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\begin{document}

\title{Stearoyl-CoA desaturase regulates membrane biogenesis and hepatic
merozoite formation in Plasmodium berghei}



\author[1]{Sunil  Kumar Narwal}%
\author[1]{Akancha Mishra}%
\author[1]{Ankit Ghosh}%
\author[1]{Hadi Hasan Choudhary}%
\author[1]{Satish Mishra}%
\affil[1]{CSIR-Central Drug Research Institute}%


\vspace{-1em}



  
  \date{June 10, 2024}


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\selectlanguage{english}
\begin{abstract}
\emph{Plasmodium} is an obligate intracellular parasite that requires
intense lipid synthesis for membrane biogenesis and survival. One of the
principal membrane components is oleic acid, which is required to
maintain the membrane's biophysical properties and fluidity. The malaria
parasite \emph{Plasmodium} can modify fatty acids, and stearoyl-CoA
\selectlanguage{greek}Δ\selectlanguage{english}9-desaturase (Scd) is an enzyme that catalyzes the synthesis of oleic
acid by desaturation of stearic acid. Scd is dispensable in \emph{P.
falciparum} blood stages; however, its role in mosquito and liver stages
remains unknown. We show that \emph{P. berghei} Scd localizes to the ER
in the blood and liver stages. Disruption of \emph{Scd} in the rodent
malaria parasite \emph{P. berghei} did not affect parasite blood stage
propagation, mosquito stage development, or early liver stage
development. However, when \emph{scd} KO sporozoites were inoculated
intravenously or by mosquito bite into mice, they failed to initiate
blood-stage infection. Immunofluorescence analysis revealed that
organelle biogenesis was impaired and merozoite formation was abolished,
which normally initiates blood-stage infections. Genetic complementation
of the KO parasites restored merozoite formation to a level similar to
that of WT parasites. Mice immunized with \emph{Scd} KO sporozoites
confer long-lasting sterile protection against infectious sporozoite
challenge. Thus, the \emph{Scd} KO parasite is an appealing candidate
for inducing protective preerythrocytic immunity.%
\end{abstract}%



\sloppy


\textbf{1. Introduction}

Malaria infections caused by \emph{Plasmodium} parasites continue to be
the most important tropical disease that affects both animals and
humans. Malaria was responsible for over 247 million cases and 619,000
deaths in 2021 (\emph{World malaria report 2022} ,
2022).\emph{Plasmodium} sporozoite infection is initiated when an
infected female \emph{Anopheles} mosquito probes for the blood meal and
deposits sporozoites in the skin. Sporozoites migrate to the liver to
invade hepatocytes and initiate a replication cycle that generates
thousands of hepatic merozoites to initiate an erythrocytic cycle
(Prud\selectlanguage{ngerman}êncio \emph{et al.} , 2006; Lindner \emph{et al.} , 2012). During
these replicative stages, the parasite rapidly increases its biomass,
which requires many nutrients, including lipids, for growth. Lipids are
the most abundant components of any living organism and are essential
for cell development and division. They are not only important for
membrane biogenesis but are also major signaling molecules necessary for
invasive stage formation (Mazumdar and Striepen, 2007).
\emph{Plasmodium} parasites can either obtain lipids from the host or
synthesize them de novo to maintain propagation and survival within the
host (Tarun \emph{et al.} , 2009; Ramakrishnan \emph{et al.} , 2013).
However, de novo fatty acid synthesis is only essential in liver stages
and is dispensable for the asexual blood stage because these stages can
survive by scavenging fatty acids from the serum (Yu \emph{et al.} ,
2008; Vaughan \emph{et al.} , 2009).

In apicomplexan parasites, the type II fatty acid synthesis (FAS II)
pathway is localized to the apicoplast and catalyzed by four key
enzymes, FabB/F, FabG, FabZ, and FabI (Marrakchi \emph{et al.} , 2002;
Ralph \emph{et al.} , 2004; van Dooren and Striepen,
2013).\emph{Plasmodium} parasites are also capable of modifying de novo
synthesized or scavenged fatty acids into long unsaturated fatty acids
(Gratraud \emph{et al.} , 2009; Ramakrishnan \emph{et al.} , 2015). An
endoplasmic reticulum (ER)-localized stearoyl-CoA desaturase (Scd) was
identified in \emph{P. falciparum} that catalyzes the formation of oleic
acid from stearic acid by the insertion of a cis double bond at the \selectlanguage{greek}Δ\selectlanguage{english}9
position of fatty acyl-CoAs (Shanklin \emph{et al.} , 1994;
Gratraud\emph{et al.} , 2009). By using a Scd inhibitor, Gratraud et al.
showed that methyl sterculate inhibits the synthesis of oleic acid and
the development of asexual blood stage parasites (Gratraud \emph{et al.}
, 2009). Recently, Scd was found to be dispensable in a \emph{P.
falciparum} genetic screen (Zhang \emph{et al.} , 2018); however, its
role in mosquito and liver stages remains unknown.

The World Health Organization (WHO) recommended that the RTS,S vaccine
(Zavala, 2022) might not be suitable for long-term usage because subunit
vaccines occasionally do not elicit a strong or long-lasting immune
response compared to whole-organism vaccines. Immunization with
radiation-attenuated sporozoites (RAS) induces greater than 90\% sterile
protection in humans and has been the gold standard (Nussenzweig\emph{et
al.} , 1967). Repeated intravenous administration of RAS vaccines can
achieve sterile protection in humans (Seder \emph{et al.} , 2013).
Immunization with live sporozoites attenuated by genetic modifications
has gathered much attention, as they have been shown to produce
protective immune responses equal to, or even greater than, those
produced by irradiated sporozoites in rodent models (Khan \emph{et al.}
, 2012; Nganou-Makamdop and Sauerwein, 2013). GAPs offer several
advantages over radiation-based attenuation, as they constitute a
homogeneous population with a distinct genetic identity, and their
attenuation is not dependent on external factors. Advances
in\emph{Plasmodium} genetics have enabled the generation of GAPs, which
have overcome the limitations of RAS being used as a whole-organism
vaccine. The obvious advantages of GAPs over RAS are that they are
strategically generated by targeting genes, which are important for
liver stage development, and that these KOs are clonal lines inducing a
uniform immune response owing to their block at one particular stage,
i.e., early to mid-liver stage (Mueller \emph{et al.} , 2005b;
Mueller\emph{et al.} , 2005a) or late liver stage (Ishino \emph{et al.}
, 2009; Dankwa \emph{et al.} , 2016). These studies were extended to
human parasites, where the success of \emph{P. falciparum} GAP was shown
(Murphy \emph{et al.} , 2022). The protection of these attenuated
sporozoite vaccines involves antibodies elicited against sporozoite
antigens that neutralize their ability to invade hepatocytes
(Seder\emph{et al.} , 2013). Moreover, the protection is mediated
through CD8+ T-cell responses that target infected hepatocytes (Epstein
\emph{et al.} , 2011).

Immunization of mice with late-arresting parasites (Butler \emph{et al.}
, 2011) resulted in a superior immune response and protection compared
with early-arresting GAP (Aly \emph{et al.} , 2008). It was shown that
immunization with \emph{P. falciparum} sporozoites under drug coverage
allows parasites to mature into the late liver stage and elicit durable
protection at lower doses compared to the \emph{P. falciparum}RAS
sporozoites vaccine (Mordm\selectlanguage{ngerman}üller \emph{et al.} , 2017). These data
suggest that late-arresting GAP will be a superior immunogen and an
ideal whole parasite vaccine. Although the arrested parasites are a
source of antigen for effective priming of the immune system, their
antigenic repertoire induces cross-stage immunity only when parasites
experience a block at the late liver stage. Since late liver stages
exhibit a subset of antigens that are common to blood stages,
identifying genes that can yield a late-arresting mutant will have a
broader impact on developing an efficacious GAP vaccine. Here, by using
a genetically tractable model of \emph{P. berghei} , we show that Scd is
expressed during the blood and liver stages and is localized to the ER.
To evaluate Scd function in mosquito and liver stages, we disrupted the
gene, which resulted in impaired late liver-stage development that
failed to initiate blood-stage infection. Immunization with \emph{Scd}KO
sporozoites protects against infectious sporozoite challenges.

\textbf{2. Results}

\textbf{2. 1 \emph{P. berghei} Scd localized primarily in the
endoplasmic reticulum}

To study the expression and localization of Scd in the \emph{P.
berghei}life cycle, the gene was endogenously tagged with 3XHA by using
double crossover homologous recombination (Fig. S1A). The correct
integration of the targeting cassette at the \emph{Scd} locus was
confirmed by diagnostic PCR (Fig. S1B). Furthermore, the expression of
Scd-3XHA was confirmed by western blotting using an anti-HA antibody
(Fig. S1C). Transgenic parasites were analyzed throughout the parasite
life cycle stages, and we found that the C-terminal tag did not affect
parasite development (Fig. S1D and E). Previous studies have
demonstrated the association of SCD with the endoplasmic reticulum
(Gratraud \emph{et al.} , 2009). By using \emph{Pf} Scd-GFP transgenic
parasites, it was shown that \emph{P. falciparum} SCD localized to the
ER in asexual blood stages. Similarly, we performed colocalization
studies with the ER marker Bip and found that \emph{P. berghei was}
expressed in blood and liver stages and primarily colocalized to the ER
(Fig. 1A and B).

\textbf{2. 2 Scd is not required for \emph{P. berghei} blood and
mosquito stage development}

To address the function of Scd, we generated \emph{Scd} KO parasites by
using double crossover homologous recombination (Fig. S2A). After
recombination, the Scd ORF was replaced by the GFP and hDHFR:yFCU
expression cassettes, and GFP-expressing KO parasites were confirmed
using fluorescence microscopy (Fig. S2B). The correct gene replacement
was confirmed by diagnostic PCR, and clonal lines were obtained by
limiting dilution of the parasites. The clonal lines were further
confirmed by diagnostic PCR, and the absence of the Scd ORF was
determined by locus PCR, which amplified 4.33 kb in WT GFP and 7.16 kb
in \emph{Scd} KO parasites (Fig. S2C). Furthermore, we confirmed the
insertion of the targeting cassette at the correct locus by Southern
blotting. The genomic DNA was digested with \emph{EcoR} V and probed
with a 5'UTR probe, which revealed a band of 3.28 kb in KO compared to
8.4 kb in WT GFP due to the presence of an \emph{EcoR} V site in the GFP
cassette (Fig. S2D). The Scd-complemented parasite line was also
generated to check the specificity of the phenotype (Fig. S2E). For
this, a Scd expression cassette consisting of the 5'UTR, ORF, and 3'UTR
was amplified and transfected into \emph{Scd} KO parasites. After
recombination, the WT locus was amplified in Scd comp parasites (Fig.
S2F). Next, we checked the effect of gene deletion on parasite growth
during asexual blood stage propagation and found a similar growth
pattern of WT GFP and KO parasites (Fig. S2G). To further understand the
role of Scd in mosquito and liver stages, we transmitted the parasites
to the mosquitoes by allowing mosquitoes to feed on infected mice.
Examination on day 14 post feeding demonstrated normal development of
oocysts and oocyst-derived sporozoites in the mosquito midgut (Fig.
S3A-D). On days 18-22 post feeding, we observed intact salivary glands
that showed a normal sporozoite load and a comparable number of salivary
gland sporozoites in KO and WT GFP parasites (Fig. S3E and F). These
results indicate that Scd is dispensable for parasite blood and mosquito
stage development.

\textbf{2. 3 \emph{Scd} KO sporozoites infect the liver but fail to
establish blood-stage infections in mice}

To evaluate the role of Scd in liver stage development, different doses
of WT and KO sporozoites were injected intravenously into C57BL/6 mice
or infected by mosquito bite, and the appearance of the parasite in the
blood was monitored by making a Giemsa blood smear. We found that all
the mice injected with WT GFP and Scd comp became patent on day 3,
whereas mice inoculated with KO sporozoites remained negative until the
observation period (Table 1). The normal prepatent period of Scd comp
parasites suggests that the lack of infectivity of KO parasites was due
to the deletion of the Scd gene. To determine the stage-specific defect
in KO parasites, we first investigated the invasion ability of KO
sporozoites. For this, sporozoites were allowed to invade HepG2 cells,
and sporozoites present inside and outside of the cells were counted,
which revealed normal invasion by KO sporozoites (Fig. S4). Post
invasion, sporozoites transform into EEFs that mature into merozoites,
which are released 63 to 70 hpi in a controlled manner in the form of
merosomes, and then the parasite biomass in the liver declines sharply
(Sturm \emph{et al.} , 2006). To analyze the progress of EEF development
in vivo, the livers of infected mice were harvested at 36, 55, and 72
hpi, and parasite biomass was quantified by measuring 18S rRNA copy
number using real-time PCR. We found that the 18S rRNA copy number was
comparable in WT GFP and KO parasites at 36 hpi, but it was
significantly lower at 55 hpi in KO parasites than in WT GFP parasites
(Fig. 2A). However, the 18S rRNA copy number at 72 hpi was significantly
lower in WT GFP than in KO (Fig. 2A), which suggested that mature WT GFP
parasites egress from the liver but that KO parasites failed to mature
and remained in the liver. We confirmed the maturation defect by
determining the merozoite surface protein 1 (MSP1) transcripts at 55 hpi
and found that it was significantly decreased in the KO parasites (Fig.
2B). These data provide evidence that \emph{Scd} KO parasites failed to
mature into hepatic merozoites and did not egress from hepatocytes.

\textbf{2. 4 \emph{Scd} KO liver stages grow normally but do not form
hepatic merozoites}

To further explore the \emph{Scd} KO phenotype, HepG2 cells were
infected with sporozoites, and the culture was fixed at different time
points and stained with anti-UIS4 antibody and Hoechst. The \emph{Scd}KO
parasites grew similarly to the WT GFP parasites during liver stage
development (Fig. 3A). We counted the EEF numbers and measured the size
at 36, 48, and 62 hpi and found comparable numbers and sizes
in\emph{Scd} KO and WT GFP parasites (Fig. 3B-G). Next, we checked the
formation of merozoites by staining with an anti-MSP1 antibody. WT GFP
showed nuclear segregation and merozoite formation, whereas there was
abnormal DNA segregation in \emph{Scd} KO parasites, and merozoite
formation was abolished (Fig. 4A). We counted the nuclei in the EEF,
which were found to be significantly fewer in \emph{Scd} KO parasites
(Fig. 4B). Next, we observed the culture for the formation of merosomes
at 65 hpi and found it only in WT GFP (Fig. 4C). We counted the
merosomes in WT GFP, and an equivalent amount of culture supernatant was
injected into Swiss mice. The prepatent period was determined by making
a Giemsa-stained blood smear. Mice injected with WT GFP merosomes became
patent, whereas \emph{Scd} KO supernatant-injected mice remained
negative until the observation period (Table 2). This result suggests
that Scd plays an important role in the maturation of hepatic merozoites
and is essential for the liver-to-blood stage transition.

\textbf{2. 5 Lack of Scd causes severe defects in organelle biogenesis}

Lack of FAS II function in \emph{Toxoplasma gondii} leads to defects in
apicoplast biogenesis (Mazumdar \emph{et al.} , 2006). To evaluate
organelle morphology in \emph{Scd} KO parasites, EEFs fixed at 62 hpi
were immunostained with anti-ACP or anti-Bip antibodies to visualize the
branching of the apicoplast and endoplasmic reticulum, respectively. We
found normal branching of the apicoplast and endoplasmic reticulum in WT
GFP parasites, whereas it was impaired in \emph{Scd} KO parasites (Fig.
5A and B). Together, these results indicate that the FAS II pathway is
essential for membrane biogenesis and parasite survival.

\textbf{2. 6 Immunization with \emph{Scd} KO sporozoites protects
against malaria}

Immunizations with sporozoites that arrest in the liver elicit a
long-term protective host response and protect against infectious
sporozoite challenge (Duffy, 2022). To study preerythrocytic protection
induced by \emph{Scd} KO parasites, groups of C57BL/6 mice were
intravenously injected with three doses of sporozoites at intervals of
two weeks and challenged with infectious sporozoites 10 days post-last
immunization. All immunized mice remained blood stage negative after
immunization, again demonstrating the lack of breakthrough blood stage
infection. We found that all the mice injected with uninfected salivary
gland debris developed blood-stage parasitemia, whereas mice immunized
with \emph{Scd} KO sporozoites were completely protected (Table 3). We
further challenged the protected mice 120 days after the last
immunization, and they remained protected (Table 3). These results
demonstrate that immunization with \emph{Scd} KO sporozoites induces
preerythrocytic immunity and protects against malaria.

\textbf{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 \emph{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 \emph{et al.} , 2009). In this study, we
demonstrate that \emph{P. berghei} Scd is not required for blood or
mosquito stage development but is essential for liver stage development.
The phenotype of \emph{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 \emph{et al.} , 2008; Vaughan \emph{et al.} , 2009). The
dispensability of the FASII pathway (Vaughan \emph{et al.} , 2009) and
desaturase enzyme Scd suggest that the \emph{Plasmodium} parasite can
scavenge lipids and saturated and unsaturated fatty acids directly from
serum (Grellier \emph{et al.} , 1991; Ofulla \emph{et al.} ,
1993).\emph{Plasmodium} parasites directly utilize some FASII-generated
fatty acids as lipid precursors without modification (Lindner \emph{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 \emph{et al.} , 2004; Mazumdar and
Striepen, 2007; Gratraud\emph{et 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 \emph{P. falciparum} blood stage (Waller \emph{et
al.} , 2003; Gratraud \emph{et al.} , 2009). However, previous reports
and this study on lipid metabolism pathway mutants in \emph{P. berghei}
,\emph{P. yoelii} , and \emph{P. falciparum} suggest that FASII and Scd
are not required for blood-stage development (Yu \emph{et al.} , 2008;
Vaughan \emph{et al.} , 2009). Recently, Scd was found to be dispensable
for the \emph{P. falciparum} blood stage in a genetic screen
(Zhang\emph{et al.} , 2018). Further investigations are needed to
determine why in vitro-maintained \emph{P. falciparum} cultures are
sensitive to FasII and Scd inhibitors. The normal development of
\emph{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 \emph{et
al.} , 2006). Deletion of the FASII enzymes fabI and fabB/F in \emph{P.
falciparum} abolished sporozoite development in mosquitoes, suggesting
the essentiality of the FASII pathway during mosquito stage development
(van Schaijk \emph{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 \emph{et al.} , 2009). The
requirement of FASII during mosquito stage development in \emph{P.
falciparum} can be explained by the number of sporozoites produced per
oocyst. Human malaria parasites \emph{P. falciparum} and \emph{P. vivax}
produce approximately 3,400 and 3,700 sporozoites per oocyst compared to
approximately 800 and 1,000 in \emph{P. berghei} and \emph{P. yoelii} ,
respectively (Rosenberg \emph{et al.} , 1990; Shimizu \emph{et al.} ,
2010; Lindner \emph{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 \emph{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 \emph{et al.} , 2007; Vaughan \emph{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 \emph{et al.} , 1996). However, whether GPI
biosynthesis is impaired in \emph{Scd} KO parasites needs further
investigation. The defects in organelles such as apicoplast and ER
morphology and biogenesis in \emph{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 \emph{T. gondii} (Ramakrishnan \emph{et al.} , 2012).

Immunizations with GAP sporozoites elicit a long-term protective host
response (Overstreet \emph{et al.} , 2008). We found that immunization
with \emph{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 \emph{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 \emph{et al.} , 2016).
Multiple gene deletion GAPs can be created to overcome these limitations
by combining them with known GAPs (Yu \emph{et al.} , 2008;
Vaughan\emph{et al.} , 2009; Dankwa \emph{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 \emph{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 \emph{Scd} KO sporozoites protects against WT
sporozoite challenge and could be used as a potential GAP vaccine.

\textbf{4 Materials and Methods}

\textbf{4. 1 Parasites and mice}

\emph{P. berghei} ANKA (MRA 311) and \emph{P. berghei} ANKA GFP (MRA 867
507 m6cl1) were obtained from BEI resources, USA. Swiss albino and
C57BL/6 mice were used for parasite infections. All animal experiments
performed in this study were approved by the Institutional Animal Ethics
Committee at CSIR-Central Drug Research Institute, India (IAEC reference
no: IAEC/2013/83).

\textbf{4. 2 Generation of Scd-3XHA transgenic parasites}

For the 3XHA tagging of the stearoyl-CoA desaturase (Scd) (\emph{Pb}
ANKA\_1110700) gene, two fragments, F1 (0.773 kb) and F2 (0.584 kb),
were amplified from \emph{P. berghei} genomic DNA using primers
1045/1055 and 1041/1042 and cloned into the pBC-3XHA-hDHFR vector at
\emph{Xho} I/\emph{Bgl} II and \emph{Not} I/\emph{Asc} I, respectively.
The construct was linearized with \emph{Xho} I/\emph{Asc} I and
transfected into \emph{P. berghei} ANKA schizonts as previously
described (Janse \emph{et al.} , 2006). The resistant parasites were
selected using pyrimethamine (Sigma, USA), and genomic DNA was isolated
using a genomic DNA isolation kit (Promega, USA). Correct 5' and 3'
site-specific integration was confirmed by diagnostic PCR using primer
pairs 1043/1218 and 1215/1044, respectively (primer sequences are given
in Table S1). For the localization of Scd in the blood stage, transgenic
Scd-3XHA-infected blood was spotted on 12-well slides (Thermo Fisher
Scientific, USA) and fixed with 4\% paraformaldehyde.

\textbf{4. 3 Western blot analysis}

For the analysis of the Scd-3XHA fusion protein in transgenic parasites,
blood-stage parasites were harvested, washed three times with PBS, and
pelleted by centrifugation. The iRBC pellet was lysed using 0.15\%
saponin, and the released parasites were pelleted, washed with PBS
containing complete protease inhibitors (Roche, Switzerland), and
resuspended in Laemmli buffer. Immunoblotting was performed as
previously described (Narwal \emph{et al.} , 2022). Briefly, samples
were resolved on SDS\selectlanguage{english}-PAGE and transferred onto nitrocellulose membranes
(Bio-Rad, USA), blocked with 5\% nonfat dry milk in PBS, and incubated
with anti-HA antibody (diluted 1:1,000, Novus Biologicals, USA) followed
by incubation with HRP-conjugated anti-rabbit IgG (diluted 1:5,000,
Amersham Biosciences, United Kingdom). The signals were detected using
ECL chemiluminescent substrate (Thermo Scientific, USA) in a ChemiDoc
XRS+ System (Bio-Rad, USA).

\textbf{4. 4 Mosquito cycles}

\emph{Anopheles stephensi} mosquitoes were maintained as previously
described (Narwal \emph{et al.} , 2022). To obtain sporozoites,
mosquitoes were allowed to feed on infected Swiss mice, and cages were
kept in an environmental chamber maintained at 190degC with 80\% relative
humidity. The development of oocysts was observed on day 14, and
salivary gland sporozoites were dissected between days 18-22
postfeeding.

\textbf{4. 5 Generation of Scd knockout (\emph{Scd} KO) and complemented
(Scd comp) parasites}

The Scd (\emph{Pb} ANKA\_1110700) targeting vector was constructed by
cloning two PCR products, F3 (578 bp) and F4 (573 bp), into the
pBC-GFP-hDHFR:yFCU plasmid at \emph{Xho} I/\emph{Sal} I and\emph{Not}
I/\emph{Asc} I, respectively. The fragments F3 and F4 were amplified
from \emph{P. berghei} ANKA genomic DNA using primer pairs 1039/1056 and
1041/1042, respectively. The final vector was digested with \emph{Xho}
I/\emph{Asc} I and transfected into \emph{P. berghei}schizonts as
previously described (Janse \emph{et al.} , 2006). Genomic DNA was
isolated from the drug-resistant GFP-expressing parasites, and correct
5' and 3' site-specific integration was confirmed by diagnostic PCR
using primers 1410/1225 and 1215/1044, respectively. Clonal lines were
obtained by limiting dilution of the parasite and again confirmed for
integration by PCR. The modified genomic locus was confirmed by PCR
using primers 1410/1044. For Southern blotting, parasite genomic DNA was
digested with E\emph{coR} V, run on a 0.7\% agarose gel, transferred to
a positively charged nylon membrane (Amersham Biosciences, United
Kingdom) and developed as previously described (Narwal \emph{et al.} ,
2022). The membrane was probed with a dioxygenin-labeled 5' probe (DIG
High Prime DNA labeling and detection kit, Roche Applied Sciences,
Switzerland). To generate a \emph{Scd} KO complemented parasite line, a
fragment consisting of the Scd 5'UTR, ORF, and 3'UTR was amplified using
primers 1410/1044 and transfected into \emph{Scd} KO parasites.
Parasites containing restored Scd loci were selected by negative
selection using a 5-fluorocytosine (5-FC) drug (Sigma, USA) as
previously described (Srivastava and Mishra, 2022). Negative selection
selects parasites lacking the hDHFR:yFCU cassette. The complemented line
was confirmed by the absence of GFP fluorescence and amplification of
the Scd expression cassette by diagnostic PCR using primers 1410/1044.

\textbf{4. 6 Determination of the \emph{Scd} KO phenotype}

Phenotypic analyses of the \emph{Scd} KO parasites were performed as
previously described (Srivastava and Mishra, 2022). Asexual blood growth
was monitored by intravenously injecting 200 \selectlanguage{greek}µ\selectlanguage{english}l of blood with 0.2\%
parasitemia into two groups of Swiss albino mice (n=4). Parasitemia was
determined by counting Giemsa-stained blood smears. The WT and\emph{Scd}
KO sporozoites were isolated from the infected mosquito's salivary
glands on days 18-22 post blood meal and counted using a hemocytometer.
For the determination of in vivo infectivity, C57BL/6 mice were either
intravenously inoculated with salivary gland sporozoites or infected by
mosquito bite, and blood-stage infection was monitored by making
Giemsa-stained blood smears. Another group of mice injected with 5,000
sporozoites was sacrificed at 36, 55, and 72 hpi, and the livers were
harvested and homogenized in TRIzol reagent (Invitrogen, USA). For the
invasion assay, HepG2 cells were infected with salivary gland
sporozoites (10,000/well) and culture fixed after 1 h and stained with
anti-CSP antibody (Yoshida \emph{et al.} , 1980) before and after
permeabilization as described (R\selectlanguage{ngerman}énia \emph{et al.} , 1988). The detailed
EEF development assay was performed as previously described (Srivastava
and Mishra, 2022). Briefly, HepG2 cells (5.5 x10\textsuperscript{4})
were seeded in a 48-well plate containing collagen-coated coverslips and
incubated overnight at 37°C in a CO\textsubscript{2} incubator. Salivary
gland sporozoites (5,000/well) were added and incubated at 37°C, and the
culture was fixed using 4\% paraformaldehyde (PFA) at different time
points. For the merosome assay, HepG2 cells (100,000/well) were seeded
in a 24-well plate, infected with salivary gland sporozoites
(40,000/well), and harvested at 65 hpi to count the merosome numbers.
Merosomes were injected into Swiss mice to check the infectivity.

\textbf{4. 7 Real-time PCR assays for quantification of parasites}

Total RNA was isolated from the infected liver, and cDNA was synthesized
as previously described (Choudhary \emph{et al.} , 2019). The parasite
burden was quantified by amplifying 18S rRNA (Bruña-Romero \emph{et al.}
, 2001) using primers 1195/1196. MSP1 transcripts were quantified using
primers 1219/1220. Liver samples were normalized to mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using primers
1193/1194. To quantify the copy number, gene-specific standards were run
alongside the unknown samples.

\textbf{4. 8 Immunofluorescence Assay}

IFA was performed as described previously (Narwal \emph{et al.} , 2022).
The PFA-fixed cells were washed twice with PBS, and blood-stage
parasites were permeabilized with 0.1\% Triton-X100 in PBS for 10 min
and liver stages in chilled methanol for 20 min at 4°C and blocked with
1\% BSA-PBS. The cells were incubated for 1 h with the following primary
antibodies: anti-HA antibody (diluted 1:1,000, Novus Biologicals, USA),
anti-BiP (diluted 1:125, MRA\selectlanguage{english}-1246, BEI resources), anti-UIS4 (diluted
1:1,000 (Mueller \emph{et al.} , 2005b), anti-merozoite surface protein
1 (MSP1) monoclonal antibody (diluted 1:5000 (Holder and Freeman, 1981),
and anti-acyl carrier protein (ACP, diluted 1:800) (Gallagher and
Prigge, 2010). The signals were visualized with secondary antibodies:
Alexa Fluor 594-conjugated anti-rabbit IgG (diluted 1:500; Invitrogen,
USA), Alexa Fluor 488-conjugated anti-mouse IgG (diluted 1:500;
Invitrogen, USA), and Alexa Fluor 488-conjugated anti-rabbit IgG
(diluted 1:500; Invitrogen, USA). The nuclei were stained with Hoechst,
and the coverslips were mounted using Prolong Diamond antifade reagent
(Thermo Scientific, USA). Images were acquired using FV1000 software on
a confocal laser scanning microscope (Olympus BX61WI) equipped with a
UPlanSAPO 100x/1.4 oil immersion objective. The EEFs were counted
manually, and the area was measured using Nikon NIS elements BR imaging
software under an Eclipse 80i fluorescence microscope/Plan Fluor
40x/0.75 objective.

\textbf{4. 9 Immunization and challenge experiment}

Female C57BL/6 mice (6-8 weeks old) were intravenously injected with
three doses of salivary gland sporozoites at an interval of two weeks.
Control groups were injected with uninfected mosquito salivary gland
debris. Both groups were challenged with 5,000 WT sporozoites 10 post
last immunization. The appearance of parasites in blood was monitored by
making Giemsa-stained blood smears.

\textbf{4. 10 Statistical analysis}

Statistical analysis was carried out in GraphPad Prism software using
two-tailed, unpaired Student's t test or one-way ANOVA.

\textbf{Acknowledgments}

We thank BEI resources, USA, for the parasite strains and plasmids. We
thank Dr. Kota Arun Kumar (University of Hyderabad, India) for the
pBC-3XHA-hDHFR vector. We thank Dr. Anthony A. Holder (The Francis Crick
Institute, UK), Drs. Photini Sinnis and Sean Prigge (Johns Hopkins
University, USA) for anti-MSP1, anti-UIS4, and anti-ACP antibodies,
respectively. We acknowledge the THUNDER (BSC0102) and MOES (GAP0118)
Intravital and Confocal microscopy facility of CSIR-CDRI. The Indian
Council of Medical Research, University Grants Commission, and Council
of Scientific and Industrial Research, Government of India research
fellowships supported SKN, AM, HHC, and AG.

\textbf{Conflict of interest}

The authors declare that they have no conflicts of interest.

\textbf{Author contributions}

SKN and SM conceived the idea, designed and performed the experiments,
analyzed the data and wrote the manuscript. AM, AG, and HHC performed
the experiments. All the authors have read and approved the manuscript.

\textbf{Declarations}

\textbf{Ethics approval}

The current study does not involve human samples. All animal procedures
conducted were approved by the Institutional Animal Ethics Committee at
CSIR-Central Drug Research Institute, India (IAEC/2013/83).

\textbf{Consent to participate}

Not applicable.

\textbf{Consent for publication}

Not applicable.

\textbf{Availability of data and material}

All data are available within this manuscript, and raw data are
available from the corresponding author upon reasonable request.
Materials generated in this study are available from the corresponding
author upon request.

\textbf{Funding}

The work was supported by the Ramalingaswami Fellowship grant
(BT/RLF/Re-entry/20/2012).

\textbf{Authors' information}

\textbf{Division of Molecular Microbiology and Immunology, CSIR-Central
Drug Research Institute, Lucknow 226031, India.}

Sunil Kumar Narwal, Akancha Mishra, Ankit Ghosh, Hadi Hasan Choudhary,
Satish Mishra

\textbf{Academy of Scientific and Innovative Research (AcSIR), Ghaziabad
201002, India.}

Akancha Mishra, Satish Mishra

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\textbf{Table 1.} Infectivity of salivary gland sporozoites in C57BL/6
mice. NA, Not applicable.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Experiment} & \textbf{Parasite} & \textbf{Number of sporozoites
inoculated} & \textbf{Mice positive/mice inoculated} & \textbf{Prepatent
period (day)}\tabularnewline
1 & WT & 5,000 & 10/10 & 3\tabularnewline
& \emph{Scd} KO & 5,000 & 0/15 & NA\tabularnewline
& \emph{Scd} comp & 5,000 & 5/5 & 3\tabularnewline
2 & WT & 10,000 & 10/10 & 3\tabularnewline
& \emph{Scd} KO & 10,000 & 0/20 & NA\tabularnewline
& \emph{Scd} KO & 20,000 & 0/10 & NA\tabularnewline
& \emph{Scd} comp & 10,000 & 5/5 & 3\tabularnewline
& WT & From 20 mosquitos & 5/5 & 3\tabularnewline
& \emph{Scd} KO & From 20 mosquitos & 0/5 & NA\tabularnewline
3 & WT & 50,000 & 5/5 & 3\tabularnewline
& \emph{Scd} KO & 50,000 & 0/10 & NA\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2.} Infectivity of merosomes in Swiss mice. NA, Not
applicable.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\textbf{Experiment} & \textbf{Parasite} & \textbf{Number of merosomes
injected} & \textbf{Mice positive/mice injected} & \textbf{Prepatent
period (day)}\tabularnewline
1 & WT & 1 & 5/5 & 6\tabularnewline
& \emph{Scd} KO & Spernatant & 0/5 & NA\tabularnewline
2 & WT & 10 & 5/5 & 2\tabularnewline
& \emph{Scd} KO & Spernatant & 0/5 & NA\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 3.} Immunization of C57BL/6 mice by intravenous injection
of \emph{Scd} KO sporozoites. All immunized mice were protected from
WT~\emph{P. berghei} sporozoite challenge. NA, Not applicable\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Experiment}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Group}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Immunization dose}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Challenge day}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Challenge dose}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{No. of patent/no. of challenged}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\textbf{Prepatent period (day)}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{1}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Control\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(3x) SG debris\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5,000\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5/5\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\emph{Scd} KO\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(1x) 5x10\textsuperscript{4} and (2x) 2.5x10\textsuperscript{4}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5,000\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
0/5\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
NA\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
\textbf{2}\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Control\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
(3x) SG debris\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
10\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5,000\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
5/5\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
3\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\emph{Scd} KO\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
(1x) 5x10\textsuperscript{4} and (2x) 2.5x10\textsuperscript{4}\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
10 120\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
5,000 5,000\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0/5 0/5\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
NA NA\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure legends}

\textbf{Fig. 1. Localization of \emph{P. berghei} Scd in the parasite
endoplasmic reticulum.} (\textbf{A} ) IFA with asexual blood stage
parasites confirmed Scd expression, which colocalized with the
endoplasmic reticulum (ER) marker Bip. (\textbf{B} ) The expression of
Scd was monitored during liver stage development at different time
points, and we found late expression of Scd at 48 and 55 hpi, which
colocalized with the ER marker BiP. The nuclei were stained with
Hoechst.

\textbf{Fig. 2.} \emph{Scd} \textbf{KO parasites display defects in late
liver stage development.} (\textbf{A} ) To determine the liver parasite
burden, sporozoites were injected intravenously into C57BL/6 mice,
livers were homogenized at 36, 55 and 72 hpi, RNA was isolated, and
transcripts were quantified using real-time PCR. The 18S rRNA copy
number was determined and normalized to mouse GAPDH transcripts.
The\emph{P. berghei} 18S rRNA copy number was comparable in WT GFP
and\emph{Scd} KO parasites at 36 hpi but significantly lower in
\emph{Scd}KO parasites than in WT GFP parasites at 55 hpi, and at 72
hpi, it was opposite and found to be significantly higher in KO
parasites, indicating that only WT GFP parasites were able to egress
from the liver. Similar results were obtained in three independent
experiments. Data represent the mean \selectlanguage{ngerman}± SD, n = 5 mice per group.
(\textbf{B} ) A lower level of MSP1 transcripts at 55 hpi in KO-infected
liver suggests a failure in the maturation of parasites. Data are
presented as the mean ± SEM from two independent experiments
(significant difference (p=0.0132), Student's t test).

\textbf{Fig. 3.} \emph{Scd} \textbf{KO liver stages grow normally.} (A)
Sporozoite-infected HepG2 cells were fixed at 12, 24, 36, 48, and 62 hpi
and immunostained with anti-UIS4 antibody. Nuclei were stained with
Hoechst. Visually, liver stage growth appeared to be comparable
in\emph{Scd} KO and WT GFP parasites. (\textbf{B, D and F} ) The EEF
numbers were found to be normal at 36, 48, and 62 hpi (p=0.3950 at 36
hpi, p= 0.9419 at 48 hpi, and p= 0.6070 at 62 hpi). (\textbf{C, E and G}
) Determination of EEF size at 36, 48, and 62 hpi. The size of EEFs was
comparable between WT GFP and \emph{Scd} KO parasites (p=0.5096 at 36
hpi, p=0.6269 at 48 hpi, and p= 0.1642 at 62 hpi). The data were pooled
from three independent experiments. Data represent the mean ± SD. P
values were determined by an unpaired, two-tailed Student's t test.

\textbf{Fig. 4.} \emph{Scd} \textbf{KO parasites arrest late during
liver stage development and do not form hepatic merozoites.}(\textbf{A}
) Infected HepG2 cells were harvested at 62 hpi, fixed, and
immunostained with anti-merozoite surface protein 1 (MSP1) antibody, and
DNA was stained with Hoechst. We found normal segregation of nuclei and
formation of merozoites in WT GFP, but it was impaired in \emph{Scd} KO
parasites. (\textbf{B} ) Counting of nuclei in the EEF revealed impaired
nuclear division in KO parasites (p\textless{}0.001, Student's t test).
The data were pooled from three independent experiments. Data represent
the mean ± SD. (\textbf{C} ) The merosome numbers were counted using a
hemocytometer. \emph{Scd} KO parasites formed EEFs that were comparable
to WT GFP parasites but failed to form merosomes, a significant
difference (p\textless{}0.0001, Student's t test).

\textbf{Fig. 5. Lack of Scd leads to defects in organelle morphology and
biogenesis.} (\textbf{A} ) Infected HepG2 cells were fixed at 62 hpi and
immunostained with apicoplast marker anti-ACP antibody. WT GFP showed
normal branching of the apicoplast, which was impaired in \emph{Scd} KO
parasites. (\textbf{B} ) Immunostaining of EEFs with an ER marker
anti-Bip antibody revealed impaired branching in \emph{Scd} KO
parasites.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fig/Fig}

\end{center}
\end{figure}

\textbf{Hosted file}

\verb`Fig.2.eps`  available at \url{https://authorea.com/users/653075/articles/660231-stearoyl-coa-desaturase-regulates-membrane-biogenesis-and-hepatic-merozoite-formation-in-plasmodium-berghei}

\textbf{Hosted file}

\verb`Fig.3.eps`  available at \url{https://authorea.com/users/653075/articles/660231-stearoyl-coa-desaturase-regulates-membrane-biogenesis-and-hepatic-merozoite-formation-in-plasmodium-berghei}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fij/Fij}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Fik/Fik}

\end{center}
\end{figure}

\selectlanguage{english}
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\end{document}

