P25 and P28 proteins of the malaria ookinete surface have multiple and partially redundant functions.

The ookinete surface proteins (P25 and P28) are proven antimalarial transmission-blocking vaccine targets, yet their biological functions are unknown. By using single (Sko) and double gene knock-out (Dko) Plasmodium berghei parasites, we show that P25 and P28 share multiple functions during ookinete/oocyst development. In the midgut of mosquitoes, the formation of ookinetes lacking both proteins (Dko parasites) is significantly inhibited due to decreased protection against lethal factors, including protease attack. In addition, Dko ookinetes have a much reduced capacity to traverse the midgut epithelium and to transform into the oocyst stage. P25 and P28 are partially redundant in these functions, since the efficiency of ookinete/oocyst development is only mildly compromised in parasites lacking either P25 or P28 (Sko parasites) compared with that of Dko parasites. The fact that Sko parasites are efficiently transmitted by the mosquito is a compelling reason for including both target antigens in transmission-blocking vaccines.

Functional equivalence of structurally distinct ribosomes in the malaria parasite, Plasmodium berghei.

Unlike most eukaryotes, many apicomplexan parasites contain only a few unlinked copies of ribosomal RNA (rRNA) genes. Based on stage-specific expression of these genes and structural differences among the rRNA molecules it has been suggested that Plasmodium spp. produce functionally different ribosomes in different developmental stages. This hypothesis was investigated through comparison of the structure of the large subunit rRNA molecules of the rodent malaria parasite, Plasmodium berghei, and by disruption of both of the rRNA gene units that are transcribed exclusively during development of this parasite in the mosquito (S-type rRNA gene units). In contrast to the human parasite, Plasmodium falciparum, we did not find evidence of structural differences in core regions of the distinct large subunit rRNAs which are known to be associated with catalytic activity including the GTPase site that varies in P. falciparum. Knockout P. berghei parasites lacking either of the S-type gene units were able to complete development in both the vertebrate and mosquito hosts. These results formally exclude the hypothesis that two functionally different ribosome types distinct from the predominantly blood stage-expressed A-type ribosomes, are required for development of all Plasmodium species in the mosquito. The maintenance of two functionally equivalent rRNA genes might now be explained as a gene dosage phenomenon.

Distinct roles for pbs21 and pbs25 in the in vitro ookinete to oocyst transformation of Plasmodium berghei.

We have developed an in vitro culture system for early sporogonic stages of Plasmodium berghei, which can be used to study developmental events normally taking place in the midgut of an infected mosquito. These include penetration of insect cells by the mature ookinete, transformation into oocysts and the early development of the latter, sustained through several rounds of nuclear division. The system, based upon co-culture of enriched ookinetes with several established insect cell lines, was used to study the development of mutant ookinetes lacking both the Pbs21 and Pbs25 surface proteins. Motility and entry of double knockout and Pbs21 single knockout ookinetes into the insect cells are normal, but the number of ookinetes successfully transforming into oocysts expressing the CSP protein are substantially reduced. Finally, using the yeast two-hybrid system we also show that Pbs25 has the capacity to homodimerise as well as to form heterodimers with Pbs21.

CTRP is essential for mosquito infection by malaria ookinetes.

The malaria parasite suffers severe population losses as it passes through its mosquito vector. Contributing factors are the essential but highly constrained developmental transitions that the parasite undergoes in the mosquito midgut, combined with the invasion of the midgut epithelium by the malaria ookinete (recently described as a principal elicitor of the innate immune response in the Plasmodium-infected insect). Little is known about the molecular organization of these midgut-stage parasites and their critical interactions with the blood meal and the mosquito vector. Elucidation of these molecules and interactions will open up new avenues for chemotherapeutic and immunological attack of parasite development. Here, using the rodent malaria parasite Plasmodium berghei, we identify and characterize the first microneme protein of the ookinete: circumsporozoite- and TRAP-related protein (CTRP). We show that transgenic parasites in which the CTRP gene is disrupted form ookinetes that have reduced motility, fail to invade the midgut epithelium, do not trigger the mosquito immune response, and do not develop further into oocysts. Thus, CTRP is the first molecule shown to be essential for ookinete infectivity and, consequently, mosquito transmission of malaria.

Isolation and characterization of recombinant antigens from Leishmania aethiopica that react with human antibodies.

A genomic expression library of Leishmania aethiopica was constructed in lambda gt11 and screened with patient sera and sera from healthy people living in an area of endemicity. Forty-five recombinant clones were isolated and partly characterized. Clone-specific antibodies were prepared and used with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis to estimate the molecular masses of the parasite-derived antigens containing the reactive epitope(s). Antigens with apparent molecular masses of 90, 85, 63, 50, 41, 25 and 24 kDa as well as several antigens with lower molecular masses were detected. The clone-specific antibodies from patients with diffuse cutaneous leishmaniasis reacted with high-molecular-weight antigens (30,000 less than Mr less than 90,000), whereas antibodies from patients with localized cutaneous leishmaniasis recognized low-molecular-weight antigens (Mr less than 25,000). Nine different purified recombinant antigens were obtained from lysogens in Escherichia coli Y1089 by immunoaffinity chromatography on anti-beta-galactosidase columns and were subsequently tested with patient sera. It is suggested that some of these recombinant antigens might be used for immunodiagnostic purposes.

Topography of the oxytocin receptor system in rat brain: an autoradiographical study with a selective radioiodinated oxytocin antagonist.

A new, highly selective radio-iodinated oxytocin receptor antagonist [( 1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid, 2-O-methyltyrosine, 4-threonine, 8-ornithine, 9-tyrosylamide]-vasotocin) was used to identify and quantitate specific binding sites for the neurohypophyseal hormone oxytocin with in vitro incubation of rat brain sections and autoradiography. Exclusively oxytocin binding sites were detected in view of the high affinity of the [125I]-labelled oxytocin antagonist for oxytocin binding sites and the negligible affinity for the vasopressin liver (V1) and kidney (V2) receptor types. The putative oxytocin receptors were abundantly present in several brain regions, where previously discrimination between oxytocin and vasopressin binding was difficult, i.e. the olfactory nucleus, the islands of Calleja, the ventromedial nucleus of the hypothalamus, the central amygdaloid nucleus and the ventral subiculum of the hippocampus. In addition oxytocin receptors were demonstrated in other areas, such as the taenia tecta, dorsolateral caudate putamen, ventral pallidum, accumbens, lateral septum, bed nucleus of the stria terminalis, thalamic paraventricular nucleus, lateral, basolateral and medial amygdala, the dorsal subiculum, perirhinal cortex and the amygdaloid-hippocampal area. The high affinity and the low detection threshold of this [125I]-labelled oxytocin antagonist permitted identification of oxytocin receptors in new regions such as the ventral part of the lateral septum, medial septum, dorsal motor nucleus of the vagus nerve and the olive nuclei in the brain stem.