A Novel Plasmodium berghei Protein, LSMP, Regulates Malaria Liver Stage Maturation

ABSTRACT

Malaria parasites undergo extensive replication in hepatocytes before initiating the pathogenic blood stage of infection. Although this clinically silent phase is essential for parasite expansion, the molecular mechanisms regulating late liver stage maturation remain incompletely understood. Here, we characterize a previously unannotated protein (PBANKA_0620800), which we designate liver stage maturation protein (LSMP). LSMP was expressed during schizont, sporozoite, and liver stages and localized predominantly to the parasite cytosol. Using reverse genetic approaches, we generated LSMP KO parasites and examined their development across the parasite life cycle. Loss of LSMP did not affect asexual blood‐stage replication or mosquito‐stage development, including ookinete formation, oocyst development, and sporozoite formation. LSMP KO sporozoites invaded hepatocytes normally but exhibited pronounced defects during late liver stage development, including reduced nuclear replication, impaired merozoite formation, and delayed rupture of the parasitophorous vacuole membrane. Together, our findings demonstrate that LSMP is important for malaria liver stage maturation.

LSMP is dispensable for blood and mosquito stages but is critical for late liver stage maturation, promoting nuclear replication, merozoite formation, and timely parasite egress. Graphical abstract was created using BioRender.

1. Introduction

Malaria remains one of the most devastating infectious diseases, caused by protozoan parasites of the genus Plasmodium. The complex life cycle of Plasmodium alternates between the mosquito vector and the vertebrate host and involves several morphologically and functionally distinct developmental stages, tightly regulated by parasite protein expression. Infection of the mammalian host begins when sporozoites are injected into the skin during the bite of an infected Anopheles mosquito. These motile parasites rapidly enter the bloodstream and migrate to the liver, where they invade hepatocytes and initiate the clinically silent liver stage of infection (Prudêncio et al. 2006; Vaughan and Kappe 2017). Within hepatocytes, sporozoites transform into trophozoites and establish residence within a parasitophorous vacuole (PV), which is surrounded by the PV membrane (PVM). During this stage, a single invading sporozoite undergoes extensive replication via exoerythrocytic schizogony, generating thousands of merozoites that initiate the erythrocytic cycle responsible for the clinical symptoms of malaria (Vaughan et al. 2008; Vaughan and Kappe 2017).

Following hepatocyte invasion, the liver stage parasites undergo a remarkable transformation characterized by rapid cell growth, repeated rounds of DNA replication, and nuclear division without immediate cytokinesis. This process results in the formation of large multinucleated exoerythrocytic forms (EEFs) that occupy a substantial portion of the host hepatocyte (Prudêncio et al. 2006). During late liver stage development, parasites initiate segmentation in which membranes invaginate to partition nuclei and organelles into individual daughter cells. This stage, known as cytomere formation, culminates in the generation of thousands of infectious merozoites. The successful completion of liver stage maturation requires coordinated expansion and segregation of parasite organelles, including the mitochondrion, endoplasmic reticulum, and apicoplast. The apicoplast, a nonphotosynthetic plastid essential for parasite metabolism undergoes extensive branching during parasite replication and must be accurately partitioned into daughter merozoites (van Dooren and Striepen 2013). Disruption of organelle biogenesis or segregation leads to defects in parasite development and reduced merozoite formation (Yu et al. 2008; Vaughan et al. 2009; Narwal et al. 2024; Devi et al. 2025). Indeed, genetic studies have identified several parasite proteins whose disruption results in impaired nuclear replication, abnormal cytomere formation, or reduced merozoite numbers during liver stage development (Stanway et al. 2019). Proteins localized to the PVM, such as UIS4 and EXP1, have been shown to be necessary for liver stage development. Disruption of these proteins results in arrest during the liver stage (Mueller, Labaied, et al. 2005; Sá E Cunha et al. 2017). The final step of liver stage development involves the coordinated rupture of the PVM, followed by the controlled release of parasites from infected hepatocytes. Mature liver stage parasites trigger disassembly of the host cell actin cytoskeleton and bud from hepatocytes within vesicular structures known as merosomes (Sturm et al. 2006). These merosomes protect parasites from immune detection as they enter the bloodstream and eventually rupture to release merozoites that invade erythrocytes. Defects in PVM rupture or merosome formation can therefore significantly impair parasite transmission from the liver to the blood stage, even when earlier developmental processes occur normally (Burda et al. 2015; Srivastava and Mishra 2022; Devi et al. 2025).

Post‐translational regulation, particularly protein phosphorylation, plays an important role in controlling developmental transitions throughout the Plasmodium life cycle. The parasite genome encodes a diverse repertoire of protein kinases that regulate essential processes such as cell cycle progression, differentiation, and host cell invasion (Doerig et al. 2015). By analysing conserved kinase domains, 66 putative eukaryotic protein kinases were identified. Reverse genetic approaches showed that 23 of these kinases are not required for asexual blood stage development (Tewari et al. 2010). However, further phenotypic analysis across multiple mosquito life cycle stages revealed a specific group of kinases that are essential for sexual development and sporogony. Importantly, this analysis revealed stage‐specific functions of several kinases, SR protein kinase (SRPK) in microgamete formation, a clathrin‐associated kinase (GAK) in ookinete development, and an SNF/KIN related kinase in sporozoite maturation, highlighting stage‐specific regulatory pathways critical for parasite transmission (Tewari et al. 2010). To date, only a limited number of protein kinases have been found to be important during Plasmodium liver stage development. In P. berghei, two mitogen‐activated protein kinases, PbMAPK1 and PbMAPK2, have been described. Although both genes are transcribed during liver stage development and PbMAPK1 localizes to the cytomere stage, disruption of PbMAPK1 does not affect parasite viability in the liver (Wierk et al. 2013). In contrast, PbMAPK2 is dispensable during asexual blood stage development but is essential for male gametogenesis (Rangarajan et al. 2005; Tewari et al. 2005), highlighting the stage‐specific roles of MAPK signaling during parasite development. In addition, a cGMP‐dependent protein kinase in P. berghei has been implicated in liver stage schizont maturation alongside its established role in ookinete differentiation and motility (Rotella et al. 2021). In addition, the serine‐threonine kinase PbSTK2 has been reported to play an important role in late liver stage development (Jillapalli et al. 2019).

While several studies have characterized parasite protein kinases during the blood stage cycle, their role in the liver stage remains largely unexplored. Among these, Casein kinase II (CK2) is a highly conserved serine/threonine kinase known to phosphorylate substrates containing the minimal consensus motif S/T‐X‐X‐D/E. CK2‐mediated phosphorylation regulates multiple cellular processes in eukaryotes, including transcriptional control, cytoskeletal organization, cell differentiation, cell proliferation, cell cycle progression, and vesicular trafficking. The mammalian CK2 enzyme typically exists as a tetrameric complex composed of two catalytic subunits (α and α′) and two regulatory subunits derived from a single gene. Interestingly, unlike most other CK2 complexes, the P. falciparum genome encodes only one catalytic α‐subunit along with two distinct genes that produce two regulatory subunits (β1 and β2). All three subunits are expressed throughout the intraerythrocytic developmental cycle and are found in both the cytoplasm and nucleus of the parasite (Hitz et al. 2021). However, the functional significance of CK2 substrates in Plasmodium development remains poorly understood, and identification and functional characterization of parasite proteins regulated by phosphorylation are essential for understanding the molecular mechanisms that coordinate parasite growth and differentiation.

The PBANKA_0620800 gene encoding the liver stage maturation protein (LSMP) was previously identified in a P. berghei gametocyte proteome (Hollin et al. 2019) and was also detected in the surface proteome of P. falciparum salivary gland sporozoites (Swearingen et al. 2016). Sequence analysis revealed that LSMP is a parasite‐specific protein and contains a repetitive domain enriched with multiple consensus motifs for CK2‐mediated phosphorylation. Its detection in gametocytes and the sporozoite surface proteome, together with its parasite‐specific conservation, suggested that LSMP plays an important role in parasite development. Since no functional characterization of this gene had been reported previously, we generated LSMP knockout parasites to investigate its role during the parasite life cycle. We demonstrate that LSMP is dispensable for blood‐stage replication and mosquito‐stage development but plays an important role during liver stage maturation. Parasites lacking LSMP exhibit defects in late liver stage development characterized by reduced nuclear numbers, decreased merozoite formation, and delayed rupture of the parasitophorous vacuole membrane.

2. Results

2.1. LSMP Is a Conserved Plasmodium Protein With Multiple Predicted Phosphorylation Sites

We first analyzed LSMP (PBANKA_0620800) through bioinformatic analysis. LSMP is highly conserved within the Plasmodium genus (Figure S1). The closest homologue is a hypothetical P. berghei protein (GenBank: SCO60006.1) sharing 92% amino acid identity with PBANKA_0620800. Homologues in other rodent malaria parasites (P. yoelii, P. vinckei, and P. chabaudi) shared 44%–67% identity, whereas those in human and non‐human primate Plasmodium species were more divergent (14%–34% identity). More distant homologues were identified in other apicomplexan parasites, including Haemoproteus majoris, Toxoplasma gondii, Besnoitia besnoiti, Cryptosporidium serpentis , and Neospora caninum, but shared only 10%–13% sequence identity and are largely uncharacterized. Pairwise comparisons showed strong conservation between closely related species (e.g., P. gaboni/P. adleri, 86%; T. gondii/N. caninum, 60%), indicating that this protein family diverges rapidly outside closely related species (Figure S1). To investigate its structural organization, we downloaded an AlphaFold model of LSMP from PlasmoDB (Figure 1). The predicted structure forms an extended parallel β‐solenoid (β‐roll)‐like fold with repeating β‐strands, a structure often involved in protein–protein interactions. Consistent with this structure, LSMP contains a repetitive region spanning residues 333–511 that comprises six tandem 28‐amino acid repeats. Each repeat contains a predicted Casein kinase II phosphorylation motif (S/T‐X‐X‐D/E), with alternating serine‐based (SNAD) and threonine‐based (TVFE) motifs throughout the repeat array (Figure 1). Phosphorylation site prediction using NetPhos 3.1a identified multiple high‐confidence serine, threonine, and tyrosine phosphorylation sites throughout LSMP, with serine residues predominating (Figure S2). A striking feature was the enrichment of predicted phosphorylation sites within the central tandem repeat region. Each repeat contains the conserved motif TVFESQRCE, in which the serine residue is consistently predicted to be phosphorylated. This motif occurs six consecutive times (S360, S388, S416, S444, S472, and S500), forming a regularly spaced cluster of potential phosphorylation sites. Additional high‐confidence phosphorylation sites were predicted throughout the N and C‐terminal regions (S71, T74, T98, S147, S200, S214, T216, T261, S302, T328, T512, S528, and S541). LSMP also lacks a predicted signal peptide and transmembrane domains, suggesting that it is a soluble protein. Together, these analyses identify LSMP as a conserved, soluble Plasmodium protein with a repeat‐rich β‐solenoid structure and a phosphorylation‐rich central domain, suggesting that phosphorylation may regulate its function during parasite development.

FIGURE 1.

Structural analysis of LSMP and identification of CK2 phosphorylation motifs. AlphaFold structure of P. berghei LSMP showing the repetitive domain composed of six tandem repeats of 28 amino acids spanning residues 333–511. Each repeat contains consensus phosphorylation motifs (S/T‐X‐X‐D/E) consistent with potential substrates of casein kinase II. Alternating serine‐based (SNAD) and threonine‐based (TVFE) motifs are indicated.

2.2. LSMP Is Expressed in Multiple Parasite Stages and Localizes to the Cytosol

To check the expression and localization of LSMP, we generated peptide‐based antibody. Western blot analysis detected a band of approximately 64.4 kDa in WT parasites, whereas no band was detected in LSMP KO parasites (Figure S3A). Immunofluorescence analysis showed specific staining of WT liver stage parasites but no signal in LSMP KO parasites (Figure S3B). Immunofluorescence assays were performed across different parasite stages. In blood‐stage schizonts, strong cytosolic staining of LSMP was observed when parasites were co‐stained with anti‐HSP70 antibodies (Figure 2A). We next examined LSMP expression in mosquito and liver stages. IFA of sporozoites revealed cytosolic localization of LSMP (Figure 2B). To analyze liver stage expression, HepG2 cells were infected with sporozoites and cultures were harvested at various time points post infection. IFA with anti‐LSMP and HSP70 antibodies revealed colocalization, confirming a cytosolic localization of LSMP during liver stage development (Figure 2C). Based on the LSMP staining pattern, we next examined its association with apicoplast using an anti‐ACP antibody. The distinct, non‐overlapping staining patterns showed that LSMP does not localize to the apicoplast (Figure 2D). To determine whether LSMP associates with parasite membranes, infected hepatocyte cultures were co‐stained with anti‐LSMP and merozoite surface protein 1 (MSP1) antibodies. The LSMP signal did not overlap with MSP1, indicating that LSMP does not localize to parasite membranes (Figure 2E). Together, these results demonstrate that LSMP is expressed throughout the parasite life cycle and localizes predominantly to the parasite cytosol.

FIGURE 2.

Expression and localization of LSMP during parasite stages. (A) P. berghei infected blood stage slides were prepared, immunostained with anti‐HSP70 and anti‐LSMP antibodies. Merged images (Merge 1) show the colocalization of LSMP with cytosolic marker HSP70 in the schizonts with PCC (Pearson correlation coefficient) values of 0.92 of 15 parasites. (B) Immunofluorescence assays of sporozoite of P. berghei were performed using antibodies against LSMP and the cytosolic marker HSP70 (PCC = 0.85 of 15 parasites). (C) HepG2 cells were infected with P. berghei sporozoites, fixed at different time points (24, 40, and 55 hpi), and immunostained with anti‐HSP70 and anti‐LSMP antibodies. LSMP shows predominantly cytosolic localization across all stages (PCC = 0.91 of 20 parasites, PCC = 0.96 of 20 parasites, PCC = 0.84 of 20 parasites), with a staining pattern overlapping with HSP70. (D) Association of LSMP with the apicoplast was examined by co‐immunostaining liver‐stage parasites with anti‐ACP and anti‐LSMP antibodies. Colocalization was not observed at both 40 hpi (PCC = 0.22 of 12 parasites) and 55 hpi (PCC = 0.33 of 12 parasites). (E) At 65 hpi, parasites were stained with anti‐MSPI and anti‐LSMP antibodies. Parasite nuclei were stained with Hoechst 33342.

2.3. LSMP Is Dispensable for Blood‐Stage Growth and Mosquito‐Stage Development

To investigate the functional role of LSMP, we generated LSMP KO parasites using double crossover homologous recombination. In the targeting construct, the LSMP open reading frame was replaced with a GFP‐hDHFR:yFCU cassette (Figure S4A). Successful integration of the targeting cassette was confirmed by GFP expression (Figure S4B). Clonal parasite lines were obtained by limiting dilution, and correct integration of the construct was verified by diagnostic PCR and Southern blot analysis (Figure S4C,D). In addition, a complemented parasite line was generated by reintroducing the LSMP expression cassette into LSMP KO parasites (Figure S4E,F). To assess asexual blood stage propagation of LSMP KO parasites, mice (5 mice per group) were intravenously injected with 3.6 × 10⁶ infected red blood cells (iRBCs), and parasite growth was monitored by Giemsa‐stained blood smears. No significant differences in blood‐stage growth rates were observed between WT GFP, LSMP comp, and LSMP KO (c1 and c2) parasites (Figure S4G). We next evaluated parasite development within the mosquito vector. Mosquitoes were allowed to feed on infected mice, and parasite development was monitored at multiple time points. At 24 h post infection, midguts were dissected to assess ookinete formation and morphology. No differences were observed between WT GFP and LSMP KO parasites (Figure S5A,B). Similarly, oocyst formation and sporogony were normal (Figure S5C–F). Salivary gland sporozoites load and numbers were also unaffected in LSMP KO parasites (Figure S5G,H). Together, these results indicate that LSMP is not required for parasite development during blood or mosquito stages.

2.4. LSMP Is Required for Efficient Liver Stage Infection In Vivo

To assess the role of LSMP during liver stage infection, C57BL/6 mice (5 mice per group) were injected intravenously with 5 × 10³ WT GFP, LSMP comp, or LSMP KO (c1 and c2) sporozoites. The onset of blood‐stage parasitemia was monitored by daily examination of Giemsa‐stained blood smears. Mice infected with WT parasites developed detectable parasitemia on day 3 post infection. In contrast, mice infected with LSMP KO parasites exhibited a delay of approximately 3 days in the prepatent period (Table 1). To identify whether this delay was associated with impaired liver stage development, parasite burden in the liver was quantified. To investigate defects during liver stage development, we selected two time points, 40 and 55 hpi. At 40 hpi, parasites remain within the liver, whereas by 55 hpi they are maturing and beginning to exit hepatocytes. Livers from infected mice were collected at both time points, and parasite burden was quantified by measuring parasite 18S rRNA. Parasite burden at 40 hpi was similar between LSMP KO and WT GFP parasites (Figure 3). Mice infected with LSMP KO parasites showed a significant reduction in liver stage parasite burden compared to WT GFP parasites at 55 hpi (Figure 3). These results indicate that LSMP is not required for early liver stage development but is important for late liver stage development and transition to the blood stage.

TABLE 1.

Infectivity of LSMP KO sporozoites in C57BL/6 mice.

Experiment	Parasite	Number of sporozoites inoculated	Mice positive/Mice inoculated	Prepatent period (days)
1	WT GFP	5000	5/5	3
	LSMP KO c1	5000	5/5	5
	LSMP KO c2	5000	5/5	6
	LSMP comp	5000	5/5	3
2	WT GFP	5000	5/5	3
	LSMP KO c1	5000	5/5	6
	LSMP KO c2	5000	5/5	6
	LSMP comp	5000	5/5	3.4
3	WT GFP	5000	5/5	3.2
	LSMP KO c1	5000	5/5	5
	LSMP KO c2	5000	5/5	6
	LSMP comp	5000	5/5	3

FIGURE 3.

LSMP KO parasites exhibit reduced liver stage parasite burden in vivo. To determine liver stage parasite burden, salivary gland sporozoites from WT and LSMP KO parasites were intravenously injected into C57BL/6 mice (n = 5 mice/group). Livers were harvested at 40 and 55 hpi, homogenized, and total RNA was isolated. Parasite burden was quantified by quantitative real‐time PCR (qRT‐PCR) by amplifying Pb18S rRNA, with transcript levels normalized to mouse GAPDH expression. Parasite burden was similar between WT GFP and LSMP KO parasites at 40 hpi (p = 0.1082) but was significantly reduced in LSMP KO infected mice at 55 hpi (*p = 0.0119, Student's t‐test). Data are presented as mean ± SEM from two independent experiments.

2.5. LSMP Is Not Required for Hepatocyte Invasion but Affects Liver Stage Development

To determine whether the reduced liver stage infection resulted from impaired invasion, sporozoite invasion assays were performed using HepG2 cells. Sporozoites (10⁴) were incubated with 6 × 10⁴ HepG2 cells, and intracellular and extracellular parasites were quantified (Figure S6A,B). We found that LSMP KO sporozoites invaded hepatocytes with efficiencies comparable to WT GFP parasites, indicating that LSMP does not play a role in host cell invasion. We next monitored intracellular parasite development at multiple time points following infection. IFA revealed that LSMP KO parasites displayed normal growth patterns during early liver stage development up to 24 hpi. However, parasite development was impaired at later stages. Although the number of EEFs was comparable between WT GFP and LSMP KO parasites, the EEF area was significantly increased in LSMP KO parasites (Figure 4A–C). To further investigate this phenotype, parasite burden was quantified in infected hepatocytes at 40 and 55 hpi by measuring parasite 18S rRNA. Consistent with the in vivo observations, LSMP KO parasites exhibited a reduced parasite burden, with the defect being more pronounced at 55 hpi (Figure 4D). These data indicate that the enlarged EEF size observed in LSMP KO parasites does not correlate with increased parasite biomass and instead likely reflects abnormal late liver stage development.

FIGURE 4.

LSMP KO parasites development during liver‐stage. (A) HepG2 cultures infected with WT GFP and LSMP KO sporozoites were fixed at 24, 48, and 65 hpi and immunostained with anti‐UIS4 antibody to visualize the parasitophorous vacuole membrane. Parasite nuclei were stained with Hoechst. Qualitative analysis indicated that KO liver stage parasite size appeared higher compared to WT GFP. (B) Quantification of EEF numbers at 24, 48, and 65 hpi showed no significant difference between WT GFP and LSMP KO parasites (p = 0.8794 at 24 hpi, p = 0.6535 at 48 hpi, p = 0.9007 at 65 hpi). Data are presented as mean ± SEM from three independent experiments performed in duplicate. One‐way ANOVA was used to assess statistical significance. (C) Measurement of EEF size at 24, 48, and 65 hpi revealed comparable parasite sizes between WT GFP and LSMP KO parasites at 24 hpi (p = 0.0699). The size was significantly increased at 48 and 65 hpi (****p < 0.0001, n = 100 EEFs per parasite strain). Data were pooled from three independent experiments performed in duplicate and are presented as mean ± SEM. Statistical significance was determined using One‐way ANOVA. (D) Parasite burden was quantified in infected HepG2 cells harvested at 40 and 55 hpi. No significant difference was observed at 40 hpi (p = 0.6152), whereas parasite burden was significantly reduced at 55 hpi (*p = 0.0494, Student's t‐test). Data are presented as mean ± SEM from three biological replicates.

2.6. LSMP KO Parasites Exhibit Impaired Apicoplast Development, Reduced Merozoite Formation and Delayed PVM Rupture

To examine liver stage maturation, parasites fixed at 65 hpi were stained with antibodies against MSP1. Cytomeres and merozoites were observed in both WT GFP and LSMP KO parasites; however, the frequency of cytomere and merozoite formation was visibly reduced in LSMP KO compared with WT GFP parasites (Figure 5A). To further assess parasite development, nuclei were quantified in WT GFP, LSMP comp, and LSMP KO (c1 and c2) EEFs. LSMP KO EEFs contained significantly fewer nuclei than WT GFP and LSMP comp parasites (Figure 5B). To analyze EEF maturation in greater detail, different developmental forms, including schizonts, cytomeres, and merozoites, were quantified. LSMP KO cultures showed a higher proportion of schizonts and a lower proportion of cytomeres and merozoites compared with WT GFP parasites (Figure 5C), indicating delayed liver stage maturation in the absence of LSMP. Consistent with maturation delay, cultures infected with LSMP KO parasites produced fewer detached cells despite comparable numbers of EEFs (Figure 5E). When EEFs were examined at a later time point at 89 hpi, an increased proportion of LSMP KO parasites had progressed to cytomere and merozoite stages and detached cell formation was also increased (Figure 5D,F), suggesting that LSMP deficiency causes a delay rather than a complete block in liver stage development. Importantly, detached cells generated by LSMP KO parasites remained fully infectious when injected into mice, indicating that the merozoites produced were functionally competent (Table 2). We next examined organelle biogenesis during liver stage development at 65 hpi. Immunostaining with antibodies against ACP, IscU, and BiP revealed impaired apicoplast branching in LSMP KO parasites, whereas mitochondrial and ER branching appeared unaffected (Figure 5G–L). Because delayed maturation resulted in reduced detached cell formation, we further assessed PVM rupture by staining parasites with antibodies against the PVM marker UIS4 and quantifying parasites with intact or ruptured PVMs (Figure 6A). At 65 hpi, a substantially higher proportion of LSMP KO parasites retained intact PVMs compared with WT GFP parasites (Figure 6B), indicating delayed PVM rupture. Previous studies have shown that Plasmodium parasites disrupt host cell actin plasma membrane interactions to facilitate egress from infected hepatocytes (Burda et al. 2017). To investigate whether delayed egress in LSMP KO parasites was associated with altered host cytoskeletal remodeling, infected cells were stained with anti‐β‐actin antibodies. LSMP KO parasite‐infected hepatocytes showed increased accumulation of host actin around the parasites compared with WT‐infected cells (Figure 6C,D), consistent with impaired host cell cytoskeletal reorganization during egress. Together, these results demonstrate that LSMP is required for efficient liver stage maturation, proper apicoplast development, timely merozoite formation, and efficient rupture of the parasitophorous vacuole membrane during hepatocyte egress.

FIGURE 5.

LSMP KO parasites exhibit a defect during late liver stage development. (A) HepG2 cultures infected with WT GFP and LSMP KO sporozoites were fixed at 65 hpi and immunostained with anti‐MSP1 antibody to visualize the merozoites. Parasite nuclei were stained with Hoechst. (B) Quantification of EEFs nuclei produced in WT and LSMP KO cultures showed a significant decrease in KO (p = 0.0016, One‐way ANOVA). We analyzed 50 EEFs per condition, pooled data from three independent experiments, and expressed as mean ± SEM. (C, D) Quantification of liver stage developmental forms at 65 and 89 hpi. At 65 hpi, LSMP KO cultures contained significantly more schizonts (***p = 0.0008) and fewer merozoites (***p = 0.0004) than WT GFP cultures, whereas cytomere numbers were comparable (p = 0.3391). At 89 hpi, LSMP KO cultures still exhibited an increased proportion of schizonts (**p = 0.0017) together with fewer cytomeres (*p = 0.0348) and more merozoites (**p = 0.0018) than 65 hpi cultures. Data were obtained from three independent experiments performed in duplicate and are presented as mean ± SEM. (E, F) Detached cells (merosomes) were collected from culture supernatants at 65 and 89 hpi and counted using a hemocytometer. WT and KO infected cultures contained comparable numbers of EEFs. LSMP KO parasites produced significantly fewer detached cells compared with WT GFP parasites (****p < 0.0001, Student's t‐test) at 65 hpi. Detached cell numbers were increased at the later time point (89 hpi; *p = 0.0138). Data are pooled from three independent experiments and presented as mean ± SEM. (G, I and K) Cultures fixed at 65 hpi were immunostained with anti‐ACP, anti‐Bip and anti‐IscU antibodies to visualize apicoplast, ER and mitochondrial branching, respectively. Nuclei were stained with Hoechst 33342. (H, J and L) Quantification of EEFs showing normal ER branching (p = 0.6254, Student's t‐test) and mitochondrial branching (p = 0.4522, Student's t‐test) in WT and KO parasites. A significant number of EEFs show impaired apicoplast branching in LSMP KO parasites (*p = 0.0344, Student's t‐test). A total of 100 EEFs were analyzed for each from two independent experiments performed in duplicate (data presented as mean ± SD).

TABLE 2.

Infectivity of detached cells in Swiss mice.

Experiment	Parasite	Number of detached cells inoculated	Mice positive/Mice inoculated	Prepatent period (days)
1	WT GFP	10	5/5	3
	LSMP KO c1	10	4/4	3
	LSMP KO c2	10	3/3	3
	LSMP comp	10	5/5	3
2	WT GFP	15	5/5	3
	LSMP KO c1	10	2/2	4
	LSMP KO c2	10	2/3	3
	LSMP comp	15	5/5	3
3	WT GFP	20	5/5	2
	LSMP KO c1	Supernatant	3/3	2
	LSMP KO c2	Supernatant	2/2	2
	LSMP comp	20	5/5	2

FIGURE 6.

LSMP KO parasites exhibit delayed PVM rupture during liver stage development. (A) HepG2 cultures infected with WT and LSMP KO sporozoites were fixed at 65 hpi and immunostained with PVM markers anti‐UIS4 antibody. Parasite nuclei were stained with Hoechst 33342. (B) Quantification of intact and broken PVMs revealed a significantly higher proportion of EEFs with intact PVMs in LSMP KO parasites compared with WT GFP parasites (****p < 0.0001). Data were obtained from three independent experiments performed in duplicate and are presented as mean ± SEM. Statistical significance was determined using one‐way ANOVA. (C) Infected cultures fixed at 65 hpi were immunostained with anti‐β‐Actin antibody to visualize host cell Actin organization. Parasite nuclei were stained with Hoechst 33342. Distinct Actin localization patterns in infected hepatocytes, condensed/dotted, absent, or associated with the plasma membrane, were categorized and quantified. (D) Quantification of Actin localization patterns revealed a significantly higher proportion of plasma membrane–associated Actin in cells infected with LSMP KO parasites compared with WT‐GFP parasites (*p = 0.0272, Student's t‐test) and a higher percentage of cells had no plasma membrane in WT in comparison to KO (**p = 0.0050, Student's t‐test) whereas the other Actin patterns were similar between the two groups (p = 0.3337, Student's t‐test). A total of 120 (WT) and 150 (LSMP KO) EEFs were analyzed. Data were pooled from two independent experiments and are presented as mean ± SD.

3. Discussion

The liver stage of malaria infection represents a critical developmental phase in the life cycle of Plasmodium parasites, during which a single invading sporozoite undergoes extensive replication to generate thousands of merozoites that initiate the pathogenic blood‐stage infection. Although significant progress has been made in identifying factors involved in hepatocyte invasion and early liver stage development (Vaughan and Kappe 2017), the molecular mechanisms that regulate parasite maturation in hepatocytes remain incompletely understood. In this study, we characterize a cytosolic protein, LSMP, in the rodent malaria parasite P. berghei and demonstrate that it plays a critical role during late liver stage development.

Our results show that LSMP is expressed throughout the parasite life cycle and predominantly localizes to the parasite cytosol. This observation contrasts with a previous surface proteome study that identified LSMP in the surface fraction of P. falciparum salivary gland sporozoites (Swearingen et al. 2016). Importantly, LSMP lacks canonical features typically associated with membrane‐targeted proteins, including a signal peptide, transmembrane domain, and GPI‐anchor motif, arguing against stable membrane localization. Given that surface proteomic approaches can detect highly abundant peripheral proteins, the presence of LSMP in the sporozoite surface proteome may reflect transient associations rather than a bona fide membrane protein. Nevertheless, we cannot exclude the possibility that LSMP associates with parasite membranes under specific conditions, and this will require further investigation. Despite the broad expression pattern, genetic disruption of LSMP revealed that the protein is dispensable for parasite replication during both blood‐stage growth and mosquito‐stage development. However, our data demonstrate that LSMP plays an important role during liver stage development. This raises the question of why LSMP is specifically required during liver stage development while appearing dispensable for asexual blood and mosquito stages, even though all of these stages involve extensive DNA replication and organelle biogenesis to generate invasive forms. One possible explanation lies in the remarkable scale of parasite replication during liver stage schizogony. A single invading sporozoite can produce tens of thousands of hepatic merozoites, whereas blood‐stage schizogony typically yields only a few dozen daughter merozoites, and mosquito sporogony produces comparatively less sporozoites per oocyst than the number of merozoites formed in the liver. Such extensive proliferative expansion likely requires tighter coordination of transcriptional, translational, and metabolic processes to sustain rapid parasite growth and organelle replication (Roques et al. 2023). Under these conditions, a distinct set of regulatory proteins may become essential, and LSMP may represent one such factor that supports efficient parasite maturation during liver stage development. In addition to nuclear replication, liver stage development imposes a substantial demand for lipid biosynthesis and membrane expansion. The formation of thousands of daughter merozoites requires extensive membrane biogenesis for the parasite plasma membrane and the inner membrane complex. Liver stage parasites therefore depend on both de novo lipid synthesis and the acquisition of host‐derived lipids to support rapid membrane formation (Roques et al. 2023). Increasing evidence indicates that protein kinases play central roles in coordinating metabolic pathways with parasite development, including the regulation of lipid synthesis and membrane biogenesis. In this context, LSMP may function within kinase‐regulated signaling networks that couple parasite replication with lipid metabolic demands, thereby ensuring efficient membrane formation and parasite proliferation during liver stage schizogony. However, whether LSMP itself is phosphorylated remains to be experimentally validated, and the kinase responsible for its phosphorylation has yet to be identified.

LSMP adds to the growing list of gene products that are specifically required for late liver stage development but are dispensable during asexual blood stage replication and mosquito stage development. Mutants lacking fatty acid synthesis type II (FASII) enzymes were shown to be essential specifically for late liver stage development (Yu et al. 2008; Vaughan et al. 2009). Other proteins implicated in late liver stage development include stearoyl‐CoA desaturase (SCD) (Narwal et al. 2024), malonyl‐CoA‐acyl carrier protein transacylase (MCAT) (Devi et al. 2025), liver‐specific proteins 1 (LISP1) (Ishino et al. 2009) and 2 (LISP2) (Orito et al. 2013), sporozoite conserved orthologous transcript 1 (Scot1) (Ghosh, Mishra, et al. 2024), the putative RNA‐binding protein Mei2 (Dankwa et al. 2016), serine–threonine kinase 2 (Jillapalli et al. 2019), and the major facilitator superfamily orphan transporter MFS6 (Kenthirapalan et al. 2016). The stage‐specific phenotypes associated with disruption of these genes highlight the distinct biological requirements of Plasmodium replication within hepatocytes, and our findings identify LSMP as another protein with a specialized role during late liver stage development. Liver stage schizogony involves repeated rounds of nuclear division followed by synchronized segmentation into thousands of daughter cells (Graewe et al. 2011). Disruption of factors that regulate nuclear replication or cell cycle progression during this phase can therefore result in reduced merozoite production without necessarily affecting the structural process of segmentation. Similar phenotypes have been observed following disruption of proteins involved in parasite replication and cell cycle regulation during liver stage development (Gubbels et al. 2021).

Although LSMP KO parasites displayed increased EEF size in vitro, their primary defect appears to be a delay in maturation, as evidenced by reduced parasite burden in vitro and in vivo. Similar enlargement of liver‐stage parasites has also been reported following treatment with takinib and HS220, indicating that increased EEF size can occur independently of successful parasite development (Raphemot et al. 2019). Consistent with these findings, the enlarged EEF phenotype of LSMP KO parasites likely reflects delayed or abnormal maturation rather than enhanced development. Whether LSMP KO parasites also exhibit increased EEF size in vivo remains to be determined. Parasite replication during liver stage schizogony requires extensive organelle biogenesis and membrane remodeling. One important phenotype observed in LSMP KO parasites was a defect in apicoplast branching. The apicoplast is an essential plastid‐derived organelle that undergoes extensive elongation and branching during schizogony to ensure its proper segregation into daughter merozoites (Yeh and DeRisi 2011). Defects in apicoplast replication or inheritance have been associated with impaired parasite growth and reduced parasite viability (Foth et al. 2005). The defect in apicoplast branching observed in LSMP‐deficient parasites may therefore contribute to the reduced efficiency of parasite replication and merozoite formation during late liver stage development. Recent work has highlighted the importance of coordination between nuclear replication and organelle segregation during parasite development, suggesting that signaling pathways linking these processes are critical for successful parasite proliferation (Gubbels et al. 2021; Elaagip et al. 2022). In addition to defects in parasite maturation, LSMP‐deficient parasites exhibited delayed PVM rupture. Despite these abnormalities, detached cells produced by LSMP KO parasites retained normal infectivity when injected into mice. This observation indicates that the merozoites that do successfully form remain fully competent to initiate blood‐stage infection. Therefore, the impaired egress observed in LSMP KO parasites is most likely a secondary consequence of defective liver stage maturation rather than an intrinsic defect in merozoite viability or infectivity.

The molecular mechanism through which LSMP regulates these processes remains to be determined. Sequence analysis revealed that LSMP contains a repetitive domain enriched in consensus motifs for phosphorylation by casein kinase II (CK2), a highly conserved serine/threonine kinase implicated in diverse cellular processes including cell cycle regulation and cytoskeletal organization (Peng et al. 2011). Although CK2 activity has been detected in Plasmodium (Dorin‐Semblat et al. 2025), only a limited number of CK2 substrates have been functionally characterized in malaria parasites. The presence of multiple CK2 consensus motifs within LSMP raises the possibility that the protein may participate in signaling pathways that coordinate late liver stage development. Cytosolic regulatory proteins often function as scaffolds that integrate signaling networks controlling cell division, organelle segregation, and membrane remodeling (DiRusso et al. 2022). Future studies aimed at identifying LSMP‐interacting proteins and determining its post‐translational regulation will therefore be important for elucidating the molecular pathways that control parasite maturation in hepatocytes.

In conclusion, we demonstrate that LSMP plays an important role during late liver stage development in P. berghei. Although LSMP is dispensable during blood and mosquito stages, it plays a critical role in coordinating efficient nuclear replication, merozoite formation, and timely rupture of the parasitophorous vacuole membrane during liver stage development. These findings highlight the importance of regulatory pathways controlling parasite maturation within hepatocytes and provide new insights into the molecular mechanisms governing the transition from liver to blood‐stage infection.

4. Experimental Procedures

4.1. Mice, Mosquitoes, Parasites, and Cell Lines

Female Swiss albino mice (6–8 weeks old) were used for parasite maintenance and mosquito transmission experiments, whereas C57BL/6 mice (6–8 weeks old) were used for P. berghei infection. Mice were maintained under controlled environmental conditions as previously described (Devi et al. 2025). Rodent malaria parasite strains P. berghei ANKA (MRA‐311), P. berghei ANKA GFP (MRA‐867, 507 m6cl1) were obtained from BEI Resources, USA. P. berghei sporozoites were generated by infecting female Anopheles stephensi mosquitoes, as previously described (Narwal et al. 2024). Mosquitoes were maintained in an environmental chamber at 28°C and 80% relative humidity. Parasite liver stage development was assessed by infecting human hepatocellular carcinoma (HepG2) cells, as described earlier (Mishra et al. 2024).

4.2. Bioinformatic Analysis of LSMP

The amino acid sequence of LSMP (PBANKA_0620800) was retrieved from PlasmoDB and analyzed using bioinformatic approaches. Homologous proteins were identified by BLASTP searches against the NCBI non‐redundant (nr) protein database using default parameters (Altschul et al. 2005). Representative homologues from Plasmodium and other apicomplexan species were selected based on sequence similarity. Pairwise amino acid sequence identities obtained from BLAST alignments were compiled into a similarity matrix and visualized as a heatmap using custom Python scripts with the Pandas, NumPy, Matplotlib, and Seaborn libraries. Putative phosphorylation sites were predicted using NetPhos 3.1a (Blom et al. 2004), and serine, threonine, and tyrosine residues with prediction scores above the default threshold (0.5) were considered potential phosphorylation sites. The AlphaFold predicted three‐dimensional structure of LSMP was retrieved from PlasmoDB and used to analyze its overall structure and predicted phosphorylation motifs.

4.3. Generation of the Antibodies

An affinity‐purified polyclonal rabbit antibody against P. berghei LSMP was generated by immunizing rabbits with the synthetic peptide CEVEPKANRPKKIKN. Antibody production and affinity purification were carried out by GenScript (Piscataway, NJ, USA). A polyclonal mouse anti‐LSMP antibody was also generated using the same synthetic peptide.

4.4. Generation of LSMP KO and Complemented Parasite Lines

The P. berghei LSMP gene (PBANKA_0620800) was disrupted by double‐crossover (DCO) homologous recombination. For gene targeting, two fragments F1 (0.62 kb) and F2 (0.56 kb) from the 5′ and 3′ untranslated regions (UTRs) were amplified using primer pairs 1579/1580 and 1581/1582, respectively. These fragments were sequentially cloned into the pBC‐GFP‐hDHFR:yFCU vector at the XhoI/SalI and NotI/AscI restriction sites. The resulting construct was linearised with XhoI/AscI and transfected into P. berghei ANKA schizonts as described previously (Janse et al. 2006). Transfected parasites were selected by oral administration of pyrimethamine drug (Sigma‐Aldrich, 46706), and clonal lines were obtained by limiting dilution. Correct 5′ and 3′ integration of the targeting cassette was verified by diagnostic PCR using primer pairs 1630/1225 and 1215/1631, respectively. Further, Southern blot was performed to validate the genomic modification as previously described (Nayak et al. 2024). Briefly, genomic DNA was digested with EcoRV and probed with a 5′ specific fragment resulting in bands of 9.7 kb and 3.6 kb in WT and LSMP KO parasites, respectively. The LSMP complemented parasite line was generated by restoring gene function. A 2.8 kb fragment (F5) containing the 5′UTR, open reading frame (ORF), and 3′UTR of LSMP was amplified using primers 1579/1582 and transfected into LSMP KO schizonts. Transfected parasites were negatively selected using 5‐fluorocytosine (5‐FC; Sigma‐Aldrich, F7129) as described previously (Srivastava and Mishra 2022). Restoration of the LSMP locus was confirmed by diagnostic PCR with primers 2241/2242.

4.5. Blood‐Stage Growth and Mosquito‐Stage Development

To assess asexual blood‐stage propagation of LSMP KO parasites, Swiss mice were intravenously inoculated with equal numbers of infected red blood cells (iRBCs), as described previously (Varshney et al. 2025). Parasitemia was monitored daily by microscopic examination of Giemsa‐stained blood smears. For mosquito transmission, female Anopheles mosquitoes were allowed to feed on infected mice. To evaluate ookinete development, mosquito midguts were dissected 24 h after the infectious blood meal. Midguts were homogenized and examined by fluorescence microscopy, and ookinete numbers were quantified using a hemocytometer as previously described (Varshney et al. 2025). To assess oocyst formation and sporogony, midguts from infected mosquitoes were dissected and imaged 14 days post‐blood meal, and oocysts were counted. For quantification of midgut sporozoites, a subset of dissected midguts was homogenized using a pestle, and sporozoites were enumerated using a hemocytometer. Salivary gland sporozoite numbers were determined by dissecting salivary glands from infected mosquitoes 18–22 days post‐blood meal, followed by enumeration of sporozoites as described previously (Ghosh, Varshney, et al. 2024).

4.6. In Vivo Sporozoite Infection and Quantification of Liver Stage Parasite Burden

C57BL/6 mice (n = 5 per group) were infected by intravenous injection of salivary gland sporozoites. The onset of blood‐stage parasitemia was monitored by daily examination of Giemsa‐stained blood smears. To quantify liver stage parasite burden, a separate set of C57BL/6 mice was intravenously inoculated with 5000 salivary gland sporozoites. Livers were harvested at 40 and 55 hpi and homogenized in RNAiso Plus (Takara, 9108). Total RNA was isolated according to the manufacturer's instructions, and complementary DNA (cDNA) was synthesized as described previously (Choudhary et al. 2019). Parasite biomass was determined by quantitative real‐time PCR (qRT‐PCR) by amplifying parasite 18S rRNA using primer pair 1195/1196 on a CFX Opus 96 Real‐Time PCR System (Bio‐Rad). Parasite 18S rRNA levels were normalized to mouse Gapdh expression amplified using primer pair 1193/1194. All primers used in this study are listed in Table S1.

4.7. Sporozoite Invasion Assay

Sporozoite invasion assay was performed as previously described (Ghosh, Varshney, et al. 2024). Briefly, HepG2 cells were infected with freshly isolated sporozoites (10,000 per well) and incubated for 90 min to allow parasite invasion. Cells were then fixed with 4% PFA, and extracellular and intracellular sporozoites were distinguished using a differential immunofluorescence assay with an antibody against the circumsporozoite protein (CSP) (Yoshida et al. 1980).

4.8. Analysis of In Vitro Liver Stage Development

HepG2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). For liver stage infection assays, 6 × 10⁴ cells per well were seeded onto collagen‐coated 48‐well plates and infected with 5 × 10³ sporozoites. Infected cultures were fixed with 4% paraformaldehyde (PFA) (Sigma‐Aldrich, HT5012) at different time points. For the detached cell (merosome) assay, 10⁵ HepG2 cells were seeded onto collagen‐coated 24‐well plates and infected with 3 × 10⁴ sporozoites per well. Culture supernatants were collected at 65 and 89 hpi, and detached cells were enumerated using a hemocytometer. To assess infectivity, equal numbers of detached cells were intravenously injected into Swiss albino mice, and the onset of blood‐stage infection was monitored by daily examination of Giemsa‐stained blood smears. To estimate parasite burden, infected HepG2 cells cultured in 24‐well plates were harvested at the indicated time points using RNAiso Plus (Takara, 9108). Total RNA was extracted, reverse‐transcribed into cDNA, and parasite transcript levels were quantified by real‐time PCR as described above. Parasite transcript levels were normalized to human β‐actin using primers 1190/1191.

4.9. Western Blotting

WT and LSMP KO blood stage parasites were solubilized in laemmli sample buffer (Bio‐Rad, 1610747), samples were resolved via 12% SDS‐PAGE and transferred onto a PVDF membrane (Bio‐Rad, 1620177) by electroblotting. The membrane was blocked with 1% BSA‐PBS for 1 h at room temperature and followed by incubation with anti‐LSMP antibody (1:500) or anti‐β actin antibodies (1:1000) (CST #4970). The blot was washed three times with 1×PBST and incubated with HRP‐conjugated anti‐rabbit (1:5000) (Amersham Biosciences # NA934V) for 1 h at room temperature. The membrane was again washed three times with 1×PBST and the signals were detected via an enhanced chemiluminescence (ECL) substrate (Bio‐Rad # 170‐5060), and images were captured via the ChemiDoc XRS+ system (Bio‐Rad, USA) as described earlier (Mishra et al. 2023).

4.10. Immunofluorescence Assays

Fixed Plasmodium infected HepG2 cultures were permeabilized with methanol for 20 min at 4°C. Following permeabilization, samples were blocked with 1% bovine serum albumin (BSA) in phosphate‐buffered saline (PBS) and incubated with primary antibodies for 1–2 h at RT. The following primary antibodies were used: anti‐CSP (1:1000) (Yoshida et al. 1980), anti‐ACP (1:1000) (Gallagher and Prigge 2010), anti‐BiP (1:125), anti‐UIS4 (1:1000) (Mueller, Camargo, et al. 2005), anti‐HSP70 (1:1000), anti‐IscU (1:100) (Mohammad et al. 2021), and anti‐MSP1 (1:5000) (Holder and Freeman 1981). Primary antibodies were detected using Alexa Fluor 488‐ or 594‐conjugated secondary antibodies (Invitrogen, 1:1000), as described previously (Mishra et al. 2025). Nuclei were counterstained with Hoechst 33342 (Sigma‐Aldrich, 41399), and coverslips were mounted using ProLong diamond antifade reagent (Invitrogen, P36970). The number of EEFs was quantified manually, and parasite area was measured using Nikon NIS‐Elements BR software on an Eclipse 80i fluorescence microscope equipped with a Plan Fluor 40×/0.75 objective. Images of asexual blood‐stage and mosquito‐stage parasites were acquired using a Leica DM3000 LED microscope. EEF images were acquired using FV1000 software on an Olympus BX61WI confocal laser‐scanning microscope.

4.11. Statistical Analysis

Statistical analyzes were performed using GraphPad Prism 9. Differences between two groups were evaluated using a two‐tailed unpaired Student's t‐test, whereas comparisons among multiple groups were analyzed using one‐way analysis of variance (ANOVA). A p value of < 0.05 was considered statistically significant.

Author Contributions

Shabeer Ali: methodology. Rohini Nandi: conceptualization, methodology, investigation, validation, writing – original draft. Satish Mishra: conceptualization, data curation, investigation, validation, formal analysis, supervision, funding acquisition, visualization, writing – review and editing. Akancha Mishra: methodology.

Disclosure

All figures, tables, and text in this manuscript are original and generated by the authors. The work does not include any copyrighted, trademarked, or previously published material that requires permission for reuse.

Ethics Statement

All animal experiments were conducted at the animal facility of the CSIR‐Central Drug Research Institute (CSIR‐CDRI), India. All experimental procedures were approved by the institutional animal ethics committee (IAEC) of CSIR‐CDRI (IAEC approval number: IAEC/2023/15) and were carried out in accordance with the guidelines of the committee for the purpose of control and supervision of experiments on animals (CPCSEA), India.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Pairwise sequence identity analysis of LSMP and homologous proteins from representative Plasmodium and other apicomplexan species. Heatmap showing the percentage amino acid sequence identity obtained from pairwise sequence comparisons. Warmer colors indicate higher sequence identity, whereas cooler colors indicate lower sequence similarity. LSMP is highly conserved among rodent Plasmodium species but shows progressively lower sequence identity with primate Plasmodium species and other apicomplexan homologues, consistent with increasing evolutionary divergence.

FIGURE S2: Predicted phosphorylation sites in LSMP. Predicted serine (red), threonine (green), and tyrosine (blue) phosphorylation sites were identified using NetPhos 3.1a and are plotted according to their phosphorylation potential scores. The horizontal magenta line indicates the default prediction threshold (0.5). Multiple serine, threonine, and tyrosine residues exceeded this threshold, indicating that LSMP is likely to undergo extensive phosphorylation.

FIGURE S3: (A) Western blot analysis of WT and LSMP KO blood stage parasite lysates. The blot was first probed with an anti‐LSMP antibody, a band of ~64.4 kDa was detected in WT parasites; no such band was detected in LSMP KO parasites. The membrane was reprobed with an anti‐β actin antibody as a loading control. (B) HepG2 cultures infected with WT GFP and LSMP KO sporozoites were fixed at 40 hpi and immunostained with anti‐LSMP antibody. A diffuse cytosolic staining pattern was observed in WT parasites, whereas no signal was detected in the KO parasites. Parasite nuclei were stained with Hoechst 33342.

FIGURE S4: LSMP is not required for asexual blood‐stage propagation. (A) Schematic representation of the LSMP gene deletion strategy. Following double‐crossover homologous recombination, the WT locus was replaced with an integration cassette containing GFP and a hDHFR:yFCU marker. (B) Representative fluorescence image showing GFP‐expressing LSMP KO blood‐stage parasites. (C) Confirmation of correct genomic integration of the targeting cassette by diagnostic PCR using primer sets 1834/1225 and 1215/1835 to verify 5′ and 3′ integration, respectively. WT genomic DNA (gDNA) was used as a negative control. The primer pair 1836/1837 was used to confirm the absence of the WT LSMP locus in the KO parasites. (D) Southern blot analysis confirmed successful modification of the LSMP locus. Genomic DNA was digested with EcoRV, and the blot was probed using a 5′ probe, yielding bands of 9.7 kb in WT parasites and 3.6 kb in LSMP KO parasites. (E) Schematic representation of the genetic complementation strategy used to reintroduce the WT LSMP gene into the KO locus. (F) Restoration of the WT LSMP locus in complemented parasites was confirmed by diagnostic PCR using primer pair 1836/1837. (G) Asexual blood‐stage growth analysis of WT, LSMP KO, and LSMP complemented parasite lines (5 mice per group). No significant difference in parasite growth was observed among the groups (p = 0.9568, one‐way ANOVA). Data from two independent experiments are presented as mean ± SEM.

FIGURE S5: LSMP KO parasites develop normally during mosquito stages. (A) Representative images showing normal ookinete morphology in WT GFP and LSMP KO parasites. (B) Quantification of ookinete numbers revealed no significant difference between WT GFP and LSMP KO parasites (p = 0.3599). Data from 90 (WT GFP), 102 (LSMP KO c1), 119 (LSMP KO c2), 89 (LSMP comp) mosquitoes are shown. (C) Representative images showing midgut oocyst development in mosquitoes infected with WT GFP and LSMP KO parasites. (D) Quantification of oocysts showed that the number of oocysts formed in LSMP KO infected mosquito midguts was comparable to that observed in WT infected midguts (p = 0.0988). (E) Representative images showing sporogony within oocysts in WT GFP and LSMP KO parasites. (F) Quantification of midgut sporozoites revealed no significant difference between WT GFP and LSMP KO parasites (p = 0.3533). A total of 111 (WT GFP), 103 (LSMP KO c1), 110 (LSMP KO c2), and 103 (LSMP comp) mosquito midguts were analyzed. (G) Representative images showing salivary gland infection in mosquitoes infected with WT GFP and LSMP KO parasites. (H) Quantification of salivary gland sporozoites showed comparable sporozoite loads between WT GFP and LSMP KO parasites (p = 0.1940). Data from 100 (WT GFP), 95 (LSMP KO c1), 93 (LSMP KO c2), and 103 (LSMP comp) mosquitoes were analyzed. Data are presented as mean ± SEM and were analyzed using One‐way ANOVA.

FIGURE S6: LSMP KO sporozoites efficiently invade hepatocytes. (A) HepG2 cultures infected with WT GFP and LSMP KO sporozoites were fixed at 1.5 h post infection (hpi). Coverslips were subjected to a differential immunofluorescence assay using an anti‐CSP antibody before and after permeabilization. Extracellular sporozoites that failed to invade hepatocytes were stained red, whereas green fluorescence represents the number of total sporozoites. (B) Quantification of invasion efficiency showed no significant difference between WT GFP and LSMP KO sporozoites (p = 0.1711, Student's t‐test). Data are presented as mean ± SD and represent pooled results from two independent experiments.

TABLE S1: List of primers used in this study.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.