissues. This involves interactions between parasite adhesins and several human endothelial receptors including CD36, ICAM1 and the glycosaminoglycan, CSA[
9,
10]. During sexual development
in vivo, mature gametocytes of
P. falciparum (Stage V) do not appear in the peripheral blood circulation until 7–15 days after the initial wave of blood stream infection appears[
11]. This is due to the sequestration of immature gametocyte forms, which develop in various host tissues including the bone marrow and spleen[
12,
13]. Although superficially analogous to the sequestration of mature asexual parasite stages, the details of interactions between developmental stages of gametocytes and host tissues are poorly understood, and if cytoadherence is involved, the host receptors responsible remain unidentified. Candidate receptors for adhesion of early gametocytes (Stage I, II) include CD36[
14] and for stage III to IV include ICAM-1, CD49c, CD164 and CD166[
15]. Candidate gametocyte-expressed parasite ligands may include variants of the multigene families
var, rif and
stevor. Of these, cytoadhesive properties have only been demonstrated for PfEMP1, which has been linked to cytoadhesion of gametocyte stages I to IIA. In the later stages III to IV, PfEMP1 was observed to be retained inside parasite cytoplasm[
16]; possibly indicating that PfEMP1 may not be involved in gametocyte cytoadherence after stage IIB. However, more recent transcriptional data suggest that certain group C
var genes are selectively transcribed during gametocytogenesis
in vitro[
17], suggesting a role for this subset of PfEMP1 in gametocytes, gametes or later parasite stages in the mosquito. Type A RIFIN has been found on the surface of developing gametocytes and type B Rifin expressed but retained inside the cell at all gametocyte stages[
18]. STEVOR proteins are localized near the developing gametocyte surface membrane, but surface exposure and any direct role in adhesion to host tissues, remains to be confirmed[
4]. However, recently, Tibùrcio and colleagues[
19] showed that cell rigidity of immature gametocyte-infected erythrocytes was associated with the expression of STEVOR proteins, potentially contributing to the sequestration of these stages by mechanical retention rather than adhesion[
20].
Although antisera have been developed which can distinguish type A from type B RIFIN sub-groups[
18], variant-specific RIFIN antibodies have not previously been described. In previous studies[
18,
21] global transcription analysis of all
rif genes in sexual and asexual development of the 3D7 parasite line revealed a unique expression pattern of the type B
rif gene PF13_0006. This gene was up-regulated in late stage schizonts, developing gametocytes and in sporozoites. To test the hypothesis that this RIFIN variant has a distinctive role in parasite development, antibodies to the protein encoded by PF13_0006 were developed and the expression was followed throughout parasite development
in vitro. Evidence is presented suggesting that this specific type B RIFIN variant is expressed on the surface of free merozoites, internally in developing gametocytes and on the surface of gametes at the point of emerging from activated, mature stage V gametocytes.
Discussion
The P. falciparum parasite uses clonal antigenic variation to evade host antibody immune responses and permit the establishment of the fairly long-lasting infections that individual clones require for their transmission back to the mosquito vector. It is difficult to know what the average clonal transit time through the human host is, but it is unlikely to be shorter than three weeks and could be significantly longer. While the important role of PfEMP1 in antigenic variation during the intra-erythrocytic multiplication stages is well established, antigenic variation during merozoite, gametocyte and gamete stages has not been formally demonstrated in P. falciparum, although a role for RIFIN and STEVOR proteins remains plausible.
A-type Rifins are found associated with the surface of infected erythrocytes and gametocytes and thus appear to follow the expression pattern of PfEMP1[
5], whereas surface exposure of STEVOR so far has only been confirmed on merozoites[
3]. The data presented here show that a specific type B RIFIN variant (PF13_0006) is expressed by free, live merozoites and gametes (activated gametocytes). Transcript analysis presented here and previously[
21,
22] unambiguously show that this RIFIN is upregulated in early ring, schizonts and late stage gametocytes. To investigate PF13_0006 protein expression, rabbit antibodies to the variable region of this protein were generated. These antibodies stained the surface of 3D7 merozoites and gametes, but not the surface of intact 3D7 gametocytes or FCR3 merozoites. Also the antibodies only detected correctly sized protein in western blots of 3D7 schizonts and gametocytes extracts and not of 3D7 rings or FCR3 schizonts. Thus, although the antibodies appear to un-specifically target a number of other proteins in the western blots, collectively the data support the expression of PF13_0006 protein on the surface of merozoites and gametes. The expression of PF13_0006 inside the parasite during schizont and gametocyte stages is in agreement with previous reports using antibodies with specificity for B-type RIFINs[
5,
18]. There is as yet no evidence that expression of RIFIN variants is mutually exclusive[
5,
36]. Transcript analyses published earlier[
21,
22] indicate that transcripts of other B1 RIFINs, in particular PFI0025c, encoding the unique sequence motif identified in this study are present in merozoites and gametes at the same time as PF13_0006. High transcript levels of
PF13_0006 were also detected in early rings[
22] but no protein was detected which could reflect a spill-over of late stages in the synchronization. It should also be noted that the method for detecting transcripts is much more sensitive than the methods used to detect protein.
A proportion of malaria exposed individuals from both East and West Africa had acquired plasma antibodies recognizing recombinant PF13_0006. The seropositive rate was slightly higher among the Gambian children, but this probably reflected that these children participated in a treatment trail and therefore had a recent exposure. The data suggest that RIFIN variants antigenically similar to the variable domain of PF13_0006 occur in the African parasite populations possibly in particular the unique sequence motif of the B1 RIFINs. These antibody responses were more common among older children than in adults. A similar decline in anti-PfEMP1 seropositivity was reported previously[
46] and probably reflect that the antibody levels to these polymorphic proteins are short lived and that adults who have acquired partial immunity have less parasite exposure than the children. The prevalence of antibodies did not vary consistently with the level of transmission and was higher in the Tanzanian samples collected in 2008 than in the previous years, despite a general decline in transmission in the area[
43]. This could reflect temporal shifts in the relative abundance of parasites carrying RIFIN variants closely related to PF13_0006, as similar fluctuations related to time and geographical location have been observed for antibody recognition of different PfEMP1 variants[
47].
Confocal imaging of live, non-fixed, non-permeabilized free merozoites is challenging due to the small size and motility of these stages. However, present data does indicate that PF13_0006 is co-localized with MSP1 on the merozoite surface. This would place RIFIN along with the other variant surface antigens such as SURFINs[
48] and STEVOR[
3] which recently have been shown to be expressed on the surface of free merozoites. These studies of diverse multigene families, taken together, suggest that the antigens they encode play a role in evading the immune response against merozoites. However, as group B RIFIN only comprise ~25% (40 genes in the 3D7 genome) of the family, and the B1 sub-group around half of this proportion[
22], indicating that there is scope for other members of the family to play different roles in the parasite life cycle. It is thus tempting to speculate that the B or B1 type RIFINs have become functionally specialized for a particular role in relation to merozoite egress or erythrocyte invasion.
Similarly, the PF13_0006 B1 RIFIN may serve a particular role either within the developing gametocyte, or in emergent gametes. Whether PF13_0006 serves a role in fertilization, e.g. locating and/or binding to a “partner” cell of opposite mating type, remains to be investigated. However, it is important to note that 3D7 sporozoites have also been found to specifically transcribe the PF13_0006
rif gene[
22]. Extrapolating observations from single parasite isolates is unsound and there is a need to investigate
rif expression on other genetic backgrounds, but the finding that PF13_0006 appear to be expressed in all free-living stages of 3D7 highlights the need for functional studies of this RIFIN in extracellular forms of the parasite.
The
rif gene family is part of the
pir (
Plasmodium interspersed repeats) gene super-family of six variant multigene families found in
Plasmodium vivax (
vir),
Plasmodium ovale (oir), Plasmodium knowlesi (
kir) and in three rodent malarias (
Plasmodium chabaudi,
cir;
Plasmodium berghei,
bir;
Plasmodium yoelii,
yir). Sequence analysis of these gene families, as for
rif genes, show compartmentalization into sub-groups/types indicative of specialized functions rather than sequence variation for antigenic variation alone[
49]. This function may be common to all
Plasmodium species which share life cycle, the common challenges of host immunity, and the need for invasion and rupture of host cells. The variable domain of PF13_0006, the domain to which the antibody was raised, seems to be semi-conserved in the C-terminal part for the B1 subtype RIFINs. This part of the protein may serve a common function for the subtype and it may be possible to generate antibodies targeting B1 RIFINs expressed by different parasite clones.
Acknowledgments
We sincerely thank the children participating in this study and parents or guardians including teams which conducted studies in Tanzania and The Gambia. We thank Richard Carter for providing 1H12 and Pfs230 antibodies, JS McBride for the gift of MSP-119, and Dr. Graeme Cowan for the gift of pneumolysin, University of Edinburgh, United Kingdom. SBM was supported during this work by DANIDA, and CWW was supported by The Danish Medical Research Council (grant reference number 271-08-0540) and Augustinus Fonden. CJS is supported by the UK Health Protection Agency. DB and DEA were supported by a Niels Bohr Foundation visiting professorship to DEA, awarded by Danmarks Grundforskningsfonds. LT received support from University of Copenhagen Program of Excellence. TL was supported by the Lundbeck Foundation and The Danish Medical Research Council.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SBM, CWW carried out molecular biology studies, analysed data and wrote the paper. CJS, TL and TGT participated in the design, coordination and analysis of the study and helped to draft the manuscript. LT designed and carried out molecular biology studies, and analysed data. DCB and BD carried out molecular biology studies and revised the manuscript. DEA and JPL participated in the coordination of the study and revised the manuscript. All authors read and approved the final manuscript.