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.