Three Members of the 6-cys Protein Family of Plasmodium Play a Role in Gamete Fertility

Abstract

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.

Epidemiology and Infectivity of Plasmodium falciparum and Plasmodium vivax Gametocytes in Relation to Malaria Control and Elimination

“…Studies of rodent malaria P. berghei have indicated that HAP2 is localized on male gametocytes and microgametes (251) and that HAP2 is not necessary for adhesion of gametes but is essential for the fusion of gamete surface membranes (251). P48/45 belongs to a family of proteins that are characterized by six positionally conserved cysteines (6-Cys proteins) (494). All 6-Cys proteins contain a signal peptide, and most contain a putative glycosylphosphatidylinositol (GPI) anchor sequence, making it plausible that they are located on the surfaces of parasites or invasion-associated organelles (473,498).…”

“…P48/45 is essential for the attachment of male microgametes to fertile female macrogametes in P. berghei (493). In addition to the Pfs48/45 gene, two more 6-Cys protein genes are expressed exclusively on P. falciparum gametes (Pfs47 and Pfs230 genes), suggesting a role in gamete recognition and fertilization (228,494,498,499,515). Pfs47 is a female-specific paralogue of Pfs48/45 located on the surfaces of female gametes following their emergence from erythrocytes, but it does not appear to be crucial for fertilization (498).…”

Structural and Biochemical Characterization of Plasmodium falciparum 12 (Pf12) Reveals a Unique Interdomain Organization and the Potential for an Antiparallel Arrangement with Pf41

“…Numerous Plasmodium surface proteins are highly polymorphic (46 -50), including a majority of the 6-Cys proteins (4,19,(51)(52)(53). To determine the evolutionary selection pressures acting on individual 6-Cys protein family members, we analyzed synonymous (dN) and non-synonymous (dS) polymorphisms among P. falciparum isolates to estimate the dN/dS ratio , which measures the strength of selection acting on a proteincoding gene, for Pf12, Pf38, Pf41, Pfs47, Pfs48/45, Pf52, Pf92, and Pfs230 (Fig.…”

N-Terminal Prodomain of Pfs230 Synthesized Using a Cell-Free System Is Sufficient To Induce Complement-Dependent Malaria Transmission-Blocking Activity

“…Pfs230 is a 360-kDa protein that contains 7 CM domains ( Fig. 1A) (6,11,36) and elicits a humoral immune response in infected individuals that can mediate transmission-blocking immunity (12,16). Consequently, Pfs230 as a component of a vaccine offers the advantage that it will elicit an immune response that is boosted by natural infection and will thus provide long-lasting immunity (3,28).…”