TY - JOUR
AU - Chen, Qijun
AB - Abstract Plasmodium falciparum being the most lethal plasmodiae is still a major cause of the disease burden and mortality in malaria endemic areas. Due to the wide spread drug resistance in combination with poor socio-economic situation in the vast majority of the endemic countries, malaria is today a great global challenge. The scientific community is, however, progressing. The 23 Mb genome of P. falciparum has been decoded and publicly available. Data of transcriptional profiling at certain developmental stages have already been generated. More than 50% of P. falciparum genes are transcribed constitutively in all the developmental stages of parasite life cycle. Functional disruption of these genes might have implications for parasite growth and development. Available microarray data indicate that P. falciparum preferentially expresses rif and stevor gene families at gametocyte and sporozoite stages while var genes are predominantly expressed at the erythrocytic stage. Gene regulation mechanisms of the variant gene families in P. falciparum are still not understood though some regulatory elements have been proposed. The occurrence of severe malaria is determined by both parasite and human host factors. Sequestration and antigenic variation are two of the evasion mechanisms utilized by P. falciparum in order to escape the human host defences. Understanding the molecular mechanisms underlying these phenomena is of a major importance and interest in malaria research. Here, we summarize and highlight the recent progress in molecular aspects of severe malaria. Malaria, Infection, Adhesion, Antigenic variation, Genome 1 Introduction Malaria, once a target for eradication, remains a major threat, especially in sub-Saharan Africa. An estimated 300–500 million clinical cases occur each year, and between one and three million deaths, primarily of children and pregnant women, are attributable to this disease. Every 40 s a child dies of malaria, resulting in a daily loss of more than 2000 young lives worldwide [1]. These estimates render malaria the pre-eminent tropical parasitic disease and one of the top three killers among communicable diseases. Plasmodium falciparum, one of the four parasite species infecting humans, is the deadliest species causing a broad spectrum of clinical symptoms. Most cases of endemic P. falciparum malaria remain uncomplicated, however some develop a number of severe complications including cerebral malaria, severe anaemia and placental malaria. Severe malaria occurs when the parasite reaches the erythrocytic phase of the infection and starts to proliferate inside the erythrocytes. A characteristic feature of P. falciparum malaria is the ability of the parasite-infected red blood cells (pRBCs) to adhere to host endothelium (cytoadherence) and to non-infected erythrocytes (rosetting). These binding events eventually lead to the occlusion of the microvasculature in various tissues and organs, such as the brain in cerebral malaria [2]; hence contributing directly to the pathogenesis of severe malaria disease. A number of host molecules have been implicated as receptors for parasite adhesion and P. falciparum erythrocyte membrane protein 1 (PfEMP1) has been suggested as the key adhesive ligand of pRBCs. Malaria is a complicated syndrome determined by both parasitic and human factors. Though P. falciparum has adapted in human being for just a few million years [3], it has already evolved diverse biological mechanisms necessary for survival in its hosts. With a highly AT-rich genome, the parasite can frequently change its genetic material for optimal adaptation to the surrounding environment. One example of immune evasive strategies of P. falciparum is the expression of variant surface antigens such as PfEMP1. By adhering to various receptors via PfEMP1, the parasite is removed from the circulation and thus avoids splenic clearance. However, the surface exposure of PfEMP1 allows for immune recognition, which is in turn circumvented by the parasite through the mechanism of antigenic variation. The exposure of a new variant antigen thus engenders changes in receptor recognition and tissue tropism of pRBCs. Despite the evasive mechanisms, if the human host survives a number of exposures, a semi-protective immunity against severe disease will develop eventually. Cross-reactive antibodies recognising a subpopulation of variant antigens (PfEMP1) appear to confer this protection [4]. Many children never reach the level of immunity required for survival and hence die. Attempts to eliminate or at least suppress malaria have been an important public health story through much of the last century. The current focus of malaria control programmes in Africa is primarily on clinical management of the disease through early treatment with effective antimalarial drugs. In some areas, insecticide-treated nets are used to prevent mosquito attacks. However, due to the continuous emergence of drug resistance in combination with the unavailability and the high cost of novel drugs such as Artemisinins and its derivates, we have now fewer tools to control malaria. The alternatives are particularly scarce in the highly endemic areas of Africa. We are thus in desperate need of new strategies in order to prevent the most severe outcomes of the disease. The releases of the parasite, its mosquito vector and the human genome data have provided the malaria research community with invaluable tools enabling in-depth dissection of the parasite biology and malaria pathogenesis. 2 Genome aspects of Plasmodium falciparum parasite After several years of tremendous efforts by several institutions, the genome of P. falciparum 3D7 clone was decoded in 2002 [5]. The genome data have enabled us to gain a deeper understanding of P. falciparum biology. Functional mapping of all genes expressed at different developmental stages is now possible due to the availability of genome-wide arrays combined with other techniques such as gene knock-in and knock-out by transfection and large scale proteomic analysis. Through comparison with genome sequences of other plasmodial parasites as well as human genome data, falciparum-specific metabolic pathways and new antigens are expected to be discovered. With such knowledge new drug targets and vaccine candidates can be fruitfully explored. The 23 million base pairs P. falciparum genome (clone 3D7) comprises 14 chromosomes, of which at least 5409 genes have been predicted [5]. Additional sequence information includes a 6-kb mitochondrial genome and a 35-kb circular plastid DNA. The genome organization possesses some similarities to other Plasmodium spp. Chromosomes of all Plasmodium spp. have the same telomere repeats. Genes encoding variant antigens associated with erythrocyte membrane are predominantly located in the subtelomeric regions of the chromosomes (Ref. [5] and Table 1). Compared to other plasmodia, the chromosome ends of P. falciparum are more complex, consisting of five unique subtelomeric blocks (SB 1–5) that constitute different functions during the parasite life cycle [5]. At least three variant gene families (var, rif and stevor families) involved in antigenic variation are predominantly located in these areas. In the subtelomeric block 4 (SB-4) regions of the 3D7 clone, arrays of genes (var–rif–stevor/rif) encoding variant antigens, displayed on the infected erythrocyte surface, are uniformly found. 36 out of 59 var genes, 149 out of 159 rif genes and all stevor genes are located in the subtelomeric ends. Table 1 Variant antigen families of different Plasmodium species Plasmodium species  Variant gene families  Gene number/genome  Variant antigens  Function  P. falciparum  var  59  PfEMP1  Antigenic variation cytoadherence  rif  149  RIFIN  Antigenic variation    Stevor  ∼28  STEVOR  Antigenic variation    P. vivax  vir      Antigenic variation  P. knowsei  SICA gene    SICA  Antigenic variation  P. yoelii  yir      Antigenic variation  P.chabaudi  cir      Antigenic variation  Plasmodium species  Variant gene families  Gene number/genome  Variant antigens  Function  P. falciparum  var  59  PfEMP1  Antigenic variation cytoadherence  rif  149  RIFIN  Antigenic variation    Stevor  ∼28  STEVOR  Antigenic variation    P. vivax  vir      Antigenic variation  P. knowsei  SICA gene    SICA  Antigenic variation  P. yoelii  yir      Antigenic variation  P.chabaudi  cir      Antigenic variation  View Large Table 1 Variant antigen families of different Plasmodium species Plasmodium species  Variant gene families  Gene number/genome  Variant antigens  Function  P. falciparum  var  59  PfEMP1  Antigenic variation cytoadherence  rif  149  RIFIN  Antigenic variation    Stevor  ∼28  STEVOR  Antigenic variation    P. vivax  vir      Antigenic variation  P. knowsei  SICA gene    SICA  Antigenic variation  P. yoelii  yir      Antigenic variation  P.chabaudi  cir      Antigenic variation  Plasmodium species  Variant gene families  Gene number/genome  Variant antigens  Function  P. falciparum  var  59  PfEMP1  Antigenic variation cytoadherence  rif  149  RIFIN  Antigenic variation    Stevor  ∼28  STEVOR  Antigenic variation    P. vivax  vir      Antigenic variation  P. knowsei  SICA gene    SICA  Antigenic variation  P. yoelii  yir      Antigenic variation  P.chabaudi  cir      Antigenic variation  View Large Chromosome ends are regions undergoing dynamic changes in eukaryotic cells. In P. falciparum, chromosome ends form clusters during mitosis in erythrocytes. Rep-20 repeats in the subtelomeric region and the binding proteins likely mediate the chromosome end cluster formation which obviously facilitates genetic exchanges between chromosomes [6,7]. In 3D7 strain, gene replication and recombination events are found in genes located in subtelomeric regions. Though some centrally located genes also show signs of both recombination and duplications [8]. Chromosome location and gene type seem to be important factors. For example var genes in subtelomeric regions and in chromosome central regions never recombine. The recombination mechanism in the centrally located genes is likely to be different from that of genes in the chromosome ends. var genes located in central regions of the chromosomes have however been suggested to be more conserved [9]. The genome sequence has provided us a platform for dissecting parasite biology. Recent publications based on the analyses of microarray hybridisation data with parasite RNAs transcribed at different developmental stages have generated some interesting data [10,11]. Genes coding for proteins of related functions seem to be activated in clusters independent of chromosome locations. For example, mRNAs encoding proteases involved in schizont disruption and erythrocyte invasion are dominantly transcribed at schizont stage (after 36 h erythrocyte invasion), while genes encoding proteinases necessary for haemoglobin digestion are predominantly activated during the trophozoite stage. The data are quite in-line with that of proteomic analysis on parasites isolated from different developmental stages [12]. With up to 60% of the genes being unknown, based on the recent findings, we might be able to predict the functions of the proteins encoded by these unknown genes (a “guilty-by-association” approach) [13]. The transcription data have also revealed that many P. falciparum genes have early activation and slow silencing process. One example is the mRNAs of some sporozoite-specific genes, such as CSP mRNA also being present in the blood stage though not expressed into protein. It is still not understood why the gene activation process is loosely controlled. Certainly, this phenomenon adds further complexity to the cluster classification suggested by Le Roch et al. [10]. Finally, decoding gene sequences and characterizing encoded proteins are relatively easier tasks than comprehending the genome organization, gene regulation and interactions of genomic regions. Parasites that proliferate faster and those that are more virulent might have unique genomic organization in certain areas. Thus, in order to unravel the genome mystery simply knowing the expression pattern of certain genes is not sufficient but further in-depth information is required. 3 Diversity of invasion pathways 3.1 Factors linked to hepatocyte invasion Before adaptation to the erythrocytic cycle, P. falciparum must pass through a developmental stage in hepatocytes. Sporozoites released from mosquito salivary gland have to target and penetrate hepatocytes efficiently. CSP and TRAP expressed on sporozoite surface [14] have been identified as two major parasite ligands for liver cell invasion and parasite development in both mosquito and liver stages, since knocking out any gene encoding either protein hampers the development of the parasite in vivo. Both CSP and TRAP bind to heparan sulfate (HS) on hepatocyte surface. CSP is believed to initiate sporozoite binding with HS receptor, while TRAP mediates cell membrane penetration process. CSP and TRAP antigens have been the two prominent antigens for developing anti-invasion vaccines. HS might not be the only receptor for hepatocyte invasion. Recently, CD81 has been reported as a receptor for both P. falciparum and rodent malaria parasites invasion in hepatocytes [15]. CD81 is a kind of tetraspanin expressed on hepatocyte surface. It has been implicated as a receptor for Hepatitis C virus invasion [16]. Interestingly anti-CD81 mAb could completely block parasite invasion into hepatocytes in the experiments with P. yoelii (265BY strain), P. berghei (NK65 strain) and P. falciparum (NF54 strain), suggesting an alternative pathway for malaria parasite invasion. The parasite ligand to CD81 has not been identified. Proteomic analysis as well as gene specific hybridization with mRNAs purified from this stages have confirmed that large numbers of the variant antigen families (var, rif and stevor) are expressed in this stage [10–12]. Though the var-gene expression at sporozoite stage is very low, the expression of certain rif genes is reasonably high [10,12]. Since these genes are not physically adjacent to the sporozoite specific genes, their expression is unlikely due to ‘leakage’ activation. The location and potential receptor-binding function of these variable proteins expressed at the sporozoite stage are becoming interesting questions. 3.2 Factors linked to erythrocyte invasion The process of erythrocyte invasion of merozoites released from ruptured liver cells involves binding of merozoite to erythrocyte surface, apical reorientation, parasite receptor discharging, junction reformation and membrane penetration. Several merozoite-related proteins such as merozoite surface proteins (MSPs), erythrocyte-binding protein (EBA-175) and apical membrane antigen-1 (AMA-1) have been extensively studied in these processes [14], but it is almost unknown which merozoite-associated molecule plays the key role in the invasion process. In order to evade the human immune responses and to optimally adapt to hosts of different genetic backgrounds, plasmodium parasites have evolved a plethora of erythrocyte binding and invasion pathways. In P. yoelii, each merozoite in a single infected RBC (schizont) can express a different member of the red blood cell-binding protein family [17]. Since these proteins are antigenically different the parasite has a better chance to circumvent invasion-blocking antibodies. In P. falciparum, the erythrocyte binding and invasion pathways, mediated by the erythrocyte-binding ligand (ebl) family, are almost as redundant as that in other plasmodium spp. [18]. Initially, sialic acid was identified as P. falciparum merozoite receptor on erythrocyte surface; later it was found that many parasite strains could invade RBC via sialic acid-independent pathways [19,20]. This led to the discovery of glycophorin B and C as invasion receptors [21,22]. Some parasite strains, expressing EBA-175 allele, use sialic acid on glycophorin A as a receptor [22], while other strains have adopted alternative invasion pathways utilizing other ebl family members as ligands. For example, EBA-140 is used by some strains to bind glycophorin C [23]. Erythrocyte invasion is a process involving many receptor–ligand interactions, most of which are far from clear. Understanding the invasion process will undoubtedly facilitate the design of invasion blocking strategies. 4 Diverse variant gene families and antigenic variation 4.1 The var-gene family In P. falciparum genome, there is a group of genes that encode the variant antigens expressed on the surface of infected erythrocyte membrane, termed P. falciparum erythrocyte membrane protein 1 (PfEMP1). The genes are collectively named var genes constituting a family of ~60 copies per genome. var genes are scattered in all chromosomes; most of which are located in the subtelomeric and some in the central regions of the chromosomes (chromosome 4, 6, 7, 8 and 12 in the 3D7 strains) [5]. var genes in the subtelomeric regions are more vulnerable to recombination and presumably undergo frequent sequence alterations. Though the centrally located var genes are relatively conserved, gene recombination events also affect their stability. For example, the five var genes (PFD0625c, 0630c, 0635c, and PFD0095c, 1000c) in the central part of chromosome 4 of 3D7 clone are either duplicated or recombined with each other [5,8]. As for the sequence polymorphism, the sizes of var genes are very diverse, ranging from 3.9 to 13 kb. The proteins, PfEMP1s, encoded by the gene family are antigenically different and responsible for major antigenic variation at the infected erythrocyte surface. Each PfEMP1 molecule is composed of several distinct domain structures. The extra-cellular part, encoded by an Exon I region of a var gene, is constituted of a variable N-terminal segment (NTS), several different types of Duffy-binding like domains (DBL, α–η) and cysteine-rich interdomain regions (CIDR, α–γ) [24]. In some PfEMP1s, a small sequence fragment is always present after the DBLβ domain forming a DBLβC2 structure. The whole molecule is believed to be anchored on the erythrocyte membrane through a transmembrane (TM) domain and an intra-cellular C-terminal acidic segment (ATS) (Fig. 1). In 3D7 genome, one var gene (PF0030c) and three var-like genes encode PfEMP1 or PfEMP1-like molecules that have uncommon domain structures [5]. The var-like genes have not attracted any study, while Pf0030c seems to be a conserved gene implicated in placental malaria [25]. PfEMP1 family has been associated with malaria pathogenesis, especially cerebral malaria. Studies on the var-gene family as well as the function of different PfEMP1 molecules are still one of the important research areas. Figure 1 View largeDownload slide Representative structures of the var, rif and stevor genes as well as the encoded PfEMP1, RIFIN and STEVOR proteins. var genes are between 3.9 and 13 kb in size. The encoded PfEMP1s are composed of different number of Duffy-binding like (DBL) and cysteine-rich interdomain region (CIDR) domains. All var genes have an intron relatively close to 3′ end and all PfEMP1s have a transmembrane region (TM) followed by an intracellular acidic terminal segment (ATS). rif and stevor genes as well as their encoded RIFIN and STEVOR proteins are similar in structure. Their intron is relatively close to 5′ end and Exon I of both genes only encode a signal peptide-like sequence. Two TM sequences are located in the middle as well as close to the C-terminus of RIFIN and STEVOR. Figure 1 View largeDownload slide Representative structures of the var, rif and stevor genes as well as the encoded PfEMP1, RIFIN and STEVOR proteins. var genes are between 3.9 and 13 kb in size. The encoded PfEMP1s are composed of different number of Duffy-binding like (DBL) and cysteine-rich interdomain region (CIDR) domains. All var genes have an intron relatively close to 3′ end and all PfEMP1s have a transmembrane region (TM) followed by an intracellular acidic terminal segment (ATS). rif and stevor genes as well as their encoded RIFIN and STEVOR proteins are similar in structure. Their intron is relatively close to 5′ end and Exon I of both genes only encode a signal peptide-like sequence. Two TM sequences are located in the middle as well as close to the C-terminus of RIFIN and STEVOR. Understanding the mechanism of malaria antigenic variation is considered to be an important step towards the development of any immune protective measure. However, the knowledge of var-gene control mechanisms is still fragmented. But with the venue of the genome data, an array of exciting new findings has emerged, providing us with an in depth understanding of essential var-related biology. 4.2 var-gene control mechanisms var-gene control involves gene activation, switching and silencing mechanisms that are both developmentally and genetically regulated. Studies on parasites in erythrocytic stage suggest that var-gene expression occurs in a mutually exclusive manner; only one gene is dominantly transcribed to full-length mRNA and expressed while the rest remain silenced [26,27]. Each var gene represents a single transcriptional unit that is capable of activation in situ. Available data indicate that var genes are differentially expressed at all developmental stages [12]. But, the expression at erythrocytic stage is more prominently relevant to parasite development in term of immune evasion. A few hours after merozoite invasion, a var-gene activation process is initiated (some var genes might be activated already in merozoites in hepotocytes). Several var genes could be transcribed in each parasite spontaneously. However, mature trophozoites (~16 h) dominantly express one PfEMP1 on the surface. Due to the absence of immune pressure the PfEMP1 expression profile of in vitro cultures can change constitutively, but default expression of the same var gene for a relatively long period has also been seen. Whereas in vivo the expression pattern could be completely different. Recent studies indicate that there is a complete changeover of the var gene expression profile after parasites pass through the mosquito vectors suggesting that the var gene-switching rate is much higher in vivo [28]. Some physically co-localized var genes (also rif and stevor genes) tend to be expressed at the same developmental stages [12,29]. Several mechanisms could be behind the selection and control processes. Analysis of the var upstream sequences have revealed three types of promoter-like sequences termed upsA, upsB and upsC [5]. Promoter types are associated with var gene location and orientation. The var genes in the subtelomeric regions with orientation towards the telomere have upsA type promoters. var genes in the subtelomeric region but with reading orientation towards the centromere have upsB type promoters. The var genes located in the central part of the chromosomes have upsC type promoters. Using DNA–protein binding and migration shift assays conserved sequence motifs termed chromosome-central var gene promoter element (CPE) and subtelomeric var gene promoter element (SPE) were encountered in these promoter regions [30]. Differential transcription of var genes at different chromosomal locations is suggested to be due to the differential expression of the promoter repressor elements. Intron-silencing mechanisms have been proposed to operate as control machinery preventing the exhaustion of the var gene repertoire [31]. Recently, an intronic sequence element was found to have promoter activity which might drive the transcription of intronic RNA [32]. Small RNA transcripts from some var introns have been shown to be able to silence the expression of the same gene in a cooperative way with the 5′-UTR sequences. Evidence supporting this is the discovery of a common, conserved var gene (varCOMMON) present in as many as 60–70% of the wild isolates, as well as in the laboratory strain 3D7 [33]. varCOMMON is constitutively transcribed in these parasites. The gene lacks an intron and ends in telomeric repeat sequence in the 3D7 strain suggesting that the intron has a silencing function. However, intron-silencing may not provide the whole picture of var gene silencing mechanisms, since the promoter motifs in the intron are similar among var genes in different chromosomal locations. This cannot explain why genes are differentially suppressed. Voss et al. [30] found that there are two types of promoter-binding proteins that might mediate var gene silencing. The two protein elements, SPE1BP and CPEBP, were found differentially expressed in 3D7 parasites. SPE1BP is a protein which is only associated with promoters of var genes in the subtelomeric regions. This type of protein(s) is detectable 16 h after invasion. While the CPEBP type element(s) is associated with centrally located var genes and is detected 24 h after invasion. This could explain the transcription time-shift between the centromerically and subtelomerically located var genes. The data of Northern blot analyses performed by different groups [30,34,35, and unpublished] also confirm these findings. Some subtelomeric var genes are most likely activated at a very early stage after merozoite invasion (0–10 h) and the transcription will be turned off when the parasites reach late trophozoite stage (after 16 h). While var genes located in the central regions reach their transcriptional peak after 16 h post-invasion. The turn-off of transcription of the two types of var genes coincides with the expression of the two var promoter-binding proteins mentioned earlier (Ref. [30] and Fig. 2). However, there are still many questions remained to be answered. Why does the parasite repress most of the var genes but one? What factors interact with the repressor proteins? Further, what factors are coordinated with the expression or transportation of PfEMP1 to the surface? Obviously, some subtelomeric var genes are transcribed rather early but the transportation or display of the protein to the surface is delayed for several hours. While transcription, expression and protein transportation of the internal var genes are all imminently connected. There is still no link between the RNA silencing factor [31,32] and the protein factors [30]. It is unlikely that the intronic transcripts function at post-transcriptional level, due to the fact that they affect promoter activity instead of mRNA stability. There is also a possibility that the protein factors proposed by Voss et al. [30] are in fact a complex of proteins and RNA, such as the RISC (RNA-induced silencing complex), commonly found in RNAi-mediated gene silencing process. Transcription of RNA polymerase III and antisense RNA molecules [10] are indeed found in the parasite, but more experimental data are required in order to elucidate their function in the gene regulation process. Figure 2 View largeDownload slide Differential var gene activation, silencing and switching. var genes are differentially activated depending independent of chromosomal locations. Either subtelomeric genes (pink) or the centromeric var genes (blue) can be activated or silenced in a particular parasite. Switching of var gene activation can occur at either subelomeric or centromeric regions. The var specific promoter suppressor proteins and the short RNA transcripts from intron sequences of each var gene cooperatively silence var gene transcription. Figure 2 View largeDownload slide Differential var gene activation, silencing and switching. var genes are differentially activated depending independent of chromosomal locations. Either subtelomeric genes (pink) or the centromeric var genes (blue) can be activated or silenced in a particular parasite. Switching of var gene activation can occur at either subelomeric or centromeric regions. The var specific promoter suppressor proteins and the short RNA transcripts from intron sequences of each var gene cooperatively silence var gene transcription. Chromatin structure changes have been speculated to be associated with var gene switching. But neither methylation nor acetylation has been proved to be associated with the expression of a specific var gene. In vitro observations indicate that certain parasites are quick switchers, such as the parasites in the ITG line [36], and some parasites keep the expression of one var for many generations such as the parasites in the FCR3 lineage. In in vivo environment, the presence of the immune pressure, the host age and gender all seem to have an impact on the selection of var gene expression. In younger children and in malaria naïve individuals, the parasites tend to express more virulent types of variant surface antigens (VSA, mainly PfEMP1) on the pRBC surface (for review see [37]). And in pregnant women, the placenta provides a subpopulation of parasites a selective growth. Parasites sequestering in the placenta presumably express a specific PfEMP1 which only binds to receptors present on the syncytiotrophoblast surface. The selection mechanism of the var gene specifically expressed in placenta is still a mystery. Nuclear hormone receptor-like motif sequences have been reported in some var promoter regions but the potential function of these sequences is still not known [30]. The underlying mechanisms of var gene selection are not established and, being an area of immense importance, there is most certainly a need for further investigations. 4.3 The rif gene family rif (repetitive interspersed family) is the largest gene family implicated in P. falciparum antigenic variation. 149 rif genes are found in the 3D7 genome [5]. Like var genes, rif genes are distributed in both subtelomeric and central regions of all chromosomes. Each rif gene is composed of two Exons (Fig. 1). The short 5′ Exon I encodes a signal peptide. After Exon I, there is a short intron and Exon II encodes the major portion of the polypeptide named RIFIN. rif genes are always adjacent to var genes in the genome implicating a functional relatedness. Some rif genes are transcribed several hours later than var genes [35]. It is not known whether the centromeric rif genes are also activated later than the subtelomeric ones. Though, RIFINs are also clonally expressed, unlike var/PfEMP1, one parasite might express several RIFINs at the surface (like FCR3S1.2) [38]. Because of the co-localization of var and rif genes, the activation and silencing process of the two gene families might be linked. Proteomic analyses of 3D7 parasites indicate that adjacent genes (both var and rif) are transcribed and expressed concurrently [12], though the transcription level of the two genes might be different. As for the var gene family, gene duplication and mutations are also frequent in the rif gene family; however, gene recombination has not been seen. Further more, subtelomric genes might be translocated to central parts of the chromosome after duplication. At certain developmental stage (such as the sporozoite stage), members of the rif gene family are more active than the var genes, implying that the two gene families might have different biological functions. RIFIN is also different from PfEMP1 in structure. RIFIN has a N-terminal signal fragment suggesting that this protein is transported to the surface. It has two putative transmembrane domains. The potential extracellular region is most variable and is composed of hydrophobic amino acids. The C-terminal cytoplasmic part of the protein is relatively conserved and composed of positive changed amino acids. Some parasites express little (like 3D7, R29 and TM284S2 clone) RIFIN at the pRBC surface [38,39], while some parasites (like FCR3S1.2) express several RIFINs on the pRBC surface. This could be due to the genetic difference among these parasite clones after long time of in vitro cultivation. The biological function of RIFINs is still not known though they have been suggested as CD31 and rosetting ligands [38,39]. The co-localization of both RIFIN and PfEMP1 in the parasitophorous vacuole in the infected erythrocyte indicates that the two molecules reach the erythrocyte surface through the same pathway [40]. However, some laboratory strains (such as TM284S2, R29 and Dd2 clones) express little RIFIN, which implies that RIFINs are not essential for parasite development in in vitro environment. RIFINs are frequently found in parasites in malaria patients and the existence of specific antibodies responses to this protein family argues for their direct interaction with human immune system [38,41]. Since similar variant antigen families are also found in other plasmodial parasites such as the vir, yir and cir in P. vivax, P. yoelii and P. chabaudi (Table 1), their main function may be to contribute to the antigenic variation at different developmental stages. 4.4 The stevor gene family stevor (subtelomeric variable open reading frame) is the third variable gene family unique to P. falciparum parasite [42]. As its name implicates, stevor genes are only located in the chromosomal subtelomeric areas adjacent to rif genes. There are around 30 stevor genes in each falciparum genome. All these genes are different in sequence and there are no obvious gene duplication and recombination in stevor gene family. The gene sequences have been proposed to be more conserved among different parasite clones [43]. stevor genes have similar structures as rif genes but are slightly shorter (Fig. 1). A short Exon I encodes a potential signal peptide. The larger Exon II encodes the major part of the polypeptide which has two transmembrane regions. This indicates that the encoded protein STEVOR might form a loop anchored on a membrane structure. Like that of RIFIN, the function of STEVOR is still not known. The transcription of some stevor genes has been found to be restricted in a tight period of 22–32 h post-erythrocyte invasion [44]. Data from experiments with monoclonal antibodies and polyclonal antibodies indicated that STEVOR is transported through PMV to Maurerás clefts (MC) and is finally located at the submembrane of the pRBC [44]. It is interesting to investigate whether the three variant antigens, PfEMP1, RIFIN and STEVOR, might form a complex structure and migrate in the same pathway to the pRBC surface. Transcription and expression of STEVOR at gametocyte stage have also been confirmed. Interestingly, only a subset of stevor genes have been proposed to be activated, the messenger RNA was alternatively spliced and the protein was smaller than that expressed at trophozoite stage [45]. It has been speculated that STEVOR might mediate gametocyte adhesion in deep tissues but currently supporting data are not available [43]. STEVORs are also expressed by sporozoites and together with RIFINs they might further contribute to the antigenic diversity. The three variant gene families of P. falciparum have attracted broad scientific interests and major investigations. Understanding the biology of these gene families as well as the functions of the encoded proteins will have great implication for the development of therapeutic and preventive measures against severe malaria. 5 Evolution of virulence Evolution of virulence factors is a common microbial survival strategy. Plasmodial parasites maintain long-term chronic infection in the human host through constant antigenic variation, which is beneficial for the further transmission of the parasite via the mosquito host. In P. falciparum malaria, two factors have been attributed to parasite virulence. One is the fast growth rate [46]. Parasites causing severe malaria potentially multiply much faster than those causing mild malaria. Factors resulting in high multiplication rates are still not known. However, parasites with high proliferation capacity alone might not be able to provoke severe disease in immunocompetent hosts. The other factor attributed to P. falciparum virulence is PfEMP1 (for reviews, see [47–49]). PfEMP1 is a family of adhesive molecules which are believed to contribute to mature stage pRBC adhesion in the microvaculature. Massive local pRBC sequestration in various organs causes severe outcomes such as cerebral malaria. Parasites that fail to adhere in deep tissues are to be eliminated by the spleen. Since PfEMP1 is directly exposed to the immune system, evolution involved in this gene family will benefit parasite survival and transmission. Apart from keeping a large var gene family in each genome, gene duplications, mutations and recombinations occur rather frequently, partially due to the transition between haploid and diploid phases. Gene duplication phenomenon has been documented since the 1930s and is thought to supply raw genetic material to increase the biological evolution and diversity [50]. In the variant gene families, both in var and rif genes, gene duplications are frequently observed in some lab strains [8,29]. Four var genes in the 3D7 strain were found duplicated once or twice and at least 8 rif genes were found duplicated from one to 4 times (unpublished). Some genes are mutated after duplication or recombine with other genes in the family and a few genes are silenced after mutation. In P. falciparum genome, the number of var fragments (pseudogenes) is more prevalent than that of actual genes indicating frequent gene deletion events in the genome. Such high frequency of genetic changes implicates that the gene products are very essential for parasite survival. However, due to the restriction of host receptors, parasites need to maintain trails of stability in terms of structure and functionality. For example, most CIDR domains encoded in the genome bind to CD36 receptor though the degree of sequence diversity in the region is very high. In holoendemic areas, a small number of children below five years of age develop severe/cerebral malaria after infection. The PfEMP1 types expressed by the parasites causing severe malaria are rarely found but commonly recognized by immune sera [51–54]. Furthermore, anti-severe malaria immunity is correlated with specific antibodies recognising these PfEMP1 variants. The data suggest that parasites in naïve hosts or individuals with premature immunity preferentially express a subset of var genes, speculated to be of a more conserved nature [52,54]. However, supportive data are missing, and the gene type or the sequence has yet to be identified from patients suffering from severe malaria. Fingerprinting these genes is obviously important for the development of anti-severe malaria vaccine. An interesting question is why the parasites keep a set of genes which can potentially kill their hosts, hence having a suicidal outcome for the parasite itself. One explanation is that the genes are physically adjacent to the chromosome centre where the occurrence of genetic alterations is rare [7,9]. The PfEMP1s encoded by these genes are more adhesive to human receptors [55], and unpublished. Preliminary data indicate that these genes are frequently activated [10]. This may explain why the PfEMP1s expressed by virulent parasites are broadly recognized. Another possibility is the genomic evolution. Due to the frequent genome-wide recombination and mutations, genes encoding more virulent (adhesive) types of PfEMP1s will be generated but in lower frequency. There is, however, no data supporting these hypotheses. Cloning and comparison analysis of the var genes expressed by the most virulent parasites from cerebral malaria patients are important steps to answer these questions. 6 The malaria pathogenesis Severe manifestations and complications due to P. falciparum malaria include a range of clinical features such as cerebral malaria, severe anaemia, severe respiratory distress, hypoglycaemia, renal failure and pulmonary oedema. Cerebral malaria and severe anaemia are, however, the most common causes of hospitalization and death, especially in malaria naïve individuals [56]. Cerebral malaria patients progressively develop coma and unconsciousness. Microvasculature occlusion by clumps of pRBCs, RBC-pRBC rosettes and other fibrillar materials are believed to be the direct causes. Parasite factors such as glycosylphosphatidylinositol (GPI)-anchored elements stimulate the production of TNF-α and IFN-γ which in turn up-regulate the expression and re-localization of endothelial receptors such as ICAM-1, PECAM-1/CD31. Hence, mature stage parasites expressing adhesins, such as PfEMP1, on the pRBC surface can adhere to the endothelium interacting with the upregulated receptors. In vitro studies have identified a range of host receptors binding to pRBCs. Different domains of PfEMP1 seem to have diverse binding affinities to different endothelial receptors (Table 2). Table 2 Host receptors and the adhesive PfEMP1 domains Host receptor  Receptor location  Parasite ligand  References  HS-like GAGs  RBC  DBL1α  [34]  CR1 (CD35)  RBC  DBL1α  [100]  Blood group antigen A  RBC  DBL1α  [101]  HS  Endothelium  DBL1α  [58]  CSA  Endothelium, syncytiotrophoblast  DBL3γ  [74,75]  HA  Syncytiotrophoblast  PfEMP1  [72]  PECAM-1/CD31  Endothelium  DBL2δ  [55,88]  ICAM-1  Endothelium  DBL2βC2  [87]  CD36  Endothelium, platelet  CIDR1α  [81,82]  TSP  Endothelium, serum  PfEMP1  [129]  VCAM-1  Endothelium  ?  [130]  E-selectin  Endothelium  ?  [130]  Non-immune Ig  Serum, syncytiotrophoblast, B-cell  CIDR1α, DBL2β  [55,73]  Host receptor  Receptor location  Parasite ligand  References  HS-like GAGs  RBC  DBL1α  [34]  CR1 (CD35)  RBC  DBL1α  [100]  Blood group antigen A  RBC  DBL1α  [101]  HS  Endothelium  DBL1α  [58]  CSA  Endothelium, syncytiotrophoblast  DBL3γ  [74,75]  HA  Syncytiotrophoblast  PfEMP1  [72]  PECAM-1/CD31  Endothelium  DBL2δ  [55,88]  ICAM-1  Endothelium  DBL2βC2  [87]  CD36  Endothelium, platelet  CIDR1α  [81,82]  TSP  Endothelium, serum  PfEMP1  [129]  VCAM-1  Endothelium  ?  [130]  E-selectin  Endothelium  ?  [130]  Non-immune Ig  Serum, syncytiotrophoblast, B-cell  CIDR1α, DBL2β  [55,73]  View Large Table 2 Host receptors and the adhesive PfEMP1 domains Host receptor  Receptor location  Parasite ligand  References  HS-like GAGs  RBC  DBL1α  [34]  CR1 (CD35)  RBC  DBL1α  [100]  Blood group antigen A  RBC  DBL1α  [101]  HS  Endothelium  DBL1α  [58]  CSA  Endothelium, syncytiotrophoblast  DBL3γ  [74,75]  HA  Syncytiotrophoblast  PfEMP1  [72]  PECAM-1/CD31  Endothelium  DBL2δ  [55,88]  ICAM-1  Endothelium  DBL2βC2  [87]  CD36  Endothelium, platelet  CIDR1α  [81,82]  TSP  Endothelium, serum  PfEMP1  [129]  VCAM-1  Endothelium  ?  [130]  E-selectin  Endothelium  ?  [130]  Non-immune Ig  Serum, syncytiotrophoblast, B-cell  CIDR1α, DBL2β  [55,73]  Host receptor  Receptor location  Parasite ligand  References  HS-like GAGs  RBC  DBL1α  [34]  CR1 (CD35)  RBC  DBL1α  [100]  Blood group antigen A  RBC  DBL1α  [101]  HS  Endothelium  DBL1α  [58]  CSA  Endothelium, syncytiotrophoblast  DBL3γ  [74,75]  HA  Syncytiotrophoblast  PfEMP1  [72]  PECAM-1/CD31  Endothelium  DBL2δ  [55,88]  ICAM-1  Endothelium  DBL2βC2  [87]  CD36  Endothelium, platelet  CIDR1α  [81,82]  TSP  Endothelium, serum  PfEMP1  [129]  VCAM-1  Endothelium  ?  [130]  E-selectin  Endothelium  ?  [130]  Non-immune Ig  Serum, syncytiotrophoblast, B-cell  CIDR1α, DBL2β  [55,73]  View Large 6.1 Cerebral malaria Progressing coma, unconsciousness and multiple convulsions are the common manifestations in cerebral malaria. The pathology of cerebral malaria was initially believed to be due to the occlusion of the blood flow by sequestered cells inside the microvasculatures. But little is known about the exact cause of the blood–brain barrier damage. There seems to be two phases of endothelial responses during malaria infection. At the beginning, parasite factors such as components of GPI molecules induce the pro-inflammatory reaction that leads to an up-regulation and production of cytokines such as TNF-α (for review see [48]). The pro-inflammatory cytokines induce profound alterations to the endothelial cell (EC) linings including up-regulation of endothelial receptors in the brain. ICAM-1 and PCAM-1/CD31 are the two receptors responding to this cytokine stimulation. Mature stage parasites sequester in the microvasculature via the interaction between parasite ligand such as PfEMP1 and host receptors like ICAM-1, PECAM-1/CD31 and heparin sulfate [57,58]. However, the inflammatory reaction with the migration of lymphocytes and macrophages in the sequestered loci has not been found to be a major event [59]. After the circulation of pRBCs to the microvasculature and the establishment of physical contact with EC, an endothelial stress process characterized by cell apoptosis is initiated. The damage to the ECs caused by the direct interaction between pRBC and EC is the main factor triggering the development of cerebral malaria [60,61]. Loss of cell junction proteins such as Zo-1 and occlusion seem to cause the leakage at the blood–brain barrier. Serum proteins and liquid diffuse in massive amounts into the brain tissue which leads to coma and further damage to the nervous system. Macrophage activation has been speculated to contribute to the loss of barrier proteins, however further studies are required. 6.2 Severe anaemia All children and pregnant women suffering from clinical malaria have some degree of anaemia. Only a small group of patients develop severe anaemia. Severe anaemia may occur both in patients with chronic malaria with low parasitemia and in patients with acute severe infections and high parasitemia. The underlying molecular background of the two scenarios is not clear. Nutritional deficiency is one of the factors contributing to the severity of anaemia [62]. But parasite and human host factors seem to play the major roles. Severe anaemia is mainly due to (1) parasite destruction of infected erythrocytes, (2) less production of erythrocytes and (3) enhanced clearance of both infected and non-infected RBCs by spleen and macrophages (Refs. [46,63,64] and summarized in Table 3). Parasites that proliferate fast in the blood will destroy more red blood cells. Parasites isolated from patients suffering from severe malaria have been found to proliferate much faster than those from mild malaria under in vitro culture conditions [46]. Another observation is that parasitic products such as free pigment released after schizont rupture and oxygen/nitrogen radicals released by macrophages in response to pro-inflammatory cytokine (such as TNF-α) stimulation have detrimental effect on the bone marrow resulting in decreased production of RBCs [63]. Also, in severe malaria patients, the half-life time of un-infected RBCs are shorter than that of healthy people. Acidosis and parasite-secreted antigens could cause red blood cell deformation with less deformability. Rigid RBCs are more likely recognized and destroyed by the spleen [64,65]. Further, circulating macrophages have been found to phagocytose non-infected RBCs [66]. Table 3 Factors associated with severe anaemia Bone marrow damage-less production:      1. TNF-α      2. Oxygen and nitrogen radicals      3. Pigments    Parasite growth:      Rupture of pRBCs during late late stage schizony    Spleen-mediated clearance:      1. pRBCs infected with mature stage parasites will be recognized by spleen      2. Uninfected RBCs with abnormal deformability (ageing) will be cleared from spleen    Macrophage-mediated clearance:      1. Macrophage phagocytes infected RBCs      2. Macrophage phagocytes uninfected RBCs  Bone marrow damage-less production:      1. TNF-α      2. Oxygen and nitrogen radicals      3. Pigments    Parasite growth:      Rupture of pRBCs during late late stage schizony    Spleen-mediated clearance:      1. pRBCs infected with mature stage parasites will be recognized by spleen      2. Uninfected RBCs with abnormal deformability (ageing) will be cleared from spleen    Macrophage-mediated clearance:      1. Macrophage phagocytes infected RBCs      2. Macrophage phagocytes uninfected RBCs  View Large Table 3 Factors associated with severe anaemia Bone marrow damage-less production:      1. TNF-α      2. Oxygen and nitrogen radicals      3. Pigments    Parasite growth:      Rupture of pRBCs during late late stage schizony    Spleen-mediated clearance:      1. pRBCs infected with mature stage parasites will be recognized by spleen      2. Uninfected RBCs with abnormal deformability (ageing) will be cleared from spleen    Macrophage-mediated clearance:      1. Macrophage phagocytes infected RBCs      2. Macrophage phagocytes uninfected RBCs  Bone marrow damage-less production:      1. TNF-α      2. Oxygen and nitrogen radicals      3. Pigments    Parasite growth:      Rupture of pRBCs during late late stage schizony    Spleen-mediated clearance:      1. pRBCs infected with mature stage parasites will be recognized by spleen      2. Uninfected RBCs with abnormal deformability (ageing) will be cleared from spleen    Macrophage-mediated clearance:      1. Macrophage phagocytes infected RBCs      2. Macrophage phagocytes uninfected RBCs  View Large Noteworthy is that anaemia is not always detrimental to the host. By reducing the blood viscosity, the circulation in the microvasculature might become more efficient; resulting in an increased nutrient and oxygen supply. One example supporting this hypothesis is the finding of a lower mortality rate in severely anaemic children in comparison to those with other severe syndromes [56]. 6.3 Placental malaria In malaria endemic areas primigravidae women are the prime victims of placental malaria. The disease affects both the mother and the foetus. Abortion or low birth weight is quite common though some women develop severe malaria at the same time. P. falciparum parasites have the ability to sequester in large numbers in the placenta that provides a plausible mechanism for the impairment of foetal nutrition and low birth weight of babies born to infected women. However, two recent studies from Thailand and India challenge the hypothesis of mechanical obstruction [67,68]. According to these studies P. vivax parasites seem capable of causing low birth weight without sequestration in placenta. Perhaps some inflammatory cytokines produced locally or systemically could be involved in the pathogenesis process. The P. falciparum parasites displaying affinity to placenta are antigenically distinct from those causing cerebral malaria. Antibodies from the mother seldom protect the new born from developing severe diseases. To date, available data suggest that the parasites infecting the placenta are antigenically or genetically different from those infecting non-pregnant individuals [69]. A malaria immune female will become vulnerable to placental infection during her first pregnancy. This strongly proposes that only synergic anti-malaria immunity to parasites at most developmental stages can be truly protective and an anti-trophozoite stage vaccine is very essential for preventing the development of placental malaria. The number of receptors associated with placental malaria is fewer compared to those in cerebral malaria. CSA, hyaluronic acid (HA), non-immune IgG and a few unknown receptors are the implicated candidates (Refs. [70–73] and Table 2). CSA is the most prevalent receptor for pregnancy malaria as suggested by several studies [70,71]. Three PfEMP1 molecules from three parasite lines have been implicated for placental binding with specificity to CSA or human non-immune immunoglobulin [73–75]. Though the CSA-binding domain has been mapped to DBLγ in the two PfEMP1s published by both Reeder et al. [74] and Buffet et al. [75], their implication in placenta binding has been challenged by recent findings. The FCR3CSA type PfEMP1 mRNA or transcripts do not seem to be restricted to the placental isolates [33,76,77]. Furthermore, Northern blot and real-time RT-PCR analyses have revealed that the transcription of FCR3CSA type mRNA is not up-regulated by panning or selecting of parasites on CSA [25,78]. The data from Salanti et al. [25] suggest that an unusual var gene-encoded PfEMP1 might be responsible for CSA binding in placental malaria. The var gene in 3D7 strain was located in chromosome 12 and a pseudogene of similar sequence in chromosome 13. The PfEMP1 encoded by this var gene is composed of domain sequences which do not fit into any DBL groups. The sequence is phylogenetically different from other var genes. It is up-regulated in parasites selected for CSA binding. So far it is not known whether and when the gene is expressed into protein. It is also not clear whether most placental isolates express this gene. On the other hand, the data from Lekana Douki et al. [79] are rather convincing. A panel of monoclonal Abs could specifically precipitate a surface labelled polypeptide from parasites binding to CSA. But, as the authors implied, the mAbs recognise conformational epitopes of the PfEMP1 expressed by the placental-binding parasites. Thus it is still possible that parasites expressing similar PfEMP1s can be recognised by the same mAbs. To solve all these controversies, proteomic analysis with the CSA-selected parasites seems very necessary. Anti-placental malaria vaccine has been predicted as the easiest vaccine to develop due to the fact that the surface ligand is presumably conserved. But this will only be possible when the correct ligand is identified. 7 Phenotypes associated with severe malaria 7.1 Adhesion to endothelial receptors To date, more than 10 endothelial receptors have been identified for P. falciparum pRBC adhesion (Table 2). But it is still not clear which receptors are involved in cerebral malaria. CD36 is the most common receptor found so far. Most laboratory strains and wild isolates can bind to this receptor. But this receptor is constitutively present in the microvasculatures and its expression is not affected by inflammatory cytokines such as TNF-α. The distribution of this receptor on endothelial surfaces in the brain is marginal and its contribution to cerebral malaria is not clear. However, one recent study has suggested that platelets could function as a bridge between pRBC and endothelial receptors. PRBCs could interact with CD36 on platelets, which have receptors on the endothelial surfaces [80]. But this finding cannot explain why only a small group of parasites can cause cerebral malaria if most parasites bind to CD36. The molecular basis for CD36 binding is uniformly targeted to a cysteine-rich motif in the CIDR1α domain of PfEMP1 family [81,82]. Interestingly, most CIDR1α domains of PfEMP1s encoded in the 3D7 P. falciparum genome bind to this receptor tested in in vitro assays [83]. ICAM-1 is an endothelial receptor which has been implicated in cerebral malaria [84]. This receptor is present on most microvasculature surfaces and is up-regulated by TNF-α and IFN-γ. However, in vitro studies have demonstrated that the affinity of most pRBCs to ICAM-1 is weak and synergic cooperation with other receptors might be necessary for a stable adhesion [85,86]. DBLβC2 domain of a PfEMP1 has been mapped as an ICAM-1-binding domain [87]. In 3D7 clone, the number of PfEMP1s with potential ICAM-1-binding domains is quite few. One could speculate that only a limited number of parasites can bind to this receptor. Whether these parasites only contribute to cerebral malaria remains to be further studied. Platelet-endothelial cell adhesion molecule 1 (PECAM-1/CD31) was recently identified as yet another endothelial receptor involved in P. falciparum pRBC adhesion [88]. CD31 belongs to the immunoglobulin superfamily and is located in the junction of endothelial cells. Increased expression and re-distribution of the receptor has been seen after IFN-γ stimulation [88]. Clinical investigation in Kenya has revealed an association between pRBC binding to this receptor and severe disease [89]. Sequence polymorphism has been observed in CD31 genes from people living in different malaria endemic areas. The significance of these polymorphisms in term of sensitivity to severe disease has not been conclusive [90,91]. One DBL2δ domain from FCR3S1.2 PfEMP1 has been suggested to bind CD31 [55]. Other parasite-derived molecules might also mediate interaction with this receptor due to the fact that trypsinization could not completely abolish the binding of FCR3S1.2 to CD31 [38]. 7.2 Rosetting and autoagglutination Rosetting is a phenomenon where two or more uninfected RBCs bind to an infected RBC. Autoagglutination refers to the aggregation of infected red blood cells in nonimmune serum. Both phenomena have been found associated with severe diseases. All Plasmodium species studied so far can form rosettes (reviewed in [92]). But most studies have focused on P. falciparum rosetting. More than 80% of the clinical isolates examined so far display different degree of rosette-formation capacity when grown in vitro. Four independent studies in Africa have suggested that rosetting is associated with severe malaria since parasites from severe malaria patients form rosettes at a significantly higher rate than that from mild malaria patients [89,93–95]. These findings indicated that parasites causing severe diseases are phenotypically different from those causing mild diseases. However, studies in Papua New Guinea and some areas in Africa failed to correlate rosetting capability and disease severity [96,97]. Human genetic factors might be the reason for the discrepancy between these studies [98]. Rosetting receptors are diverse. Heparan sulfate-like glycosaminoglycans, ABO blood group antigens and complement receptor 1 (CR1) on human erythrocyte surface have been characterised as rosetting receptors (Refs. [34,55,99,100] and Table 2). However, stable rosettes require participation of multiple serum components such as non-immune human immunoglobulins (IgG and IgM) as well as other serum proteins [101,102]. DBL1α domains from two PfEMP1s expressed by two parasite clones (R29 and FCR3S1.2) have been characterized as rosetting ligands [34,55,100]. Due to human genetic diversity, the number of rosetting ligands could be more. Some P. falciparum strains form autoagglutinates in in vitro culture, a phenomenon where several infected RBCs aggregate together. Serum proteins and parasite-derived ligand (most likely PfEMP1) are thought to mediate the binding though there is no clear clue to this phenomenon. Autoagglutinates are distinct from parasite aggregates (agglutination) mediated by immune sera reflecting an anti-parasite immune response. In in vitro condition, the tendency of forming autoagglutinates is higher when the parasitemia is high though some parasites form autoagglutinates at lower parasitemia as well (for review, see [92]). 7.3 Clumps formation and platelet activation Another phenomenon termed clumping was reported recently by Pain et al. [103]. When platelets were added to some parasite cultures, clumps of only infected RBCs were observed. The phenomenon was obviously associated with the CD36 receptor on platelets. But the parasite ligand is not clear. CD36-binding parasites do not necessarily form clumps. It has been suggested that parasite ability to form clumps is associated with disease severity, but examination of biopsies from malaria patients has revealed that platelet accumulation in cerebral malaria is not always found together with pRBCs [104]. In severe malaria, platelet activation in the microvasculature seems to be common, and there are observations that platelet alone can sequester in the microvasculature following an induction by TNF-α and IFN-γ[105]. 7.4 Multiple adhesions Studies on clinical isolates directly from malaria patients have found that parasites from those suffering from severe diseases were more adhesive and could bind to several human receptors [89]. Though there are relatively few studies in this perspective, multi-adhesion phenotype could be an important feature of virulent parasites. There are several reasons that a parasite is multi-adhesive. Human genetic diversity is obviously an important factor (see next section). To be able to sequester in human hosts of different genetic background, the parasites might need to have affinity to several receptors simultaneously. If the parasites could only adhere to one receptor, they may be eliminated soon due to the poor affinity to that receptor in certain distinct genetic host populations. Having a big arsenal of adhesive protein family in the genome is certainly important, but to be able to adhere simultaneously to several receptors is of revolutionary advantage to the parasites assuring their survival in the hosts. One example is the finding that rosetting parasites are more frequently found in patients of severe malaria and most rosetting parasites are indeed multiple adhesive. At least one of the molecular determinants of the multiple adhesion phenotype is PfEMP1 expressed on the pRBC surface. One PfEMP1 domain can bind to similar receptor on different cell surface. For instance, the rosetting domain of FCR3S1.2 PfEMP1 has been shown to be able to bind to HS-like glycosaminoglycans on erythrocyte and HS on endothelium [55,58]. The CIDR1α domain of A4var PfEMP1 can bind to CD36 on endothelial cell, dendritic cell and platelet surface [103,106,107]. Further, domains of a PfEMP1 can bind simultaneously to different receptors. CIDR1α and DBL2δ domains of FCR3S1.2 PfEMP1 could bind to CD36 and CD31, respectively [55], while CIDR1α and DBL2β C2 of A4var PfEMP1 bind CD36 and ICAM-1 [87,106]. To date, multiple adhesion is not necessarily a phenotype of every parasite, but the ability to be able to bind to several human receptors certainly give the parasites a possibility to hide in the postcapillary regions where they can escape from clearance by the spleen before initiating next erythrocytic cycle. 8 Human genetic factors Malaria infections have varying outcome in different individuals. While some become severely ill, others recover without any complications. Human genetic factors account partly for these variations. Several susceptibility and resistance determinants involving polymorphisms of erythrocytes, endothelial receptors or the immune system have already been defined. In many studies red blood cell polymorphisms (e.g. ABO blood group, sickle cell trait, G6PD deficiency, ovalocytosis) have been proposed as protective factors against severe malaria (for reviews, see [108,109]). More recently polymorphisms of components of the inflammatory response have been investigated, and among them, polymorphisms in the promoter region of the TNF-α gene are the most widely studied [110–113]. Results from different study sites have varied but in all studies polymorphisms in the promoter region of TNF-α seem to be associated with severe malarial disease. In one of the recent studies, undertaken in Western Kenya, an association between a TNF-α polymorphism (TNF-2 homozygosity) and premature birth was shown, and among those born prematurely, this allele was associated with higher mortality [110]. Assuming that this polymorphism causes an increased TNF-α production, one of the mechanisms by which higher levels of TNF-α could accelerate pre-term labour is via the up-regulation of cyclooxygenase (COX)-2 enzyme-mediated prostaglandin production [114,115]. In addition, TNF-α is known to enhance the matrix metalloproteinase production resulting in collagen degradation of foetal membranes and maternal tissues, which in turn may lead to premature rupture of the membranes and cervical dilation [116]. However, these explanations are not established facts and the underlying mechanisms for the observed associations require further in-depth investigations. The genetic polymorphism of human immune systems is believed to be directly associated with susceptibility to malaria infections (for review, see [117,118]). HLA genotypes have been extensively investigated and trials of HLA genotypes being associated with susceptibility (HLA-B*5301 type) or resistance (HLA-DRB1*0101 type) have been reported [118,119]. In a recent study, it was found that the genotype of the NK cell receptor (the killer Ig-like receptor, KIR) was at least one of the determinants involved in the interaction between NK cell and P. falciparum-infected erythrocytes and the secretion of IFN-γ[120]. However, the study on the genetic variation of KIR receptor in malaria endemic areas requires further investigation. Discovering the immunogenetic factors associated with malaria disease is important for understanding the pathogenesis and for rational design of malaria vaccines with higher affinity to MHC molecules. The polymorphism of endothelial receptors associated with increased susceptibility to severe malaria has recently been described. A high-frequency coding polymorphism in the N-terminal domain of ICAM-1 was found to be associated with susceptibility to cerebral malaria in Kenya but not in the Gambia [121,122]. Malaria has played a major role in selecting and maintaining protective polymorphism in endemic regions. Since cytoadhesion of pRBCs have been associated with severe disease and ICAM-1 has been implicated as one of the important receptors involved in these interactions one would assume that protective mutations in the N-terminal domain of ICAM-1, which also contains the binding site for pRBCs, could occur in malaria-endemic regions. However this was not the case in the Kenyan study, where individuals with the mutation (ICAM-1Kilifi) were more at risk for cerebral malaria. In order to understand how such polymorphism can be maintained and why it has differential effect on malaria susceptibility in different African populations Craig et al. and Adams et al. address the issue by investigating the binding properties of two different P. falciparum lines, ITO4-A4 (A4) and ItG-ICAM, to the two allelic forms of ICAM-1, the mutated (ICAM-1Kilifi) and the reference (ICAM-1Ref) ICAM-1 proteins [123,124]. A4 and ItG parasites are low and high ICAM-1 binders respectively. In summary, the authors demonstrate that the total adhesion of ItG-infected RBCs to the two ICAM-1s is greater than that of A4-infected RBCs. Also pRBCs from both lines show reduced binding to ICAM-1Kilifi. However, under flow conditions the dynamic rolling of the pRBCs on ICAM-1Kilifi differs between the strains. The percentage and the velocity of A4-infected RBCs rolling on ICAM-1Kilifi are markedly higher compared to that on ICAM-1Ref whereas the rolling velocity of ItG-infected RBCs is similar on both proteins. How a decreased binding to ICAM-1Kilifi could be consistent with increased risk of cerebral malaria in ICAM-1Kilifi homozygotes is not clear. Although simplified, one could hypothesize that this allele was originally selected for as a protective polymorphism via reduced binding of pRBCs to ICAM-1, thus reducing the risk for cerebral malaria. However, due to evolutionary process between the host and the parasite some compensatory mutations in PfEMP-1 could have been selected in parasites for high avidity ICAM-1 binding in high transmission areas, such as Kenya. Hence, the ICAM-1Kilifi allele has become a cerebral malaria susceptibility determinant. But, in low transmission areas, such as The Gambia, the allele may still be protective. However, ICAM-1 was analysed as a single receptor in those studies. The parasites might be able to bind to other receptors as well. The genetic determimants in this aspect are far from clear. Furthermore, reports on malaria susceptibility in different ethnic groups in Burkina Faso, West Africa have revealed that the Fulani population is less susceptible and more immunologically responsive to malaria than their neighbouring ethnic groups [125–128]. According to former reports this is not due to malaria exposure or socio-cultural circumstances [125,126]. Neither does it seem to involve other known genetic determinants. Recently, Luoni et al. [125] reported that an IL-4 polymorphism occurs at high frequency in the Fulani, which seem to be associated with the elevated anti-malarial antibody levels. To date, many genetic factors have been defined and it is likely that many more have yet to be discovered. Efforts at present are aimed at understanding the functional basis of known associations. For optimal design of any drug or vaccine, disease associated host genetic factors have to be taken into consideration. It is thus important to further elucidate the importance of these factors in disease and health and to understand the underlying mechanisms. 9 Concluding remarks Disease severity in P. falciparum infections is partly dependent on host genetic factors and to a large extent on the parasite and its efficient immuno-evasive mechanisms. The severe symptoms of malaria are manifested in the erythrocytic stage of the parasite life cycle through the process of sequestration. Instrumental in this process is the expression of parasite-derived proteins on the pRBC surface which promote cytoadhesion and rosetting. Disruption of these adhesive events should therefore prevent the pathology of severe disease. Thus, identifying the proteins involved in these events is fruitful in the development of anti-severe malaria interventions. The most challenging obstacle is, however, the antigenic diversity of malaria antigens. As discussed earlier, all the surface exposed antigens (including antigens on the free sporozoite and merozoite surface) display certain degree of polymorphism. In terms of vaccine development, sterile immunity is an unachievable goal. However, the molecule types which cause both placental and cerebral malaria are rare. Hence development of anti-severe disease interventions could be possible if there only exist a few PfEMP1 types, which are truly associated with the diseases. Currently, we have no optimal strategy to sustainably reduce or eliminate the burden of malarial disease. There is no vaccine and with a worrisome ever-increasing drug resistance our chances to win the battle against malaria are diminishing. Nevertheless, we should be optimistic. We have now access to genome sequences, bioinformatic tools and high-throughput technologies which will ultimately provide an integrated picture of parasite biology and malaria pathogenesis and hopefully facilitate the target identification for therapeutic interventions. Analysis of the genome and the assessment of expression profiles at different stages of parasite life cycle have already amassed a great body of information. However, expression profiles at the mRNA level only will not provide a comprehensive picture. Immune responses recognize proteins not mRNA, and in many instances, such as alternative splice sites or post-translational modifications, mRNA transcription data are not adequate. Thus a confirmation of the gene expression profiles at the protein level is fundamental. Advances in proteomics together with recent developments in gene-targeting technologies will therefore be crucial for the final identification of the most promising drug targets and vaccine candidates. 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TI - Molecular aspects of malaria pathogenesis
JF - Journal of the Endocrine Society
DO - 10.1016/j.femsim.2004.01.010
DA - 2004-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/molecular-aspects-of-malaria-pathogenesis-PL5uRspNto
SP - 9
EP - 26
VL - 41
IS - 1
DP - DeepDyve
ER -