Distinct Prominent Roles for Enzymes of Plasmodium berghei Heme Biosynthesis in Sporozoite and Liver Stage Maturation

Abstract

Malarial parasites have evolved complex regulation of heme supply and disposal to adjust to heme-rich and -deprived host environments. In addition to its own pathway for heme biosynthesis, Plasmodium likely harbors mechanisms for heme scavenging from host erythrocytes. Elaborate compartmentalization of de novo heme synthesis into three subcellular locations, including the vestigial plastid organelle, indicates critical roles in life cycle progression. In this study, we systematically profile the essentiality of heme biosynthesis by targeted gene deletion of enzymes in early steps of this pathway. We show that disruption of endogenous heme biosynthesis leads to a first detectable defect in oocyst maturation and sporogony in the Anopheles vector, whereas blood stage propagation, colonization of mosquito midguts, or initiation of oocyst development occurs indistinguishably from that of wild-type parasites. Although sporozoites are produced by parasites lacking an intact pathway for heme biosynthesis, they are absent from mosquito salivary glands, indicative of a vital role for heme biosynthesis only in sporozoite maturation. Rescue of the first defect in sporogony permitted analysis of potential roles in liver stages. We show that liver stage parasites benefit from but do not strictly depend upon their own aminolevulinic acid synthase and that they can scavenge aminolevulinic acid from the host environment. Together, our experimental genetics analysis of Plasmodium enzymes for heme biosynthesis exemplifies remarkable shifts between the use of endogenous and host resources during life cycle progression. Heme is a ubiquitous iron-porphyrin complex that serves as a cofactor of hemoproteins, such as globins, catalases, and cytochromes (1). The iron component of heme permits the binding of diatomic gases, as well as unique enzymatic redox reactions based on its reduction potential from the oxidized ferric (Fe 3ϩ ) to the reduced ferrous (Fe 2ϩ ) state (1). Thus, heme mediates fundamental biological processes, including oxygen transport, antioxidant responses, and cellular respiration, and is, therefore, indispensable for virtually all living organisms. Exceptions, like the parasitic kinetoplastid flagellate Phytomonas serpens that can survive in the complete absence of heme (2), are rare. As a result of this nearly universal dependence on heme, two complementary strategies for heme acquisition have evolved; heme scavenging and de novo heme biosynthesis.Plasmodium parasites are the causative agents of malaria and have adapted to an intraerythrocytic lifestyle during blood infection, the exclusive pathogenic phase of the parasite life cycle. Erythrocytes make up the largest pool of heme in the human body, as they are rich in hemoglobin. In turn, hemoglobin constitutes the major source of amino acids for developing blood stage parasites, which import and digest almost all host hemoglobin, liberating large amounts of heme (3). Free heme acts as a biological Fenton reagent and can thus damage organic molecules by oxidation...

A novel genetic technique in Plasmodium berghei allows liver stage analysis of genes required for mosquito stage development and demonstrates that de novo heme synthesis is essential for liver stage development in the malaria parasite

“…This confirms previous findings [12, 15] that the parasite heme synthesis enzymes do not play an essential role in the early mosquito life stages in P . berghei .…”

RNA-Seq Analysis Illuminates the Early Stages of Plasmodium Liver Infection

“…For example, we found that LYTB is upregulated at 2 and 4 hpi, indicating isoprenoids may be important at this time. Although the FASII and de novo heme biosynthesis pathways have been genetically and chemically validated as essential to the late liver stages, less is known about isoprenoid biosynthesis during the early liver stages (34,35). When intracellular sporozoites metamorphosize to replication-competent trophozoites, most organelles are expelled at the exclusion of the nucleus, mitochondrion, and apicoplast (10).…”

Vitamin and cofactor acquisition in apicomplexans: Synthesis versus salvage

“…Whereas protein-driven hemozoin formation has been postulated before (101), lipid-driven mechanisms (102,103) and an autocatalytic process have also been proposed (104). Unsurprisingly, heme synthesis is not essential for Plasmodium during the intraerythrocytic development, but the pathway becomes fitness-conferring during liver stages and is essential for development in the mosquito (105)(106)(107)(108)(109). Specifically, the loss of FC impairs male gamete formation and ablates oocyst formation in mosquitoes, indicating that Plasmodium can utilize salvaged heme but relies on its synthesis when levels of exogeneous heme become limiting within the insect vector (105,106).…”