Three members of the 6-cys protein family of Plasmodium play a role in gamete fertility.

The process of fertilization is critically dependent on the mutual recognition of gametes and in Plasmodium, the male gamete surface protein P48/45 is vital to this process. This protein belongs to a family of 10 structurally related proteins, the so called 6-cys family. To identify the role of additional members of this family in Plasmodium fertilisation, we performed genetic and functional analysis on the five members of the 6-cys family that are transcribed during the gametocyte stage of P. berghei. This analysis revealed that in addition to P48/45, two members (P230 and P47) also play an essential role in the process of parasite fertilization. Mating studies between parasites lacking P230, P48/45 or P47 demonstrate that P230, like P48/45, is a male fertility factor, consistent with the previous demonstration of a protein complex containing both P48/45 and P230. In contrast, disruption of P47 results in a strong reduction of female fertility, while males remain unaffected. Further analysis revealed that gametes of mutants lacking expression of p48/45 or p230 or p47 are unable to either recognise or attach to each other. Disruption of the paralog of p230, p230p, also specifically expressed in gametocytes, had no observable effect on fertilization. These results indicate that the P. berghei 6-cys family contains a number of proteins that are either male or female specific ligands that play an important role in gamete recognition and/or attachment. The implications of low levels of fertilisation that exist even in the absence of these proteins, indicating alternative pathways of fertilisation, as well as positive selection acting on these proteins, are discussed in the context of targeting these proteins as transmission blocking vaccine candidates.

Transmission of Plasmodium falciparum in urban Yaoundé, Cameroon, is seasonal and age-dependent.

Data on malaria transmission intensity and prevalences of asexual parasites and of gametocytes were obtained in an urban district of Yaoundé, Cameroon. The transmission level from mosquito to human was determined by indoor night capture of mosquitoes on human volunteers, revealing a calculated entomological inoculation rate of 34 infectious bites per person per year. Only Anopheles gambiae and A. funestus contributed to malaria transmission and their distribution was seasonal. Cross-sectional surveys every 2 months from July 1999 to May 2000 (n = 965) showed average annual prevalences of 35% Plasmodium falciparum asexual parasites (range 29-38%) and 4.4% gametocytes (range 0-6.7%). Prevalence of high parasitaemia (> 400 parasites/microL) and of gametocytes was seasonal. Prevalence of asexual parasitaemias and of gametocytaemias was age-dependent. The potential infectious reservoir in this area is dominated by the age group 0-15 years, representing 75% of carriers of asexual parasites (P < 0.001), 85% of carriers of high parasitaemias (P < 0.001), and 83% of gametocyte carriers (P = 0.03). Full year logistic models developed from the available data accurately predicted parasite prevalences in subsequent analyses, thus permitting a precise determination of study samples for intervention and seroepidemiology studies, and analysis of the infectious reservoir in this area.

Plasmodium cysteine repeat modular proteins 1-4: complex proteins with roles throughout the malaria parasite life cycle.

The Cysteine Repeat Modular Proteins (PCRMP1-4) of Plasmodium, are encoded by a small gene family that is conserved in malaria and other Apicomplexan parasites. They are very large, predicted surface proteins with multipass transmembrane domains containing motifs that are conserved within families of cysteine-rich, predicted surface proteins in a range of unicellular eukaryotes, and a unique combination of protein-binding motifs, including a >100 kDa cysteine-rich modular region, an epidermal growth factor-like domain and a Kringle domain. PCRMP1 and 2 are expressed in life cycle stages in both the mosquito and vertebrate. They colocalize with PfEMP1 (P. falciparum Erythrocyte Membrane Antigen-1) during its export from P. falciparum blood-stage parasites and are exposed on the surface of haemolymph- and salivary gland-sporozoites in the mosquito, consistent with a role in host tissue targeting and invasion. Gene disruption of pcrmp1 and 2 in the rodent malaria model, P. berghei, demonstrated that both are essential for transmission of the parasite from the mosquito to the mouse and has established their discrete and important roles in sporozoite targeting to the mosquito salivary gland. The unprecedented expression pattern and structural features of the PCRMPs thus suggest a variety of roles mediating host-parasite interactions throughout the parasite life cycle.

Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry.

The annotated genomes of organisms define a 'blueprint' of their possible gene products. Post-genome analyses attempt to confirm and modify the annotation and impose a sense of the spatial, temporal and developmental usage of genetic information by the organism. Here we describe a large-scale, high-accuracy (average deviation less than 0.02 Da at 1,000 Da) mass spectrometric proteome analysis of selected stages of the human malaria parasite Plasmodium falciparum. The analysis revealed 1,289 proteins of which 714 proteins were identified in asexual blood stages, 931 in gametocytes and 645 in gametes. The last two groups provide insights into the biology of the sexual stages of the parasite, and include conserved, stage-specific, secreted and membrane-associated proteins. A subset of these proteins contain domains that indicate a role in cell-cell interactions, and therefore can be evaluated as potential components of a malaria vaccine formulation. We also report a set of peptides with significant matches in the parasite genome but not in the protein set predicted by computational methods.