Female Inheritance of Malarial lap Genes Is Essential for Mosquito Transmission

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Abstract
Members of the LCCL/lectin adhesive-like protein (LAP) family, a family of six putative secreted proteins with predicted adhesive extracellular domains, have all been detected in the sexual and sporogonic stages of Plasmodium and have previously been predicted to play a role in parasite–mosquito interactions and/or immunomodulation. In this study we have investigated the function of PbLAP1, 2, 4, and 6. Through phenotypic analysis of Plasmodium berghei loss-of-function mutants, we have demonstrated that PbLAP2, 4, and 6, as previously shown for PbLAP1, are critical for oocyst maturation and sporozoite formation, and essential for transmission from mosquitoes to mice. Sporozoite formation was rescued by a genetic cross with wild-type parasites, which results in the production of heterokaryotic polyploid ookinetes and oocysts, and ultimately infective Δpblap sporozoites, but not if the individual Δpblap parasite lines were crossed amongst each other. Genetic crosses with female-deficient (Δpbs47) and male-deficient (Δpbs48/45) parasites show that the lethal phenotype is only rescued when the wild-type pblap gene is inherited from a female gametocyte, thus explaining the failure to rescue in the crosses between different Δpblap parasite lines. We conclude that the functions of PbLAPs1, 2, 4, and 6 are critical prior to the expression of the male-derived gene after microgametogenesis, fertilization, and meiosis, possibly in the gametocyte-to-ookinete period of differentiation. The phenotypes detectable by cytological methods in the oocyst some 10 d after the critical period of activity suggests key roles of the LAPs or LAP-dependent processes in the regulation of the cell cycle, possibly in the regulation of cytoplasm-to-nuclear ratio, and, importantly, in the events of cytokinesis at sporozoite formation. This phenotype is not seen in the other dividing forms of the mutant parasite lines in the liver and blood stages. Malaria parasites are transmitted between human hosts by female mosquitoes. Following fertilization between male and female gametes in the blood meal, zygotes develop into motile ookinetes that, 24 hours later, cross the mosquito midgut epithelium and encyst on the midgut wall. During this development, parasite numbers fall dramatically and as such, this may be an ideal point at which to disrupt transmission, but first essential parasite targets need to be identified. A protein family implicated in the interactions between parasites and mosquitoes is the LCCL/lectin adhesive-like protein (LAP) family. LAPs are highly expressed in the sexual and ookinete stages, yet when we removed genes encoding each of four LAPs from the genome of a rodent model malaria parasite, a developmental defect was only observed in the oocyst some ten days after the protein was first expressed. These “knockout” parasites did not undergo normal replication and consequently could not be transmitted to mice. Through genetic crosses with parasite mutants producing exclusively either female or male gametes, we demonstrate that parasites can only complete their development successfully if a wild-type lap gene is inherited through the female cell. These data throw new light on the regulation of parasite development in the mosquito, suggesting that initial development is maternally controlled, and that the LAPs may be candidates for intervention.