The malaria parasite Plasmodium falciparum undergoes antigenic variation to evade host immune responses through switching expression of variant surface proteins encoded by the var gene family. We demonstrate that both a subtelomeric transgene and var genes are subject to reversible gene silencing. Var gene silencing involves the SIR complex as gene disruption of PfSIR2 results in activation of this gene family. We also demonstrate that perinuclear gene activation involves chromatin alterations and repositioning into a location that may be permissive for transcription. Together, this implies that locus repositioning and heterochromatic silencing play important roles in the epigenetic regulation of virulence genes in P. falciparum.

Mono-allelic expression of gene families is used by many organisms to mediate phenotypic variation of surface proteins. In the apicomplexan parasite Plasmodium falciparum, responsible for the severe form of malaria in humans, this is exemplified by antigenic variation of the highly polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1, encoded by the 60-member var gene family, represents a major virulence factor due to its central role in immune evasion and intravascular parasite sequestration. Mutually exclusive expression of PfEMP1 is controlled by epigenetic mechanisms involving chromatin modification and perinuclear var locus repositioning. Here we show that a var promoter mediates the nucleation and spreading of stably inherited silenced chromatin. Transcriptional activation of this promoter occurs at the nuclear periphery in association with chromosome-end clusters. Additionally, the var promoter sequence is sufficient to infiltrate a transgene into the allelic exclusion programme of var gene expression, as transcriptional activation of this transgene results in silencing of endogenous var gene transcription. These results show that a var promoter is sufficient for epigenetic silencing and mono-allelic transcription of this virulence gene family, and are fundamental for our understanding of antigenic variation in P. falciparum. Furthermore, the PfEMP1 knockdown parasites obtained in this study will be important tools to increase our understanding of P. falciparum-mediated virulence and immune evasion.

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Two chloroquine-resistant cloned isolates of Plasmodiumfalciparum were subjected to mefloquine selection to test if this resulted in alterations in chloroquine sensitivity and amplification of the pfmdrl gene. The mefloquine-resistant lines derived by this selection were shown to have amplified and overexpressed the pfmdrl gene and its protein product (Pghl).Macrorestriction maps of chromosome 5, where pfindrl is encoded, showed that this chromosome has increased in size in response to mefloquine selection, indicating the presence of a gene(s) in this area of the genome that confers a selective advantage in the presence of mefloquine. Concomitant with the increase in mefloquine resistance was a corresponding increase in the level ofresistance to halofantrine and quinine, suggesting a true multidrug-resistance phenotype. The mefloquineselected parasite lines also showed an inverse relationship between the level of chloroquine resistance and increased pfmdrl gene copy number. These results have important implications for the derivation of amplified copies of the pfmdrl gene in field isolates, as they suggest that quinine pressure may be involved.Plasmodium falciparum is the causative agent of the most severe form of human malaria, and the ability of this parasite to develop resistance to antimalarial agents, such as chloroquine, makes it difficult to select appropriate drugs for both prophylaxis and treatment. Chloroquine resistance in P. falciparum involves a decrease in chloroquine concentration in the parasite, and the rate of chloroquine efflux has been shown to be 40-to 50-fold more than in sensitive isolates (1). Other studies have suggested that decreased influx of chloroquine in resistant parasites is responsible for the phenotype (2).A P-glycoprotein homologue 1 (Pghl) encoded by the pfmdrl gene (3, 4) has been suggested to be involved in the chloroquine-resistance phenotype (5). The Pghl protein is localized on the membrane of the digestive vacuole of P. falciparum (6), and heterologous expression of this protein in Chinese hamster ovary (CHO) cells confers a chloroquinesensitive phenotype involving increased accumulation of the drug in the lysosomes (H. Van Es, S. Karcz, F. Chu, A.F.C., P. Gros, and E. Schurr, unpublished work). These results have suggested that Pghl is involved either directly or indirectly with the concentration of chloroquine in the P. falciparum food vacuole.The possible role of Pghl in concentrating chloroquine in the digestive vacuole is consistent with results of the selection for increased levels of chloroquine resistance obtained with three cloned lines of P. falciparum (7). The chloroquine pressure caused deamplification of the pfmdrl gene and decreased expression of the protein. It was also found that concomitant with the increase in chloroquine resistance was a decrease in the level of mefloquine resistance. In another study, selection for mefloquine resistance in the W2 isolate of P. falciparum showed a decrease in chloroquine resistance (8) and amplification of the...

The development of effective malaria vaccines and immune biomarkers of malaria is a high priority for malaria control and elimination. Ags expressed by merozoites of Plasmodium falciparum are likely to be important targets of human immunity and are promising vaccine candidates, but very few Ags have been studied. We developed an approach to assess Ab responses to a comprehensive repertoire of merozoite proteins and investigate whether they are targets of protective Abs. We expressed 91 recombinant proteins, located on the merozoite surface or within invasion organelles, and screened them for quality and reactivity to human Abs. Subsequently, Abs to 46 proteins were studied in a longitudinal cohort of 206 Papua New Guinean children to define Ab acquisition and associations with protective immunity. Ab responses were higher among older children and those with active parasitemia. High-level Ab responses to rhoptry and microneme proteins that function in erythrocyte invasion were identified as being most strongly associated with protective immunity compared with other Ags. Additionally, Abs to new or understudied Ags were more strongly associated with protection than were Abs to current vaccine candidates that have progressed to phase 1 or 2 vaccine trials. Combinations of Ab responses were identified that were more strongly associated with protective immunity than responses to their single-Ag components. This study identifies Ags that are likely to be key targets of protective human immunity and facilitates the prioritization of Ags for further evaluation as vaccine candidates and/or for use as biomarkers of immunity in malaria surveillance and control.

Highlights d Specific compounds against P. falciparum Plasmepsin IX and X were identified d PMIX and PMX are modulators of parasite proteins for egress, invasion, and development d Anti-PMIX and anti-PMX compounds inhibit liver, blood, and mosquito stages of Plasmodium d One compound, WM382, can clear mouse models of P. berghei and P. falciparum parasites

The EBAs appear important targets of acquired protective immunity. These findings support their further development as vaccine candidates.

Erythrocyte invasion by Plasmodium requires molecules present both on the merozoite surface and within the specialized organelles of the apical complex. The Plasmodium erythrocyte binding protein family includes the Plasmodium falciparum sialic acidbinding protein, EBA-175 (erythrocyte binding antigen-175), which binds sialic acid present on glycophorin A of human erythrocytes. We address the role of the conserved 3-cysteine rich region, the transmembrane, and cytoplasmic domains through targeted gene disruption. Truncation of EBA-175 had no measurable effect on either the level of EBA-175 protein expression or its subcellular localization. Similarly, there appears to be no impairment in the ability of soluble EBA-175 to be released into the culture supernatant after schizont rupture. Additionally, the 3-cys rich region, transmembrane, and cytoplasmic domains of EBA-175 are apparently non-essential for merozoite invasion. In contrast, erythrocyte invasion via the EBA-175͞glycophorin A route appears to have been disrupted to such a degree that the mutant lines have undergone a stable switch in invasion phenotype. As such, EBA-175 appears to have been functionally inactivated within the truncation mutants. The sialic acid-independent invasion pathway within the mutant parasites accounts for approximately 85% of invasion into normal erythrocytes. These data demonstrate the ability of P. falciparum to utilize alternate pathways for invasion of red blood cells, a property that most likely provides a substantial survival advantage in terms of overcoming host receptor heterogeneity and͞or immune pressure.