Tomasino Pace

Egress of Plasmodium berghei gametes from their host erythrocyte is mediated by the MDV-1/PEG3 protein

Malaria parasites invade erythrocytes of their host both for asexual multiplication and for differentiation to male and female gametocytes – the precursor cells of Plasmodium gametes. For further development the parasite is dependent on efficient release of the asexual daughter cells and of the gametes from the host erythrocyte. How malarial parasites exit their host cells remains largely unknown. We here report the characterization of a Plasmodium berghei protein that is involved in egress of both male and female gametes from the host erythrocyte. Protein MDV‐1/PEG3, like its Plasmodium falciparum orthologue, is present in gametocytes of both sexes, but more abundant in the female, where it is associated with dense granular organelles, the osmiophilic bodies. Δmdv‐1/peg3 parasites in which MDV‐1/PEG3 production was abolished by gene disruption had a strongly reduced capacity to form zygotes resulting from a reduced capability of both the male and female gametes to disrupt the surrounding parasitophorous vacuole and to egress from the host erythrocyte. These data demonstrate that emergence from the host cell of male and female gametes relies on a common, MDV‐1/PEG3‐dependent mechanism that is distinct from mechanisms used by asexual parasites.

Generation of chromosome size polymorphism during in vivo mitotic multiplication of Plasmodium berghei involves both loss and addition of subtelomeric repeat sequences.

Extensive chromosome size polymorphism arises in Plasmodium berghei during in vivo mitotic multiplication. Size differences between homologous chromosomes involve rearrangements occurring in the subtelomeric portions while internal chromosomal regions do not contribute significantly to chromosome size polymorphism. Differences in the copy number of a 2.3-kb subtelomeric repeated unit are shown to correlate with size variations, and in at least one case to account completely for the size difference between two variants of the same chromosome.

Set regulation in asexual and sexual Plasmodium parasites reveals a novel mechanism of stage‐specific expression

Transmission of the malaria parasite depends on specialized gamete precursors (gametocytes) that develop in the bloodstream of a vertebrate host. Gametocyte/gamete differentiation requires controlled patterns of gene expression and regulation not only of stage and gender‐specific genes but also of genes associated with DNA replication and mitosis. Once taken up by mosquito, male gametocytes undergo three mitotic cycles within few minutes to produce eight motile gametes. Here we analysed, in two Plasmodium species, the expression of SET, a conserved nuclear protein involved in chromatin dynamics. SET is expressed in both asexual and sexual blood stages but strongly accumulates in male gametocytes. We demonstrated functionally the presence of two distinct promoters upstream of the set open reading frame, the one active in all blood stage parasites while the other active only in gametocytes and in a fraction of schizonts possibly committed to sexual differentiation. In ookinetes both promoters exhibit a basal activity, while in the oocysts the gametocyte‐specific promoter is silent and the reporter gene is only transcribed from the constitutive promoter. This transcriptional control, described for the first time in Plasmodium, provides a mechanism by which single‐copy genes can be differently modulated during parasite development. In male gametocytes an overexpression of SET might contribute to a prompt entry and execution of S/M phases within mosquito vector.

A gene-family encoding small exported proteins is conserved across Plasmodium genus

A gene-family, named sep, encoding small exported proteins conserved across Plasmodium species has been identified. SEP proteins (13-16 kDa) contain a predicted signal peptide at the NH(2)-terminus, an internal hydrophobic region and a polymorphic, low-complexity region at the carboxy-terminus. One member of the Plasmodium berghei family, Pbsep1, encodes an integral membrane protein expressed along the entire erythrocytic cycle. Immunolocalisation results indicated that PbSEP1 is targeted to the membrane of the parasitophorous vacuole up to the early phases of schizogony, while, in late schizonts, it re-locates in structures within the syncitium. After erythrocyte rupture, PbSEP1 is still detectable in free merozoites thus suggesting its involvement in the early steps of parasite invasion. Seven members of the sep-family in Plasmodium falciparum have been identified. Two of them correspond to previously reported gene sequences included in a family of early transcribed membrane proteins (etramp). Structural, functional and phylogenetic features of the sep family, shown in the present work, supercede this previous classification. PfSEP proteins are exported beyond the parasite membrane and translocated, early after invasion, to the host cell compartment in association with vesicle-like structures. Colocalisation results indicated that PfSEP-specific fluorescence overlaps, at the stage of trophozoite, with that of Pf332, a protein associated with Maurers clefts, membranous structures in the cytosol of parasitised red blood cells, most probably involved in trafficking of parasite proteins. The specific signals necessary to direct SEP proteins to the vacuolar membrane in P. berghei or to the host cell compartment in P. falciparum remain to be determined.

Organization of subtelomeric repeats in Plasmodium berghei.

Several (but not all) Plasmodium berghei chromosomes bear in the subtelomeric position a cluster of 2.3-kilobase (kb) tandem repeats. The 2.3-kb unit contains 160 base pairs of telomeric sequence. The resulting subtelomeric structure is one in which stretches of telomeric sequences are periodically spaced by a 2.1-kb reiterated sequence. This periodic organization of internal telomeric sequences might be related to chromosome-size polymorphisms involving the loss or addition of subtelomeric 2.3-kb units.

Plasmodium lipid rafts contain proteins implicated in vesicular trafficking and signalling as well as members of the PIR superfamily, potentially implicated in host immune system interactions

Plasmodium parasites, the causal agents of malaria, dramatically modify the infected erythrocyte by exporting parasite proteins into one or multiple erythrocyte compartments, the cytoplasm and the plasma membrane or beyond. Despite advances in defining signals and specific cellular compartments implicated in protein trafficking in Plasmodium‐infected erythrocytes, the contribution of lipid‐mediated sorting to this cellular process has been poorly investigated. In this study, we examined the proteome of cholesterol‐rich membrane microdomains or lipid rafts, purified from erythrocytes infected by the rodent parasite Plasmodium berghei. Besides structural proteins associated with invasive forms, we detected chaperones, proteins implicated in vesicular trafficking, membrane fusion events and signalling. Interestingly, the raft proteome of mixed P. berghei blood stages included proteins encoded by members of a large family (bir) of putative variant antigens potentially implicated in host immune system interactions and targeted to the surface of the host erythrocytes. The generation of transgenic parasites expressing BIR/GFP fusions confirmed the dynamic association of members of this protein family with membrane microdomains. Our results indicated that lipid rafts in Plasmodium‐infected erythrocytes might constitute a route to sort and fold parasite proteins directed to various host cell compartments including the cell surface.

Telomeric motifs are present in a highly repetitive element in the Plasmodium berghei genome

Using as probes the subfragments of the telomeric sequence previously cloned by us from Plasmodium berghei DNA, we identified and cloned a 2.3 kb repeat, largely overlapping the original telomeric insert. Restriction mapping indicated that cloned inserts (2.3 kb in length) represented circularly permutated versions of a rather well conserved repeated element, at least in part organized in tandem. The 2.3 kb repeat family with a copy number of about 300 occupies about 4% of the whole genome. The copies are unevenly distributed among the chromosome-sized molecules revealed by pulsed field gradient electrophoresis. Complete sequence determination of the 2.3 kb element revealed that telomere-related motifs are present with a characteristic pattern in a set of tandem repeats, 27 bp long. The perfect conservation of these motifs as well as the pattern of chromosomal distribution suggest that we are dealing with a specialised structure subject to selective mechanisms of amplification and maintenance.

Long insertions within telomeres contribute to chromosome size polymorphism in Plasmodium berghei.

During prolonged in vivo mitotic multiplication of a Plasmodium berghei ANKA clone (8417HP), parasites that contained an enlarged version of chromosome 4 were observed. Restriction mapping and hybridization results demonstrated that the extra DNA present in the enlarged chromosome consists of 2.3-kb tandem repeats, known to be normally located in subtelomeric position at several chromosomal ends but absent in the original chromosome. The inserted 2.3-kb units appeared to interrupt one of the original telomeres and to create an internal (approximately 1-kb-long) telomeric sequence.

Homologous telomeric sequences are present in different species of the genus Plasmodium

The telomeric sequence cloned from Plasmodium berghei (see M. Ponzi et al. (1985) EMBO J. 4, 2991-2995) was tested for species specificity. A telomeric and a subtelomeric fragment of the cloned insert served as separate, labelled probes on pulsed field gradient electrophoretical patterns and on genomic digests from the rodent malarias Plasmodium yoelii, Plasmodium chabaudi and from the human malaria Plasmodium falciparum. Results indicate that the subtelomeric fragment, abundantly represented in two chromosomes of P. berghei, is not present in the other DNA tested, while the telomeric fragment is present in every chromosome-sized molecule in all the species tested. The telomeric location in the other genomes of the sequences homologous to the P. berghei telomeric probe is confirmed by experiments with Bal 31 exonuclease. In all cases, the TaqI site appears to delimit the common telomeric portion.

Erythrocyte Remodeling in Plasmodium berghei Infection: The Contribution of SEP Family Members

The malaria parasite Plasmodium largely modifies the infected erythrocyte through the export of proteins to multiple sites within the host cell. This remodeling is crucial for pathology and translocation of virulence factors to the erythrocyte surface. In this study, we investigated localization and export of small exported proteins/early transcribed membrane proteins (SEP/ETRAMPs), conserved within Plasmodium genus. This protein family is characterized by a predicted signal peptide, a short lysine‐rich stretch, an internal transmembrane domain and a highly charged C‐terminal region of variable length. We show here that members of the rodent Plasmodium berghei family are components of the parasitophorous vacuole membrane (PVM), which surrounds the parasite throughout the erythrocytic cycle. During P. berghei development, vesicle‐like structures containing these proteins detach from the PVM en route to the host cytosol. These SEP‐containing vesicles remain associated with the infected erythrocyte ghosts most probably anchored to the membrane skeleton. Transgenic lines expressing the green fluorescent protein appended to different portions of sep‐coding region allowed us to define motifs required for protein export. The highly charged terminal region appears to be involved in protein–protein interactions.