3 Discussion
The malaria parasite is an obligate intracellular parasite that scavenges the nutrients required to support its growth and replication from its host but also harbors enzymatic pathways for de novo synthesis of macromolecules. During its life cycle at different developmental stages, the parasite relies on its own biosynthetic machinery for cellular growth and proliferation. Lipid metabolism has emerged as a key regulator of cellular function and involves the synthesis of saturated fatty acids, which are further modified by desaturation and elongation (Shears et al. , 2015). Scd is an ER-localized enzyme involved in the desaturation of stearic acid into oleic acid to form unsaturated fatty acids (Gratraud et al. , 2009). In this study, we demonstrate that P. berghei Scd is not required for blood or mosquito stage development but is essential for liver stage development. The phenotype of Scd KO is similar to that of apicoplast-localized FASII pathway mutants, which were found to be essential for liver-stage development but not for blood-stage replication (Yu et al. , 2008; Vaughan et al. , 2009). The dispensability of the FASII pathway (Vaughan et al. , 2009) and desaturase enzyme Scd suggest that the Plasmodium parasite can scavenge lipids and saturated and unsaturated fatty acids directly from serum (Grellier et al. , 1991; Ofulla et al. , 1993).Plasmodium parasites directly utilize some FASII-generated fatty acids as lipid precursors without modification (Lindner et al. , 2014). However, the identification of apicoplast fatty acid export and the presence of desaturases and elongases in other cellular compartments suggested modification of fatty acids prior to incorporation into lipids (Ralph et al. , 2004; Mazumdar and Striepen, 2007; Gratraudet al. , 2009). This indicates that FASII-synthesized fatty acids are converted into a wide range of lipids for essential cellular function and that desaturation of fatty acids is an essential process for parasite development.
The FASII pathway enzymes and Scd were reported to be potential drug targets against the P. falciparum blood stage (Waller et al. , 2003; Gratraud et al. , 2009). However, previous reports and this study on lipid metabolism pathway mutants in P. berghei ,P. yoelii , and P. falciparum suggest that FASII and Scd are not required for blood-stage development (Yu et al. , 2008; Vaughan et al. , 2009). Recently, Scd was found to be dispensable for the P. falciparum blood stage in a genetic screen (Zhanget al. , 2018). Further investigations are needed to determine why in vitro-maintained P. falciparum cultures are sensitive to FasII and Scd inhibitors. The normal development of Scd KO parasites in mosquitoes suggests that parasites can fulfill lipid requirements from this host as well. It is currently not known how the parasite utilizes mosquito lipids, but evidence suggests that host serum fatty acids are utilized by the parasite directly during blood stage development (Krishnegowda and Gowda, 2003; Mi-Ichi et al. , 2006). Deletion of the FASII enzymes fabI and fabB/F in P. falciparum abolished sporozoite development in mosquitoes, suggesting the essentiality of the FASII pathway during mosquito stage development (van Schaijk et al. , 2014). This may be an important distinction between human and rodent malaria parasites, where the FASII pathway is only implicated in liver stages (Vaughan et al. , 2009). The requirement of FASII during mosquito stage development in P. falciparum can be explained by the number of sporozoites produced per oocyst. Human malaria parasites P. falciparum and P. vivax produce approximately 3,400 and 3,700 sporozoites per oocyst compared to approximately 800 and 1,000 in P. berghei and P. yoelii , respectively (Rosenberg et al. , 1990; Shimizu et al. , 2010; Lindner et al. , 2013). Possibly, fatty acids required for the higher numbers of sporozoite production in human malaria parasites cannot be fulfilled by mosquitoes, and the parasite relies on its own biosynthetic machinery.
Similar to FAS II, Scd is essential for late liver-stage development and hepatic merozoite formation. It was documented that PVM protein UIS3 disruption leads to early attenuation of the parasite in the liver. UIS3 interacts with the hepatocyte lipid carrier liver-fatty acid binding protein (Mikolajczak et al. , 2007), and parasites may take up lipids through UIS3 from the host during early liver stage development. However, when the lipid requirement is high for membrane biogenesis during late liver stage development, import through UIS3 fails to fulfill the requirement (Baer et al. , 2007; Vaughan et al. , 2009), and the parasite switches to its own biosynthetic machinery. Alternatively, the Scd-derived oleic acid supply cannot be met by the host required for GPI biosynthesis for MSP1, which is a GPI-anchored protein (Gerold et al. , 1996). However, whether GPI biosynthesis is impaired in Scd KO parasites needs further investigation. The defects in organelles such as apicoplast and ER morphology and biogenesis in Scd KO parasites were possibly due to a lack of long-chain fatty acids. It was shown that both the apicoplast and the ER cooperate in the synthesis of very long fatty acids in T. gondii (Ramakrishnan et al. , 2012).
Immunizations with GAP sporozoites elicit a long-term protective host response (Overstreet et al. , 2008). We found that immunization with Scd KO sporozoites in mice conferred complete protection against WT sporozoite challenge. Interestingly, GAPs that arrest late in the liver stage provide superior protection compared to early-arresting GAPs (Butler et al. , 2011). Because immunization using late-liver-stage arresting GAP expresses late-stage antigens and blood-stage overlapping antigens, whether Scd GAP induces superior protection needs further investigation. Despite the discovery of a growing list of GAPs, there have been some occasional breakthrough infections in a few GAPs. Breakthrough infections have been reported with previously reported GAP sporozoites (Kumar et al. , 2016). Multiple gene deletion GAPs can be created to overcome these limitations by combining them with known GAPs (Yu et al. , 2008; Vaughanet al. , 2009; Dankwa et al. , 2016). Multiple gene deletions prevent breakthrough infections that engender these parasites with a higher degree of attenuation to prevent any possible breakthrough infection (Mikolajczak et al. , 2014).
In conclusion, we have provided evidence that Scd is essential for the liver-to-blood stage transition. Scd disruption results in impaired organelle biogenesis and complete loss of hepatic merozoite formation. Immunization with Scd KO sporozoites protects against WT sporozoite challenge and could be used as a potential GAP vaccine.